Appendices of the Final Screening Assessment

Bacillus amyloliquefaciens 13563-0
Bacillus atrophaeus 18250-7
Bacillus licheniformis American Type Culture Collection (ATCC) 12713
Bacillus subtilis ATCC 6051A (=ATCC 6051a)
Bacillus subtilis ATCC 55405
Bacillus subtilis subspecies subtilis ATCC 6051^T
Bacillus subtilis subspecies inaquosorum ATCC 55406
Bacillus species 16970-5
Bacillus species 2 18118-1
Bacillus species 4 18121-4
Bacillus species 7 18129-3

Environment Canada
Health Canada
August 2015

Appendices

Appendix 1: Colony Morphologies of DSL B. licheniformis/subtilis Group Members
Appendix 2: Characteristics of Domestic Substances List (DSL) B. licheniformis/subtilis Group Members – 16S Ribosomal ribosomal ribonucleic acid (RNA) Gene Sequence Analysis
Appendix 3: Characteristics of DSL B. licheniformis/subtilis Group Members – Fatty Acids Methyl Ester (FAME) Analysis
Appendix 4: Cellular Content of Select Fatty Acids
Appendix 5: List of Some Mobile Elements and Associated Traits Identified in Certain Isolates of the B. subtilis Complex
Appendix 6: Virulence Genes
Appendix 7: Virulence and Pathogenicity Testing of DSL B. licheniformis/subtilis Strains: Hemolytic Activity
Appendix 8: Virulence and Pathogenicity Testing of DSL B. licheniformis/subtilis Strains: Catalase Production
Appendix 9: Antimicrobial Compound, Other Metabolites and Toxins Produced by certain isolates of the B. subtilis Complex
Appendix 10: Virulence and Pathogenicity Testing of DSL B. licheniformis/subtilis Strains: Cytotoxicity
Appendix 11: Pathogenicity, Toxicity and Irritation Testing Results for strains of the B. subtilisComplex on Terrestrial and Aquatic Vertebrates, Invertebrates and Plants
Appendix 12: Virulence and Pathogenicity Testing of the DSL B. licheniformis/subtilisStrains
Appendix 13: Food Poisoning Outbreaks
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Appendix 1: Colony Morphologies of Domestic Substances List (DSL) B. licheniformis/subtilis group members

**Table A-1: Colony morphologies *of B. amyloliquefaciens* 13563-0**
Characteristic	Tryptone Soya Broth (TSB) agar after 7 days of growth at room temperaturea	Spizizen potato agar or broth (ATCC Medium 423) at 37°C verified at 24 hours (for solid medium, add 1.5% agar) ^{Footnote Appendix 1 Table A1 [a]}
Shape	Irregular	Irregular
Size (mm) diameter	5	5
Margin	Undulate	Spreading, irregular edge
Elevation	Flat	No data
Colour/pigment	Off-white	White
Texture	Dull	Smooth, dull
Opacity	Opaque	No data

Footnote Appendix 1 Table A1 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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**Table A-2: Colony morphologies of *B. atrophaeus* 18250-7**
Characteristic	TSB agar after 24 hours of growth at room temperature^{Footnote Appendix 1 Table A2 [a]}	TSB agar after 7 days of growth at room temperature^[a]	Nutrient agar at 30°C for 24 hours^[a]	Nutrient agar or broth (ATCC medium #3) at 30°C after 24 hours under aerobic conditions^{Footnote Appendix 1 Table A2[b]}	Nutrient agar or broth (ATCC medium #3) at 30°C after 24 hours under aerobic conditions^[b]	Nutrient agar or broth (ATCC medium #3) at 30°C after 24 hours under aerobic conditions^[b]
Shape	Circular	Irregular	Circular	Circular	Circular	Irregular
Size (mm) diameter	2	5-10	0.5	No data	No data	No data
Margin	Entire	Undulate	Entire	Entire	Entire	Undulate
Elevation	Flat	Flat	No data	Low convex	Low convex	Flat
Colour/ pigmentation^[a]	White	Off-white/beige	White	Orange	No data	White
Texture	Smooth, moist	Smooth, moist	No data	Glistening	Glistening	No data
Opacity	Opaque	Opaque	No data	No data	Opaque	No data

Footnote Appendix A Table A2 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A2 b

ATCC description, multiple colony morphologies

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**Table A-3: Colony morphologies of *B. licheniformis* ATCC 12713**
Characteristic	TSB agar after 7 days of growth at room temperature^{Footnote Appendix A Table A3 [a]}, ^{Footnote Appendix A Table A3 [b]}	TSB agar after 7 days of growth at room temperature^[a],^[b]	Nutrient agar at 30°C for 24 hours^[a]	Nutrient agar or broth (ATCC medium #3) at 30°C for 24 hours under aerobic conditions^{Footnote Appendix A Table A3[c]}	Nutrient agar or broth (ATCC medium #3) at 30°C for 24 hours under aerobic conditions^[c]
Shape	Circular	Irregular	Irregular	No data	Irregular
Size (mm) diameter	5-7	5-7	2	No data	No data
Margin	Undulate	Undulate-lobate	Undulate, filiform	No data	No data
Elevation	Flat	Umbonate	Raised	Raised	Convex
Colour/pigment	Beige/off-white	Beige/off-white	No data	No data	No data
Texture	Moist, smooth	Wrinkled, dry	Dry	Dry, wrinkled	Mucoid
Opacity	Semi-translucent	Opaque	Opaque	No data	No data

Footnote Appendix A Table A3 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A3 b

Colonies stick to agar, multiple colony morphologies

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Footnote Appendix A Table A3 c

ATCC description, multiple colony morphologies

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**Table A-4: Colony morphologies of *B. subtilis* ATCC 6051A**
Characteristic	TSB agar after 7 days of growth at room temperature^{Footnote Appendix A Table A4 [a]}	Nutrient agar at 30°C for 24 hours^[a]	Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions^{Footnote Appendix A Table A4 [b]}	Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions^[b]
Shape	Circular to irregular	Irregular	Irregular and spreading with age	Irregular and spreading with age
Size (mm) diameter	6 to 25	2	Larger	Smaller
Margin	Undulate	Entire	Erose	Entire
Elevation	Flat	Flat	Flat	Flat
Colour/pigment	Off-white	Off-white	Beige/cream	Beige/cream
Texture	Moist	Dry	Dull, rougher	Dull, smoother
Opacity	Translucent	Semi-translucent	Opaque	Opaque

Footnote Appendix A Table A4 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A4 b

ATCC description, multiple colony morphologies

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**Table A-5: Colony morphologiesof *B. subtilis* ATCC 55405**
Characteristic	TSB agar after 7 days of growth at room temperature^{Footnote Appendix A Table A5 [a]},^{Footnote Appendix A Table A5[b]}	TSB agar after 7 days of growth at room temperature^[a],^[b]	TSB agar after 7 days of growth at room temperature^[a],^[b]	Nutrient agar at 30°C for 24 hours^[a]	Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions^{Footnote Appendix A Table A5 [c]}
Shape	Irregular to circular	Circular	Irregular to circular	2	Circular
Size (mm) diameter	4	4	4	No data	No data
Margin	Entire	Undulate	Lobate	Entire	Entire
Elevation	Convex	Flat	Raise	Convex	Convex
Colour/pigment	Colourless to off-white	Off-white	White	Colourless	No data
Texture	Glossy, mucoid	Matte, dry	Flat, brittle, dry	Mucoid	Mucoid, glistening
Opacity	Opaque	Translucent to opaque	Opaque	No data	Opaque

Footnote Appendix A Table A5 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A5 b

Multiple colony morphologies

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Footnote Appendix A Table A5 c

ATCC description

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**Table A-6: Colony morphologiesof *B. subtilis* subsp. *subtilis* ATCC 6051**
Characteristic	TSB agar after 7 days of growth at room temperature^{Footnote Appendix A Table A6 [a]}	Nutrient agar at 30°C for 24 hours^[a]	Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions^{Footnote Appendix A Table A6 [b]}	Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions^[b]
Shape	Irregular	Irregular	Circular	Irregular
Size (mm) diameter	20	No data	No data	No data
Margin	Undulate	Undulate	Entire	No data
Elevation	Raised	ND	Low convex	Flat
Colour/pigment	Off-white	Off-white	No data	No data
Texture	Dry	No data	Shiny	Rough
Opacity	Opaque	Opaque	Opaque	Opaque

Footnote Appendix A Table A6 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A6 b

ATCC description, multiple colony morphologies

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**Table A-7: Colony morphologies of *B.subtilis* subsp. *inaquosorum* ATCC 55406**
Characteristic	TSB agar after 7 days of growth at room temperature^{Footnote Appendix A Table A7 [a]},^{Footnote Appendix A Table A7[b]}	Nutrient agar at 30°C for 24 hours^[a]	Nutrient agar (ATCC medium #3) at 30°C verified at 24 hours^{Footnote Appendix A Table A7 [c]},^{Footnote Appendix A Table A7 [d]}	Nutrient agar (ATCC medium #3) at 30°C verified at 24 hours^[c],^[d]
Shape	Circular	Irregular	Circular	Circular
Size (mm) diameter	5	1	No data	No data
Margin	Undulate	No data	Erose	No data
Elevation	Flat	No data	Flat	No data
Colour/pigment	White	Colourless	Cream	White
Texture	Dull	No data	No data	Smooth
Opacity	Opaque	Opaque	Opaque	Opaque

Footnote Appendix A Table A7 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A7 b

Colonies grow into agar

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Footnote Appendix A Table A7 c

ATCC description, multiple colony morphologies

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Footnote Appendix A Table A7 d

Colonies dig into agar; this organism grows better on solid media than in a broth

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**Table A-8: Colony morphologies of *Bacillus* species 16970-5**
Characteristic	TSB agar after 7 days of growth at room temperature^{Footnote Appendix A Table A8 [a]}	Nutrient agar at 30°C for 24 hours^[a]	Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions^{Footnote Appendix A Table A8 [b]}
Shape	Irregular	Irregular	Circular
Size (mm) diameter	12 to 20	2	No data
Margin	Undulate	No data	Some with lobate margins
Elevation	Flat	Flat	No data
Colour	Off-white	Off-white	No data
Texture	Dull	Dry	Shiny, smooth
Opacity	Opaque	Opaque	Opaque

Footnote Appendix A Table A8 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A8 b

ATCC description

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**Table A-9: Colony morphologies of *Bacillus* species 2 18118-1**
Characteristic	TSB agar after 7 days of growth at room temperature^{Footnote Appendix A Table A9 [a]}	Spizizen potato agar or broth (ATCC Medium 423) at 37°C verified at 24 hours (for solid medium, add 1.5% agar)^[a]	Spizizen potato agar or broth (ATCC Medium 423) at 37°C verified at 24 hours (for solid medium, add 1.5% agar)^{Footnote Appendix A Table A9 [b]}	Spizizen potato agar or broth (ATCC Medium 423) at 37°C verified at 24 hours (for solid medium, add 1.5% agar)^[b]
Shape	Irregular	Circular	Circular	No data
Size (mm) diameter	5-12	5	No data	No data
Margin	Undulate	Slightly irregular edge	Slightly irregular	Dull spreading irregular edge
Elevation	Umbonate, raised	No data	No data	No data
Colour/pigment	Whitish	No data	No data	No data
Texture	Moist, shiny and dull	ND	Smooth, glistening	Glistening, smooth center
Opacity	Opaque	Opaque	Opaque	No data

Footnote Appendix A Table A9 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A9 b

ATCC description, multiple colony morphologies

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**Table A-10: Colony morphologies of *Bacillus* species 4 18121-4**
Characteristic	TSB agar after 7 days of growth at room temperature^{Footnote Appendix A Table A10 [a]}	TSB agar after 7 days of growth at room temperature^[a]	Nutrient agar at 30°C for 24 hours^[a]	Nutrient agar or broth (ATCC medium #3) at 37°C for 24 hours under aerobic conditions^{Footnote Appendix A Table A10[b]}	Nutrient agar or broth (ATCC medium #3) at 37°C for 24 hours under aerobic conditions^[b]
Shape	Circular	Elliptical	Irregular	Irregular	Rhizoid
Size (mm) diameter	5-7	5-7	2	No data	No data
Margin	Undulate	Undulate	Undulate, filiform	Undulate	Filamentous
Elevation	Imperfect-umbonate	Flat	Raised	Convex	Raised
Colour	Beige/off-white	Beige/off-white	No data	Translucent	No data
Texture	Moist	Matte	Dry	Smooth, glistening	Rough
Opacity	Opaque	Opaque	Opaque	No data	Opaque

Footnote Appendix A Table A10 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A10 b

ATCC description, multiple colony morphologies

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**Table A-11: Colony morphologies of *Bacillus* species 7 18129-3**
Characteristic	TSB agar after 7 days of growth at room temperature^{Footnote Appendix A Table A11 [a]}	Nutrient agar at 30°C for 24 hours^[a]	Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions^{Footnote Appendix A Table A11 [b]}	Nutrient agar or broth (ATCC medium #3) at 30°C verified at 24 hours and up to one week under aerobic conditions^[b]
Shape	Irregular	Irregular	Circular	Irregular
Size (mm) diameter	20	No data	No data	No data
Margin	Undulate	Undulate	Entire	No data
Elevation	Raised	No data	Low convex	Flat
Colour	Off-white	Off-white	No data	No data
Texture	Dry	No data	Shiny	Rough
Opacity	Opaque	Opaque	Opaque	Opaque

Footnote Appendix A Table A11 a

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch

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Footnote Appendix A Table A11 b

ATCC description, multiple colony morphologies

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Appendix 2: Characteristics of DSL B. licheniformis/subtilis group members – 16S ribosomal RNA gene sequence analysis

16S ribosomal RNA gene sequence data generated by Health Canada's Healthy Environments and Consumer Safety Branch. Restriction fragment length polymorphisms from within the V3 region and between the V4 and V5 region were identified according to Jeyaram et al. 2011. The 16S ribosomal RNA gene sequences were compared to the Ribosomal Database project release 11 (https://rdp.cme.msu.edu/) and top 10 matches are shown. The match hit format is: identification code, similarity score (if reference strain is specified), S ab score, unique common oligomers and sequence full name.

**Table A-12: Results of 16S Ribosomal RNA Gene Sequence Analysis of *B. amyloliquefaciens* 13563^{Footnote Appendix 2 Table A-12 [a]}**
Short identification	Similarity score	S_ab score	Unique common oligomers	Sequence full name
S001153538	1.000	1.000	1364	Bacillus sp. XI; EU779996
S001550906	1.000	1.000	1371	Bacillus subtilis; Y2; GQ148813
S001588402	1.000	1.000	1374	Bacillus amyloliquefaciens; PBT; FJ169495
S001745899	1.000	1.000	1393	Bacillus amyloliquefaciens; IMAU80205; GU125623
S002038639	1.000	1.000	1354	Bacillus amyloliquefaciens; HK1; AB279736
S002222255	1.000	1.000	1447	Bacillus amyloliquefaciens; DSM 7; DSM7; FN597644
S002222257	1.000	1.000	1447	Bacillus amyloliquefaciens; DSM 7; DSM7; FN597644
S002222259	1.000	1.000	1447	Bacillus amyloliquefaciens; DSM 7; DSM7; FN597644
S002222261	1.000	1.000	1447	Bacillus amyloliquefaciens; DSM 7; DSM7; FN597644
S002222263	1.000	1.000	1447	Bacillus amyloliquefaciens; DSM 7; DSM7; FN597644

Footnote Appendix 2 Table A12 a

The HinfI site is present and the RsaI sites are absent indicating the micro-organisms is B. amyloliquefaciens as opposed to B. subtilisto which it is closely related (Jeyaram et al. 2011).

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**Table A-13: Results of 16S Ribosomal RNA Gene Sequence Analysis of *B. atrophaeus* 18250-7^{Footnote Appendix 2 Table A-13 [a]}**
Short identification	Similarity score	S_ab score	Unique common oligomers	Sequence full name
S000382399	Not calculated	1.000	1432	Bacillus sp.; SSA3; AB017587
S000644416	Not calculated	1.000	1436	Bacillus atrophaeus; SCH0408; AY881241
S000980555	Not calculated	1.000	1337	Bacillus atrophaeus; K01-03; EU326483
S001872424	Not calculated	1.000	1411	Bacillus subtilis; JAM A-6-10; AB542912
S002035172	Not calculated	1.000	1375	Bacillus atrophaeus; NMTD54; GU568183
S002035195	Not calculated	1.000	1379	Bacillus atrophaeus; GBSC56; GU568206
S002166857	Not calculated	1.000	1376	Bacillus atrophaeus; RJGP16; GU969134
S002167105	Not calculated	1.000	1372	Bacillus atrophaeus; LSSC3; GU994860
S002221550	Not calculated	1.000	1464	Bacillus atrophaeus 1942; CP002207
S002221552	Not calculated	1.000	1464	Bacillus atrophaeus 1942; CP002207

Footnote Appendix 2 Table A13 a

B. atrophaeus 18250-7 ribosomal RNA gene sequence matches B. atrophaeus and Bacillus sp. sequences and the HinfI siteis present for the B. subtilis/licheniformis group(Jeyaram et al. 2011).

Return to footnote Appendix 2 Table A13 a referrer

**Table A-14: Results of 16S Ribosomal RNA Gene Sequence Analysis of *B. licheniformis* ATCC 12713^{Footnote Appendix 2 Table A-14 [a]}**
Short identification	Similarity score	S_ab score	Unique common oligomers	Sequence full name
S000392549	Not calculated	1.000	1427	Bacillus licheniformis; Mo1; AF372616
S000615411	Not calculated	1.000	1409	Bacillus licheniformis; ACO1; DQ228696
S000647676	Not calculated	1.000	1421	Bacillus licheniformis; K19; DQ351932
S000736754	Not calculated	1.000	1448	Bacillus licheniformis; BCRC 15413; DQ993676
S000752038	Not calculated	1.000	1409	Bacillus licheniformis; EF059752
S000824918	Not calculated	1.000	1422	Bacillus licheniformis; BCRC 12826; EF423608
S000843501	Not calculated	1.000	1442	Bacillus sp. J24; EF471917
S000901702	Not calculated	1.000	1389	Bacillus licheniformis; NBRC 12107; AB354236
S000941823	Not calculated	1.000	1389	Bacillus licheniformis; NBRC 12202; AB363734
S001153503	Not calculated	1.000	1319	Bacillus licheniformis; SVD1; EU770587

Footnote Appendix 2 Table A14 a

Bacillus licheniformis ATCC 12713 16S ribosomal RNA gene sequence matches mainly B. licheniformis ribosomal RNA gene sequences. The restriction fragment length polymorphism (RFLP) pattern (RsaI sites in V3; HinfI and CfoI site between V4 and V5) is consistent with B. licheniformis sp. (Jeyaram et al. 2011).

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**Table A-15: Results of 16S Ribosomal RNA Gene Sequence Analysis of *B. subtilis* ATCC 6051A^{Footnote Appendix 2 Table A-15 [a]}**
Short identification	Similarity score	S_ab score	Unique common oligomers	Sequence full name
S000003473	Not calculated	1.000	1423	Bacillus subtilis (T); DSM10; AJ276351
S000365537	Not calculated	1.000	1446	Bacillus sp. TUT1206; AB188212
S000383767	Not calculated	1.000	1412	Bacillus sp. CH4-4; AB055846
S000383768	Not calculated	1.000	1412	Bacillus sp. CH4-5; AB055848
S000383769	Not calculated	1.000	1412	Bacillus sp. CH15-2; AB055849
S000383770	Not calculated	1.000	1412	Bacillus sp. CH19-3; AB055850
S000383771	Not calculated	1.000	1412	Bacillus sp. CH20-1; AB055851
S000383772	Not calculated	1.000	1412	Bacillus sp. CH7-1; AB055852
S000383773	Not calculated	1.000	1412	Bacillus sp. CH10-1; AB055853
S000434646	Not calculated	1.000	1401	Bacillus subtilis; KL-073; AY030330

Footnote Appendix 2 Table A15 a

B. subtilis ATCC6051A 16S ribosomal RNA gene sequence matches Bacillus subtilis and Bacillus sp. ribosomal RNA gene sequence. The RFLP pattern (RsaI sites in V3; HinfI site between V4 and V5) is consistent for B. subtilis sp. (Jeyaram et al. 2011). However, the first putative RsaI site requires verification as it contains an ambiguous base (the dominant peak appears to correspond to A).

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**Table A-16: Results of 16S Ribosomal RNA Gene Sequence Analysis of *B. subtilis* ATCC 55405^{Footnote Appendix 2 Table A-16 [a]}**
Short identification	Similarity score	S_ab score	Unique common oligomers	Sequence full name
S000870716	Not calculated	1.000	1391	Bacillus amyloliquefaciens; NBRC 14141; AB325582
S001745899	Not calculated	1.000	1393	Bacillus amyloliquefaciens; IMAU80205; GU125623
S002038639	Not calculated	1.000	1354	Bacillus amyloliquefaciens; HK1; AB279736
S002222255	Not calculated	1.000	1447	Bacillus amyloliquefaciens DSM 7; DSM7; FN597644
S002222257	Not calculated	1.000	1447	Bacillus amyloliquefaciens DSM 7; DSM7; FN597644
S002222259	Not calculated	1.000	1447	Bacillus amyloliquefaciens DSM 7; DSM7; FN597644
S002228859	Not calculated	1.000	1224	Bacillus amyloliquefaciens; BAC3048; HM355639
S003280603	Not calculated	1.000	1329	Bacillus amyloliquefaciens; BSS5; JQ407053
S003285855	Not calculated	1.000	1305	Bacillus sp. SE18; JQ714100
S003313087	Not calculated	1.000	1342	Bacillus amyloliquefaciens; KU-8; JQ696827

Footnote Appendix 2 Table A16 a

B. subtilis ATCC 55405 16S ribosomal RNA gene sequence matches mainly B. amyloliquefacienssequences and the HinfI RFLP identified by for B. amyloliquefaciens is present (Jeyaram et al. 2011).

Return to footnote Appendix 2 Table A16 a referrer

**Table A-17: Results of 16S Ribosomal RNA Gene Sequence Analysis of *B. subtilis subsp. subtilis* ATCC 6051^{Footnote Appendix 2 Table A-17 [a]}**
Short identification	Similarity score	S_ab score	Unique common oligomers	Sequence full name
S000398967	Not calculated	1.000	1383	Bacillus subtilis; BHP6-1; AY162131
S001020073	Not calculated	1.000	1422	Bacillus subtilis; B1-33; EU435361
S001096330	Not calculated	1.000	1435	Bacillus sp. zh161; EU526087
S002038710	Not calculated	1.000	1388	Bacillus sp. PT401; AB374305
S002199724	Not calculated	1.000	1420	Uncultured Bacillus sp.; CapF3B.16; HM152583
S002199742	Not calculated	1.000	1386	Uncultured Bacillus sp.; Filt.13; HM152601
S002199744	Not calculated	1.000	1386	Uncultured Bacillus sp.; Filt.15; HM152603
S002199761	Not calculated	1.000	1386	Uncultured Bacillus sp.; Filt.32; HM152620
S002199769	Not calculated	1.000	1385	Uncultured Bacillus sp.; Filt.40; HM152628
S002199775	Not calculated	1.000	1386	Uncultured Bacillus sp.; Filt.46; HM152634

Footnote Appendix 2 Table A17 a

Bacillus species 6051 16S ribosomal RNA gene sequence matches Bacillus subtilis and Bacillus sp. ribosomal RNA gene sequence. The RFLP pattern(two RsaIsites in V3; HinfI site between V4 and V5) is consistent B. subtilis sp. (Jeyaram et al. 2011).

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**Table A-18: Results of 16S Ribosomal RNA Gene Sequence Analysis of *B. subtilis subsp. subtilis* ATCC 6051^{Footnote Appendix 2 Table A-18 [a]}**
Short identification	Similarity score	S_ab score	Unique common oligomers	Sequence full name
S001020073	Not calculated	0.946	1422	Bacillus subtilis; B1-33; EU435361
S001096330	Not calculated	0.946	1435	Bacillus sp. zh161; EU526087
S002038710	Not calculated	0.946	1388	Bacillus sp. PT401; AB374305
S002199724	Not calculated	0.946	1420	Uncultured Bacillus sp.; CapF3B.16; HM152583
S002199860	Not calculated	0.946	1421	Uncultured Bacillus sp.; Filt.132; HM152719
S002199880	Not calculated	0.946	1421	Uncultured Bacillus sp.; Filt.152; HM152739
S002410934	Not calculated	0.946	1413	Bacillus subtilis; MB5 NIOT; HQ858061
S003257857	Not calculated	0.946	1446	Bacillus subtilis subsp. inaquosorum; type strain: DSM 22148; HE582781
S003261902	Not calculated	0.946	1388	Bacillus subtilis; NBRC 3108; AB680011
S003264071	Not calculated	0.946	1388	Bacillus subtilis; NBRC 104440; AB682180

Footnote Appendix 2 Table A18 a

The 16S matches for B. subtilis subsp. inaquosorum ATCC 55406 included both Bacillus sp. and B. subtilis subsp. inaquosorum. The pattern of restriction sites within B. subtilis subsp. inaquosorum ATCC 55406 is different compared to B. licheniformis strengthening the argument that it was misnamed previously.

Return to footnote Appendix 2 Table A18 a referrer

**Table A-19: Results of 16S Ribosomal RNA Gene Sequence Analysis of Bacillus species 16970-5**
The results of the 16S rRNA gene sequencing analysis confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 16970-5 and closest similarity matches using the Ribosomal Database cannot be disclosed.

**Table A-20: Results of 16S Ribosomal RNA Gene Sequence Analysis of Bacillus species 2 18118-1**
The results of the 16S rRNA gene sequencing analysis confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 2 18118-1and closest similarity matches using the Ribosomal Database cannot be disclosed.

**Table A-21: Results of 16S Ribosomal RNA Gene Sequence Analysis of Bacillus species 4 18121-4**
The results of the 16S rRNA gene sequencing analysis confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 4 18121-4 and closest similarity matches using the Ribosomal Database cannot be disclosed.

**Table A-22: Results of 16S Ribosomal RNA Gene Sequence Analysis of Bacillus species 7 18129-3**
The results of the 16S rRNA gene sequencing analysis confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity Bacillus species 7 18129-3 and closest similarity matches using the Ribosomal Database cannot be disclosed.

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Appendix 3: Characteristics of DSL B. licheniformis/subtilis group members – Fatty acids methyl ester (FAME) analysis

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch shows the best match between the sample and the environmental and clinical MIDI databases and the fatty acid profile similarity index (average of all matches) along with the number of matches (number of matches/total number of tests, parentheses). For methods and additional details, visit the Website MIDI Labs. As a general rule of thumb, samples that cluster within a Euclidian distance of 2.5, 6 and 10 represent samples derived from the same strain, subspecies and species, respectively.

**Table A-23: FAME analysis of *B. amyloliquefaciens* 13563-0**
Environmental	Clinical
B. amyloliquefaciens 0.598 (10/15) (B. subtilis group)	B. subtilis 0.735 (12/17) GC subgroup A
B. subtilis 0.441 (3/15)	B. subtilis 0.729 (5/17) GC subgroup B
B. atrophaeus 0.801 (1/15) GC subgroup A	Not applicable
Staphylococcus lutrae 0.490 (1/15) (coag+)	Not applicable

**Table A-24: FAME analysis of *B. atrophaeus* 18250-7**
Environmental	Clinical
B. atrophaeus 0.877 (6/10)	B. atrophaeus 0.814 (5/6) GC subgroup B
Analysis not good enough (4/10) for library search	B. atrophaeus 0.853 (1/6) GC subgroup A

**Table A-25: FAME analysis of *B. licheniformis* ATCC 12713**
Environmental	Clinical
B. licheniformis 0.808 (13/14) (B. subtilis group)	B. licheniformis 0.674 (6/17) (B. subtilis group)
B. megaterium 0.719 (1/14) GC subgroup A	Staphylococcus schleiferi 0.418 (6/17)
Not applicable	B. pumilis-GC subgroup A 0.669 (2/17)
Not applicable	B. pumilis-GC subgroup B 0.468 (2/17)
Not applicable	B. subtilis 0.233 (1/17)

**Table A-26: FAME analysis of *B. subtilis*ATCC 6051A**
Environmental	Clinical
B. subtilis 0.876 (16/17)	B. subtilis 0.872 (13/13)
B. amyloliquefaciens 0.798 (1/17)	Not applicable

**Table A-27: FAME analysis of *B. subtilis*ATCC 55405**
Environmental	Clinical
B. subtilis 0.753 (10/15)	B. subtilis 0.662 (12/12)
B. amyloliquefaciens 0.736 (5/15) (B. subtilis group)	Not applicable

**Table A-28: FAME analysis of *B. subtilis*subsp. *subtilis* ATCC 6051**
Environmental	Clinical
B. subtilis 0.911 (14/14)	B. subtilis 0.760 (9/12)
Not applicable	Analysis not good enough 2/12 for library search
Not applicable	Micrococcus lylae 0.292 (1/12) GC subgroup B

**Table A-29: FAME analysis of *B. subtilis*subsp. *inaquosorum* ATCC 55406**
Environmental	Clinical
B. subtilis 0.803 (10/25)	B. subtilis 0.727 (12/12)
B. amyloliquefaciens 0.793 (9/25) (B. subtilis group)	Not applicable
B. megaterium 0.602 (4/25) GC subgroup A	Not applicable
No match 2/25	Not applicable

**Table A-30: FAME analysis of *Bacillus*species 16970-5**
Environmental	Clinical
The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 16970-5 cannot be disclosed.	The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 16970-5 cannot be disclosed.

**Table A-31: FAME analysis of *Bacillus*species 2 18118-1**
Environmental	Clinical
The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 2 18118-1 cannot be disclosed.	The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 2 18118-1 cannot be disclosed.

**Table A-32: FAME analysis of *Bacillus*species 4 18121-4**
Environmental	Clinical
The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 4 18121-4 cannot be disclosed.	The results generated from the FAME clinical database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 4 18121-4 cannot be disclosed.

**Table A-33: FAME analysis of *Bacillus*species 7 18129-3**
Environmental	Clinical
The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 7 18129-3 cannot be disclosed.	The results generated from the FAME environmental database confirms the identity of this strain as it was purported to be when it was nominated to the Domestic Substances List. However, due to confidentiality claims the identity of Bacillus species 7 18129-3 cannot be disclosed.

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Appendix 4: Cellular content of select fatty acids

**Table A-34: Cellular Content of Select Fatty Acids in DSL *B. licheniformis/subtilis* Group Members^{Footnote Appendix 4 Table A34 [a]}**
DSL Strain	C16:O (%)	Iso-C17:1ω10c (%)
B. amyloliquefaciens 13563-0	3.17	2.4
B. atrophaeus 18250-7	3.07	1.5
B. licheniformis ATCC 12713	2.89	1.56
B. subtilis subsp. subtilis ATCC 6051	4.34	1.95
B. subtilis ATCC 6051A	2.55	2.65
B. subtilis ATCC 55405	3.05	2.34
B. subtilis subsp. inaquosorum ATCC 55406	3.3	1.16
Bacillus species 16970-5	3.52	1.81
Bacillus species 2 18118-1	6.09	1.77
Bacillus species 4 18121-4	3.41	1.11
Bacillus species 7 18129-3	4.34	1.95

Footnote Appendix 4 Table A34 a

Unpublished data generated by Health Canada's Healthy Environments and Consumer Safety Branch

Return to footnote Appendix 4 Table A34 a referrer

Appendix 5: List of some mobile elements and associated traits identified in certain isolates of the B. subtilis complex

Table A-35: List of Some Mobile Elements and Associated Traits Identified in some strains of *B. licheniformis*
Element Name	Associated Traits	References
Plasmid (unknown name)	Dimethoate resistance and additional genes for antibiotic and heavy metal resistance (Na, Er, Ch, Cz, Cf, Ba2+ and Zn2+)	(Mandal et al. 2005)
Plasmid (pBL1, pBL10, pBL2)	Not specified	(Zawadzki et al. 1996)
Insertion element (IS3Bli1)	Encodes two predicted overlapping protein coding sequences, designated orfA and orfB in relative translational reading frames of 0 and 1. Eight of these elements lie in intergenic regions and one interrupts the comPgene	(Rey et al. 2004)
Prophase sequences (NZP1 and NZP3)	Codes for the large subunit of terminase, a signature protein that is highly conserved among prophages	(Rey et al. 2004)

Table A-36: List of Some Mobile Elements and Associated Traits Identified in some strains of *B. subtilis*
Element Name	Associated Traits	References
Transposon (Tn917)	Used in transposition mutagenesis (rapid cloning and construction of transcriptional gene fusions and the characterization of genes which are over-expressed)	(Pragai et al. 1994)
Plasmid (pLS20)	Promotes transfer of tetracycline resistance plasmid pBC16 from B. subtilis (natto) to the Bacillus species B. anthracis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis and B. thuringiensis.	(Koehler and Thorne, 1987)
Transposon (Tn5)	Neomycin phosphotransferase gene	(Sprengel et al. 1985)
Rolling circle replication (RCR) plasmid pTA family (pTA1015, pTA1040, pTA1050 and pTA1060)	Contains genes encoding a type I signal peptidase and genes encoding proteins belonging to the family of response regulator aspartate phosphatases likely to be involved in the regulation of post-exponential phase processes	(Meijer et al. 1998)
Plasmid (pBS4, pBS12, pBS7, pBS8, pBS6)	Not specified	(Zawadzki et al. 1996)
Transposon (integrative and conjugative elements class: ICEBs1)	Integrated into the trnS-leu2 gene is regulated by the SOS response and the RapI-PhrI cell-cell peptide signaling system When DNA damage occurs or high concentrations of potential mating partners that lack the element, ICEBs1 is excised from the chromosome and transferred to recipients.	(Auchtung et al. 2005)
Tn916 (transposon)	Implicated in the horizontal transfer of antibiotic resistance genes in many species of Gram positive bacteria Transfer of this element may increase the presence of tetracycline	(Celli and Trieu-Cuot, 1998; Marra and Scott, 1999)
Tn5397 (transposon)	Originates from Clostridium difficile; element transfers to and from B. subtilis Encodes a conjugation system that is similar to that of Tn916 Contains a group II intron	(Roberts et al. 2001; Wang and Mullany, 2000)
Tn5398 (transposon)	Originates from C. difficile Facilitates the transfer ofan MLS resistance gene (ermBZ)	(Mullany et al. 1995)
IS4Bsu1 (mobile element)	Spontaneously translocates to the swrA gene in B. subtilis natto; causes a defect in poly-gamma- glutamic acid (gamma-PGA) synthesis	(Kimura et al. 2011)

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Appendix 6: Virulence genes

**Table A-37: List of Some Virulence Genes Identified in Certain Isolates of the *B. subtilis*Complex**
Species	Virulence Gene(s)	Associated Traits	References
B. amyloliquefaciens	HblC, HblD, HblA, NheB, NheA	Enterotoxin production and discontinuous beta-hemolysis	(Phelps and McKillip, 2002)
B. licheniformis	cesA	Cereulide synthase	(Nieminen et al. 2007)
B. licheniformis	lchAA, lchAB, lchAC	Lichenysin synthase	(Nieminen et al. 2007)
B. licheniformis	bceT, hblC, hblA, hblD	Hbl enterotoxin, Bacillus hemolytic enterotoxin	(Oguntoyinbo and Sanni, 2007; Rowan et al. 2001)
B. licheniformis	lchAA, lchAB and lchAC (lichenysin synthase genes)	Surfactant lichenysin Heat-stable cyclic lipopeptide toxins; immobilizes boar sperm Structurally similar to cereulide, but the toxic activity appears to be different; it has the potential to form ion channels in host cell membranes and has a surfactant effect	(Logan, 2012; Mikkola et al. 2000; Nieminen et al. 2007; Peypoux et al. 1999)
B. licheniformis	bacA, bacB, bacC (bacitracin synthetases genes)	Cyclic polipeptides; interferes with cell wall and peptidoglycan synthesis of Gram positive and negative bacteria Possibly/indirectly related to erythromycin resistance	(Ishihara et al. 2002)
B. licheniformis and B. subtilis	bceT, hblC, hblA, hblD	Diarrhoeagenic enterotoxin production	(Rowan et al. 2001)

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Appendix 7: Virulence and Pathogenicity Testing of DSL B. licheniformis/subtilis Strains: Hemolytic Activity

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch. Strains were streaked onto 5% sheep blood agar and incubated for 37°C for 24 hours. Hemolysis was judged by clearing zones around colonies which indicate lysis of red blood cells.

**Table A-38: Hemolytic activity of DSL *B. licheniformis/subtilis* strains**
DSL Strain	Hemolytic Activity
B. amyloliquefaciens 13563-0	+^{Footnote Appendix 7 Table A38 [a]},^{Footnote Appendix 7 Table A38 [b]}
B. atrophaeus 18250-7	-^{Footnote Appendix 7 Table A38 [c]}
B. licheniformis ATCC 12713	+^{Footnote Appendix 7 Table A38 [d]}
B. subtilis ATCC 6051A	w^{Footnote Appendix 7 Table A38 [e]}
B. subtilis ATCC 55405	w
B. subtilis subsp. subtilis ATCC 6051	w
B. subtilis subsp. inaquosorum ATCC 55406	+
Bacillus species 16970-5	w
Bacillus species 2 18118-1	w
Bacillus species 4 18121-4	-
Bacillus species 7 18129-3	w

Footnote Appendix 7 Table A38 a

+, hemolytic activity

Return to footnote Appendix 7 Table A38 a referrer

Footnote Appendix 7 Table A38 b

Hemolysis was seen in 5 to 10% of colonies

Return to footnote Appendix 7 Table A38 b referrer

Footnote Appendix 7 Table A38 c

-, no hemolytic activity

Return to footnote Appendix 7 Table A38 c referrer

Footnote Appendix 7 Table A38 d

Hemolysis was seen in 70 to 80% of colonies

Return to footnote Appendix 7 Table A38 d referrer

Footnote Appendix 7 Table A38 e

w, weak hemolytic activity – clearing zones do not extend past colony margin

Return to footnote Appendix 7 Table A38 e referrer

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Appendix 8: Virulence and pathogenicity testing of DSL B. licheniformis/subtilis strains: Catalase production

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch. Bacteria were propagated on TSB agar at 28°C for 48 hours. Hydrogen peroxide dropped on to colony to determine conversion to water and hydrogen. Catalase positive reaction indicated that a given bacteria has the capacity to protect itself from reactive oxygen-induced killing from immune cells.

**Table A-39: Catalase production of DSL *B. licheniformis/subtilis* strains**
DSL Strain	Catalase Production
B. amyloliquefaciens 13563-0	+ (positive)
B. atrophaeus 18250-7	+
B. licheniformis ATCC 12713	+
B. subtilis ATCC 6051A	+
B. subtilis ATCC 55405	+
B. subtilis subsp. subtilis ATCC 6051	+
B. subtilis subsp. inaquosorum ATCC 55406	+
Bacillus species 16970-5	+
Bacillus species 2 18118-1	+
Bacillus species 4 18121-4	+
Bacillus species 7 18129-3	+

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Appendix 9: Antimicrobial Compound, Other Metabolites and Toxins Produced by certain isolates of the B. subtilis Complex

Table A-40: Antimicrobial compounds produced in some strains of *B. amyloliquefaciens*
Substance Name	Activity	References
Bacteriocin-like peptides	Broad antibacterial spectrum with activity against Gram positive bacteria	(Reviewed in Abriouel et al. 2011; Smitha and Bhat, 2012)
BLIS^{Footnote Appendix 9 Table A40 [a]}5940, BLIS RC-2, BLIS 5006	Antibacterial and antifungal activity	(Reviewed in Abriouel et al. 2011)
Surfactin, iturin, bacillomycine, azalomycin F, acivicin, arthrobactin, rhodutorola acid, valinomycin, stenothricin, enterochelin, nocardamin	Antibacterial and antifungal activity, inhibition of growth	(Wulff et al. 2002)
Subtilosin A	Bacteriocin with bactericidal activity against Gram negative bacteria Spermicidal activity against boar, bovine, horse, rat and human spermatozoa	(Reviewed in Abriouel et al. 2011)

Footnote Appendix 9 Table A40 a

BLIS: bacteriocin-like inhibitory substances

Return to footnote Appendix 9 Table A40 a referrer

Table A-41: Antimicrobial compounds produced in some strains of *B. atrophaeus*
Substance Name	Activity	References
Subtilosin A	Bacteriocin with bactericidal activity against Gram negative bacteria Spermicidal activity against boar, bovine, horse, rat and human spermatozoa	(Reviewed in Abriouel et al. 2011)

Table A-42: Antimicrobial compounds produced in some strains of *B. licheniformis*
Substance Name	Activity	References
Amoebicins (A12-A and A12-B)	Amoebolytic activity against Naegkriafowkri Antibiotic activity against yeasts (Saccharomyces heterogenicus and Cryptococcus neoformans) and several fungal species	(Galvez et al. 1993)
Amoebicin (m4-A)	Bactericidal and bacteriolytic activity on Bacilus megaterium GR10	(Lebbadi et al. 1994)
Antibiotics (bacitracin, licheniformin, proticin)	Antibiotics which are secreted and may inhibit competing organisms including Gram positive and Gram negative bacteria, yeasts and molds	(Reviewed in Katz and Demain, 1977)
Bacillocin, BLIS P40, BLIS ZJU12, BLIS MKU3, peptide A-12 C	Antibacterial and antifungal activity	(Reviewed in Abriouel et al. 2011)
Bacteriocin BL8	Thermostable, broad pH range Antibacterial against Gram positive bacteria	(Smitha and Bhat, 2012)
Bacteriocin-like peptide	Broad spectrum antagonistic activity activities against fungal pathogens and Gram positive bacteria but not most Gram negative bacteria	(He et al. 2006)
Ieodoglucomides A and B	Cytotoxic activity against lung and stomach cancer cells lines Moderate antimicrobial activity	(reviewed in Tareq et al. 2012)
Lichenicidin (α, β)	Antibacterial activity associated with the cell surface	(Reviewed in Abriouel et al. 2011; Begley et al. 2009; Dischinger et al. 2009)
Lichenin	Bacteriocin produced under anaerobic conditions	(Reviewed in Abriouel et al. 2011)
Lichenysin	Lipopeptide that acts as an anionic biosurfactin as well as an antimicrobial	(Li et al. 2010; Nerurkar, 2010; Nieminen et al. 2007)
Surfactin, iturin, acivicin, arthrobactin, rhodutorola acid, valinomycin	Antibacterial and antifungal activity, inhibition of growth	(Wulff et al. 2002)

Table A-43: Antimicrobial compounds produced in some strains of *B. subtilis*
Substance Name	Activity	Reference
Anti-bacterials (ericin S and A, sublancin 168, subtilin B, subtilosin A and A1, mersacidin, betacin, MJP1, Bac 14B, LFB112)	Bacterial activity against bacterial pathogens	(Reviewed in Abriouel et al. 2011)
Antibiotics (mycobacillin, subtilin, bacilysin, bacillomycin, fungistatin, bulbiformin, bacillin, subsporin, bacillocin, mycosubtilin, fungocin, iturin, neocidin, eumycin)	Antibiotics which may inhibit competing organisms including Gram positive and negative bacteria as well as yeasts and molds	(reviewed in Katz and Demain, 1977)
Antibiotics (subtilin, ericin, mersacidin, sublancin 168, subtilosin, surfactin, iturin, bacillomycin, mycosubtilin, fengycin plipastatin, corneybactin, bacilysin, bacilysocin, amicoumacin, mycobacillin, TL-119, rhizocticin, difficidin, 3,3'-neotrehalos-adiamine 168)	Anti-microbial activity, biofilm and swarming development, pheromones in quorum sensing and 'killing factor'.	(reviewed in Stein, 2005)
Heat-stable, protease resistant antimicrobial substance	Inhibits growth of many bacteria	(reviewed in Sorokulova et al. 2008)
Subtilin	Antimicrobial peptide Affects pore formation in the cytoplasmic membrane Produced in higher amounts under starvation conditions to eliminate competing species and increase available nutrients	(Reviewed in Abriouel et al. 2011)
Surfactin	Lipopeptide that acts as a biosurfactin and a potent antimicrobial	(Li et al. 2010)

Table A-44: Toxic metabolites produced by some strains of *B. amyloliquefaciens*
Substance Name	Activity	References
Amylosin	Inhibits boar sperm motility Cytotoxic to feline lung cells	(Mikkola et al. 2007)

Table A-45: Toxic metabolites produced by some strains of *B. licheniformis*
Substance Name	Activity	References
Non-emetic heat stable cytotoxic component	Cytotoxicity activity	(De Jonghe et al. 2010)
Heat labile cytotoxic substance	Cytotoxicity activity	(De Jonghe et al. 2010)
B. cereus-like protein toxin	Reduction in cellular metabolic activity Cytotoxic activity	(Beattie and Williams, 1999)
Emetic toxin	Induces vomiting if ingested Ionophoric uptake of K+ resulting in the dissipation of the transmembrane potential, stimulating swelling and respiration in mitochondria which leads to their inactivation	(Biesta-Peters et al. 2010; Reviewed in From et al. 2005)
Hemolysin BL (Hbl) enterotoxin	Causes diarrhea	(Rowan et al. 2001)
Heat labile B. cereus diarrheal-like toxin	Cytotoxic to McCoy cells causing leaky membranes, disrupts cell surfaces and decreases metabolic activity	(Lindsay et al. 2000)
Lichenysin	Inhibits sperm motility Synthesized in both aerobic and anaerobic condition during growth Species specific variations (A, B, C, D, G and surfactant BL86)	(Li et al. 2010; Nerurkar, 2010; Nieminen et al. 2007)
Lichenysin A	Causes loss of motility, damage to plasma membrane and acrosome, loss of cellular NADH and ATP in boar spermatozoa Toxic towards natural (non-malignant) mammalian cells May be produce aerobically and anaerobically More powerful compared to surfactin and lichenysin B	(Mikkola et al. 2000; Yakimov et al. 1996)
Non-proteinaceous, heat-stable, sperm toxic agent	Inhibits sperm motility and swells acrosome Damages cell membrane integrity Depletes cellular ATP Beta-hemolytic activity	(Salkinoja-Salonen et al. 1999)
NucB	Degrades extracellular DNA that is an essential building block of both single species and mixed biofilms Nontoxic deoxyribonuclease Sporulation-specific enzyme	(Rajarajan et al. 2013; Shakir et al. 2012)
Surfactin	Inhibits phytopathogenic fungi	(Nerurkar, 2010)

Table A-46: Toxic metabolites produced by some strains of *B. subtilis*
Substance Name	Activity	References
Hemolysin BL (Hbl) enterotoxin	Causes diarrhea	(Rowan et al. 2001)
Non-emetic heat stable cytotoxic component and a heat labile cytotoxic substance	Cytotoxicity activity	(De Jonghe et al. 2010)
Protolytic and lipolytic substances	Lysis of proteins and lipids	(De Jonghe et al. 2010)
Putative emetic toxin	Causes nausea and vomiting	(From et al. 2005)

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Appendix 10: Virulence and pathogenicity testing of DSL B. licheniformis/subtilis strains: Cytotoxicity

Data generated by Health Canada's Healthy Environments and Consumer Safety Branch. The MTT Assay was used to determine the cytotoxic potential of the strains towards HT29 (colonic epithelial cells) and J774A.1 (macrophage cells). MTT is a yellow, soluble bromide salt which is reduced to a purple, insoluble formazan crystal by dehydrogenase enzymes of living cells (indicating mitochondrial activity). In the crystal state after reduction, it is trapped inside the cell. DMSO or another solvent such as isopropanol or mineral oil can be used to solubilize the formazan, which can then exit the cell, turning the solvent a purple colour that is detectable with a spectrophotometer. This assay is suitable for animal cells that are adherent. Metabolically active bacterial cells can also reduce MTT also. Since most animal cells are not adherent bacteria and their formazan contribution can be rinsed away with PBS prior to solubilisation.

HT29 and J774A.1 were incubated at 37°C in the presence of 5% carbon dioxide. Mammalian cells were dosed with 10⁶CFU/well of vegetative bacteria for 2, 4 and 24 hours. Dose cells were washed twice with PBS before adding MTT.

Loss in bioreduction activity of the cell lines toward MTT was measured to determine the cytotoxic potential of the DSL B. licheniformis/subtilis group strains. Cytotoxicity is related to increased losses in bioreduction activity of the cell lines.

**Table A-47: Cyototoxic potential of DSL *B. licheniformis/subtilis* strains towards HT29 cells with gentamicin at 2, 4 and 24 hours**
DSL Strain^{Footnote Appendix 10 Table A47[a]}	2 hours	4 hours	24 hours
B. amyloliquefaciens 13563-0	w^{Footnote Appendix 10 Table A47 [b]}	w	w
B. atrophaeus 18250-7	w^{Footnote Appendix 10 Table A47 [c]}	w	w
B. licheniformis ATCC 12713	w	w	w
B. subtilis subsp. inaquosorum ATCC 55406	w	-	-
Bacillus species 16970-5	w	-	w
Bacillus species 2 18118-1	-	-	-
Bacillus species 4 18121-4	w	w	w

Footnote Appendix 10 Table A47 a

No data available for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051^T and Bacillusspecies 7 18129-3

Return to footnote Appendix 10 Table A47 a referrer

Footnote Appendix 10 Table A47 b

w, weak cytotoxic activity (5-50% bioreduction loss)

Return to footnote Appendix 10 Table A47 b referrer

Footnote Appendix 10 Table A47 c

Related to structural components

Return to footnote Appendix 10 Table A47 c referrer

**Table A-48: Cyototoxic potential of DSL *B. licheniformis/subtilis* strains towards HT29 cells without gentamicin at 2, 4 and 24 hours**
DSL Strain	2 hours	4 hours	24 hours
B. amyloliquefaciens 13563-0	w^{Footnote Appendix 10 Table A48 [a]}	w	w
B. atrophaeus 18250-7	w^{Footnote Appendix 10 Table A48 [b]}	w	w
B. licheniformis ATCC 12713	w	w	+^{Footnote Appendix 10 Table A48 [c]},^{Footnote Appendix 10 Table A48 [d]}
B. subtilis ATCC 6051A	ND^{Footnote Appendix 10 Table A48[e]}	w	w
B. subtilis ATCC 55405	ND	-^{Footnote Appendix 10 Table A48 [f]}	-
B. subtilis subsp. subtilis ATCC 6051^T	ND	w	w
B. subtilis subsp. inaquosorum ATCC 55406	w	w	+
Bacillus species 16970-5	w	w	+
Bacillus species 2 18118-1	w	w	+^[c]
Bacillus species 4 18121-4	w	w	w
Bacillus species 7 18129-3	ND	w	w

Footnote Appendix 10 Table A48 a

w, weak cytotoxic activity (5-50% bioreduction loss)

Return to footnote Appendix 10 Table A48 a referrer

Footnote Appendix 10 Table A48 b

Related to structural components

Return to footnote Appendix 10 Table A48 b referrer

Footnote Appendix 10 Table A48 c

+, cytotoxic activity ( greater than 50% bioreduction loss)

Return to footnote Appendix 10 Table A48 c referrer

Footnote Appendix 10 Table A48 d

Footnote Appendix 10 Table A48 e

ND, no data

Return to footnote Appendix 10 Table A48 e referrer

Footnote Appendix 10 Table A48 f

-, no cytotoxic activity ( less than 5% bioreduction loss)

Return to footnote Appendix 10 Table A48 f referrer

**Table A-49: Cyototoxic potential of DSL *B. licheniformis/subtilis* strains towards J774A.1 cells with gentamicin at 2, 4 and 24 hours**
DSL Strain^{Footnote Appendix 10 Table A49[a]}	2 hours	4 hours	24 hours
B. amyloliquefaciens 13563-0	-^{Footnote Appendix 10 Table A49 [b]}	-	-
B. atrophaeus 18250-7	w^{Footnote Appendix 10 Table A49 [c]},^{Footnote Appendix 10 Table A49 [d]}	w	w
B. licheniformis ATCC 12713	-	-	w
B. subtilis subsp. inaquosorum ATCC 55406	w	w	w
Bacillus species 16970-5	w	w	w
Bacillus species 2 18118-1	-	-	-
Bacillus species 4 18121-4	-	w	w

Footnote Appendix 10 Table A49 a

No data available for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051^T and Bacillusspecies 7 18129-3

Return to footnote Appendix 10 Table A49 a referrer

Footnote Appendix 10 Table A49 b

-, no cytotoxic activity ( less than 5% bioreduction loss)

Return to footnote Appendix 10 Table A49 b referrer

Footnote Appendix 10 Table A49 c

w, weak cytotoxic activity (5-50% bioreduction loss)

Return to footnote Appendix 10 Table A49 c referrer

Footnote Appendix 10 Table A49 d

Related to structural components

Return to footnote Appendix 10 Table A49 d referrer

**Table A-50: Cyototoxic potential of DSL *B. licheniformis/subtilis* strains towards J774A.1 cells without gentamicin at 2, 4 and 24 hours**
DSL Strain	2 hours	4 hours	24 hours
B. amyloliquefaciens 13563-0	-^{Footnote Appendix 10 Table A50 [a]}	-	-
B. atrophaeus 18250-7	w^{Footnote Appendix 10 Table A50 [b]},^{Footnote Appendix 10 Table A50 [c]}	w	w
B. licheniformis ATCC 12713	w	-	w
B. subtilis ATCC 6051A	ND^{Footnote Appendix 10 Table A50[d]}	w	w
B. subtilis ATCC 55405	ND	-	w
B. subtilis subsp. subtilis ATCC 6051^T	ND	-	w
B. subtilis subsp. inaquosorum ATCC 55406	-	-	+^{Footnote Appendix 10 Table A50 [e]}
Bacillus species 16970-5	-	-	w
Bacillus species 2 18118-1	-	-	+^{Footnote Appendix 10 Table A50 [f]}
Bacillus species 4 18121-4	-	-	w
Bacillus species 7 18129-3	ND	-	w

Footnote Appendix 10 Table A50 a

-, no cytotoxic activity ( less than 5% bioreduction loss)

Return to footnote Appendix 10 Table A50 a referrer

Footnote Appendix 10 Table A50 b

w, weak cytotoxic activity (5-50% bioreduction loss)

Return to footnote Appendix 10 Table A50 b referrer

Footnote Appendix 10 Table A50 c

Cytotoxic activity related to structural components

Return to footnote Appendix 10 Table A50 c referrer

Footnote Appendix 10 Table A50 d

ND, No data

Return to footnote Appendix 10 Table A50 d referrer

Footnote Appendix 10 Table A50 e

+, cytotoxic activity ( greater than 50% bioreduction loss)

Return to footnote Appendix 10 Table A50 e referrer

Footnote Appendix 10 Table A50 f

Cytotoxic activity related to growth

Return to footnote Appendix 10 Table A50 f referrer

Top of Page

Appendix 11: Pathogenicity, toxicity and irritation testing results for strains of the B. subtilis complex on terrestrial and aquatic vertebrates, invertebrates and plants

**Table A-51: Pathogenicity, toxicity and irritation testing results for *B. amyloliquefaciens* strain FZB24^{Footnote Appendix 11 Table A51 [a]}**
Study Type	Target organism	Dose concentration	Outcome
Acute oral toxicity and infectivity	CD rats	1.3 × 10⁸ CFU/animal	Low toxicity, not pathogenic LD₅₀ greater than 1.3 × 10⁸ CFU/animal
Acute pulmonary toxicity and infectivity	CD rats	0.1 mL of at least 1.4 × 10⁸ viable CFU/animal	Low toxicity, not pathogenic LD₅₀ greater than 1.4 × 10⁸ CFU/animal
Acute pulmonary toxicity and infectivity	Rats	1.3 × 10⁸ CFU	Not toxic/ Not pathogenic
Acute intravenous infectivity	CD rats	0.5 mL of at least 1.0 × 10⁷ CFU/animal	Not pathogenic LD₅₀ greater than 1.0 × 10⁷ CFU/animal
Acute intravenous infectivity	Rats	1.7 × 10⁸ CFU	Not toxic/ Not pathogenic
Acute dermal toxicity	Rabbits	2000 mg/kg BW^{Footnote Appendix 11 Table A51 [b]}(1.5 to 2 × 10¹² CFU/animal)	Low toxicity but severely irritating LD₅₀ greater than 2000 mg/kg
Primary dermal irritation	Rabbits	0.5 g granular test substance (7.0 × 10¹⁰ CFU/g) and 0.5 mL of 1.5% w.v suspension	No irritation observed
Primary eye irritation	Rabbit	3.6 × 10¹⁰ CFU	Eye irritant
Acute dermal irritation	New Zealand white rabbit	2000 mg/kg/BW (1.5 to 2 × 10¹² CFU/animal)	Low toxicity and severely irritating LD₅₀ greater than 2000 mg/kg/BW
Avian oral toxicity	Northern bobwhite (Colinus virginianus)	10 mL/kg BW or 1.0 × 10⁹ CFU/g BW	NOEL^{Footnote Appendix 11 Table A51 [c]}: 1.0 × 10⁹ CFU/animal
Terrestrial arthropod toxicity	Adult bees (Apis meelifera)	10⁵ CFU/mL	No signs of toxicity or pathogenicity LC₅₀ greater than 1.0 × 10⁶ CFU/mL
Terrestrial arthropod toxicity	Larva (Apis meelifera)	6.0 × 10³CFU/larva	LC₅₀ greater than 6.0 ×10³CFU/larva
Terrestrial non-arthropod invertebrates	Worm (Eisenia fetida)	6.0 × 10¹¹ CFU/kg soil	NOEC^{Footnote Appendix 11 Table A51 [d]}: 6.0 × 10¹¹ CFU/kg soil
Freshwater fish toxicity/pathogenicity	Rainbow trout (Oncorhynchus mykiss)	1.85 × 10⁹ CFU/kg to 1.85 × 10¹⁰ CFU/L (active ingredient)	LC₅₀ greater than 1.85 × 10¹⁰CFU/L NOEC: 1.85 × 10¹⁰ CFU/L
Freshwater invertebrate toxicity/pathogenicity	Daphnia magna	Up to 1.85 × 10¹⁰ CFU/L	LC₅₀ greater than 1.85 × 10¹⁰CFU/L NOEC: 1.85 × 10¹⁰ CFU/L

Footnote Appendix 11 Table A51 a

Studies done with the technical grade active ingredient and not the end-use product containing the micro-organism, (PMRA-HC, 2012; U.S. EPA, 2012)

Return to footnote Appendix 11 Table A51 a referrer

Footnote Appendix 11 Table A51 b

BW, body weight

Return to footnote Appendix 11 Table A51 b referrer

Footnote Appendix 11 Table A51 c

NOEL, no observed effect level is the highest dose of the test substance in the test substrate at which no statistically significant effect on the test organism was observed, relative to the control

Return to footnote Appendix 11 Table A51 c referrer

Footnote Appendix 11 Table A51 d

NOEC, no observed effect concentration is the highest concentration of the test substance in the test substrate at which no statistically significant effect on the test organism was observed, relative to the control

Return to footnote Appendix 11 Table A51 d referrer

**Table A-52 : Pathogenicity, toxicity and irritation testing results for *B. amyloliquefaciens* strain D747^{Footnote Appendix 11 Table A52 [a]}**
Study Type	Target organism	Dose concentration	Outcome
Acute oral toxicity	Sprague-Dawley rats (5 week old, male and female)	10⁸ CFU/animal	Not toxic, infective or pathogenic LD₅₀ greater than 5000 mg/kg
Acute pulmonary toxicity	Sprague-Dawley rats (5 week old, male and female)	10⁷ CFU/animal	Not toxic or pathogenic LC₅₀ greater than 2.18 mg/L
Acute injection	Sprague-Dawley rats (5 week old, male and female)	10⁷ CFU/animal	Not toxic, infective, or pathogenic LD₅₀ greater than 5050 mg/kg
Acute eye irritation	New Zealand white rabbits	0.1 mL of the end-use product	Eye irritant
Primary dermal irritation	New Zealand white rabbits	500 mg of the end-use product	No evidence of irritation
Avian oral toxicity	Northern bobwhite quail (Colinus virginianus)	8.9 × 10⁹ spores/bird	Not toxic LD₅₀ greater than 4.5 × 10¹¹ spores/kg BW or greater than 8.0 × 10¹⁰ spores/bird
Freshwater fish toxicity/pathogenicity	Rainbow trout (Oncorhynchus mykiss)	1.7 × 10⁸ CFU/L	LC₅₀: 8.1 × 10¹⁰ CFU/L NOEC: 1.44 × 10¹⁰ CFU/L
Freshwater invertebrate toxicity/pathogenicity	Daphnia magna	1.7 × 10⁸ CFU/L	EC₅₀: 3.7 × 10¹⁰ CFU/L NOEC: 2.84 × 10⁸ CFU/L

Footnote Appendix 11 Table A52 a

Studies done with the technical grade active ingredient and not the end-use product containing the micro-organism, (U.S. EPA, 2011)

Return to footnote Appendix 11 Table A52 a referrer

**Table A-53: Pathogenicity, toxicity and irritation testing results for *B. licheniformis* strain SB3086^{Footnote Appendix 11 Table A53 [a]}**
Study Type	Target organism	Dose concentration	Outcome
Acute oral toxicity/pathogenicity	Rats	1.0 × 10⁸ CFU/animal^{Footnote Appendix 11 Table A53 [b]}	Not toxic, infective, or pathogenic LD₅₀ greater than 5000 mg/kg
Acute pulmonary toxicity/pathogenicity	Rats	1.1 × 10⁸CFU/animal^[b]	Not toxic, infective, or pathogenic
Acute intravenous toxicity/pathogenicity	Rats	1.0 × 10⁷CFU/animal^[b]	Not toxic, infective, or pathogenic
Acute dermal toxicity	New Zealand white rabbits	ND^{Footnote Appendix 11 Table A53[c]},^{Footnote Appendix 11 Table A53[d]}	LD₅₀ greater than 5050 mg/kg
Primary eye irritation	New Zealand white rabbits	0.1 mL/animal (concentration not provided)d	Non-irritating
Delayed contact hypersensitivity	Guinea Pigs	ND^[d]	Not a dermal sensitizer
Avian oral toxicity/pathogenicity	Young mallards (Anas platyrhynchos)	4.5 × 10¹⁰ CFU/kg of BW^[b]	No signs of illness or abnormal behaviour observed
Fresh water fish toxicity/pathogenicity	Rainbow trout (Oncorhynchus mykiss)	LC₅₀ (of Formula 710-132) greater than 1.1 × 10⁶ CFU/mL^[b]	No effects as the result of the active microbial agent observed
Fresh water aquatic invertebrate toxicity/pathogenicity (21-day renewal life-cycle)	Daphnia magna	1 × 10⁴ CFU/mL^[b]	LC₅₀: 1.8 × 10⁶ CFU/mL NOAEC^[d]:1.2 × 10⁶CFU/mL^[b],^{Footnote Appendix 11 Table A53 [e]} 2 daphnids died at the end of the test at 1 × 10⁷CFU/mL (1000 times the expected environmental concentration)
Invertebrate toxicity/pathogenicity	Honeybee larvae (Apis mellifera L.)	1.6 × 10⁶ CFU/mL (of Formula 710-132)^{Footnote Appendix 11 Table A53 [f]}	No statistically significant effects on larvae survival, adverse behaviour or developmental abnormalities observed

Footnote Appendix 11 Table A53 a

(U.S. EPA, 2001)

Return to footnote Appendix 11 Table A53 a referrer

Footnote Appendix 11 Table A53 b

TGAI, technical grade active ingredient

Return to footnote Appendix 11 Table A53 b referrer

Footnote Appendix 11 Table A53 c

ND, no data

Return to footnote Appendix 11 Table A53 c referrer

Footnote Appendix 11 Table A53 d

NOAEC, no observed adverse effect concentration

Return to footnote Appendix 11 Table A53 d referrer

Footnote Appendix 11 Table A53 e

Formulation ingredients are known aquatic toxicants at high concentrations

Return to footnote Appendix 11 Table A53 e referrer

Footnote Appendix 11 Table A53 f

EP, end product

Return to footnote Appendix 11 Table A53 f referrer

Table A-54: Pathogenicity, toxicity and irritation testing results for several strains of *B. licheniformis*and *B. subtilis*
Study Type	Target organism	Dose concentration	Outcome
Experimental infection (intravenous inoculation)^{Footnote Appendix 11 Table A54 [a]}	Immune depressed BALB/c mice	4 × 10⁷ CFU	Numerous and larger lesions in many organs and more severe infection with lesions occurring in more organs compared to healthy/normal mice used in the study
Experimental infection (intravenous inoculation)^[a]	Normal mice	4 × 10⁷ CFU	Lesions observed in liver and kidneys
Experimental infection (intravenous inoculation)^{Footnote Appendix 11 Table A54 [b]}	Immune depressed BALB/c mice	Intravenous doses of less than 10⁶ to 10¹⁰ CFU	Only brain and pulmonic lesions could be definitely attributed to B. licheniformis. Mice were able to clear high numbers of bacteria within 1 week
Experimental infection (intravenous inoculation)^{Footnote Appendix 11 Table A54 [c]}	Pregnant crossbred Red Danish Dairy X American Brown Swiss cows (6-8 months of gestation, n=8) and their calves	Intravenous doses of less than 10⁹ to 10¹² CFU (once or on 4 consecutive days)	Demonstration of the abortifacient potential of B. licheniformis and the tropism for the bovine placenta. Lesions in the fetal membranes, the fetal side of the placentomes, necrosis in the fetal compartment of the placenta and inflammation in some calves. Two abortions were observed
Cytotoxic Activity^{Footnote Appendix 11 Table A54[d]}	Boar sperm motility inhibition	1-10 μg/mL	EC₅₀: 20-30 μg/mL
Acute eye irritation study^{Footnote Appendix 11 Table A54 [e]}	Male albino rabbits	0.1 g of 1.1 × 10¹¹ CFU/kg BW	No irritation or negative symptoms in the cornea or iris
Acute skin irritation study^[e]	Male albino rabbits	0.5 g of 1.1 × 10¹³ CFU/kg BW	No clinical signs of erythema or oedema
Acute oral toxicity (14-day) ^[e]	Adult male albino Wistar rats	1.1 × 10¹¹ CFU/kg BW	No treatment-related changes
Subchronic oral toxicity (13-week)^[e]	Male and female Wistar rats	1.1 × 10¹¹ CFU/kg BW and 1.1 × 10¹¹CFU/kg BW	NOAEL^{Footnote Appendix 11 Table A54 [f]}greater than 1.1 × 10¹¹ CFU/kg BW
Micronucleus assay (2 days at 24 hour intervals)^[e]	Adult male and female Swiss albino mice (CFT strain)	1.1 × 10¹⁰ CFU/kg BW and 1.1 × 10¹¹CFU/kg BW	No signs of bone marrow cytotoxicity and no observed genocyctotoxicity
Oral Pathogenicity and Toxicity Study^{Footnote Appendix 11 Table A54 [g]}	Bobwhite quails (Colinus virginianus)	3,333 kg/mg daily for 5 days	LD₅₀ greater than 2,000 mg/kg

Footnote Appendix 11 Table A54 a

B. licheniformis ATCC 14580, (Agerholm et al. 1995)

Return to footnote Appendix 11 Table A54 a referrer

Footnote Appendix 11 Table A54 b

13 strains of B. licheniformis, (Agerholm et al. 1997)

Return to footnote Appendix 11 Table A54 b referrer

Footnote Appendix 11 Table A54 c

B. licheniformis strain DVL 9315323, (Agerholm et al. 1999)

Return to footnote Appendix 11 Table A54 c referrer

Footnote Appendix 11 Table A54 d

B. licheniformis strains NR 5160 and NR 6768 (toxic heat-stable non-protein substance), (Nieminen et al. 2007)

Return to footnote Appendix 11 Table A54 d referrer

Footnote Appendix 11 Table A54 e

B. licheniformis strain Me I, The concentration used in the study corresponds to 77 × 10¹¹ CFUs for an average 70 kg human being and thus the concentration used can be considered to be 2566 to 77000 times safe for human consumption (suggested human dose range: 1 × 10⁸ to 3 × 10⁹ CFUs) (Nithya et al. 2012)

Return to footnote Appendix 11 Table A54 e referrer

Footnote Appendix 11 Table A54 f

NOAEL, no observed adverse effect level

Return to footnote Appendix 11 Table A54 f referrer

Footnote Appendix 11 Table A54 g

B. subtilis strain GB03, (U.S. EPA, 1993)

Return to footnote Appendix 11 Table A54 g referrer

**Table A-55: Pathogenicity, toxicity and irritation testing results for a mixture containing two strains of *B. licheniformis* and *B. subtilis*^{Footnote Appendix 11 Table A55 [a]}**
Study Type	Target organism	Dose concentration	Outcome
Acute toxicity	BALB/c mice	5 × 10⁷ to 2 × 10¹¹ CFU/mouse (oral administration)	No changes in tissues, organs or weight
Acute toxicity	BALB/c mice	5 × 10⁷ to 5 × 10⁹ CFU/mouse (IP^{Footnote Appendix 11 Table A55 [b]} or IV^{Footnote Appendix 11 Table A55 [c]} administration)	No changes in tissues, organs or weight
Chronic toxicity	Mice	1 × 10⁶ CFU/day	No effect on health status
Chronic toxicity	Rabbits	1 × 10⁹ CFU/day	No effect on health status
Chronic toxicity	Piglets	1 × 10⁹ CFU/day	No effect on health status

Footnote Appendix 11 Table A55 a

B. licheniformis strain VKPM B2336 and B. subtilis strain VKPM B2335, (Sorokulova et al. 2008)

Return to footnote Appendix 11 Table A55 a referrer

Footnote Appendix 11 Table A55 b

IP, intraperitoneal

Return to footnote Appendix 11 Table A55 b referrer

Footnote Appendix 11 Table A55 c

IV, intravenous

Return to footnote Appendix 11 Table A55 c referrer

**Table A-56: Pathogenicity, toxicity and irritation testing results for *B. subtilis* strain QST 713^{Footnote Appendix 11 Table A56 [a]}**
Study Type	Target organism	Dose concentration	Outcome
Acute oral toxicity and infectivity	CD rats	1.13 × 10⁸ CFU/animal	Non-toxic and not infective LD₅₀ greater than 1.13 × 10⁸ CFU/animal
Acute pulmonary toxicity and infectivity	CD rats	1.2 × 10⁸ CFU/animal	Non-toxic and not infective LD₅₀ greater than 1.2 × 10⁸ CFU/animal
Intravenous Infectivity	CD rats	9.4 × 10⁸ CFU/animal	Non-infective
Acute dermal toxicity	CD rats	2 g/kg BW (2.3 × 10¹¹ to 2.73 × 10¹¹CFU/animal	Low toxicity LD₅₀ greater than 2g/kg BW
Eye irritation	Rabbits	0.1 ml (4.8 × 10⁹ CFU/animal)	Minimally irritating
Dermal irritation	Rabbits	500 mg (2.4 × 10¹⁰ CFU/animal)	Slightly irritating
Avian oral toxicity	Northern bobwhite quail (Colinus virginianus)	1 × 10⁸ CFU/g BW/day (5000 mg/kg BW/day)	LD₅₀ greater than 5000 mg/kg BW
Freshwater fish	Rainbow trout (Oncorhynchus mykiss)	Max dose: 1.4 × 10⁷ CFU/mL	LC₅₀: 1.4 × 10⁷ CFU/mL
Freshwater aquatic invertebrate (48-hour)	Daphnia magna	5 × 10⁵ CFU/mL, 1 × 10⁶ CFU/mL, 2 × 10⁶ CFU/mL and 4 × 10⁶ CFU/mL	Mortality of 15, 15, 45 and 85% respectively and lethargy of surviving daphnids in the 100mg/L treatment
Freshwater aquatic invertebrate (21-day)	Daphnia magna	5 × 10⁵ CFU/mL, 1 × 10⁶ CFU/mL, 2 × 10⁶ CFU/mL and 4 × 10⁶ CFU/mL	LC₅₀ ~ 3 × 10⁵ CFU/mL NOEC: 7.5 × 10³CFU/mL
Freshwater aquatic invertebrate (21-day)	Daphnia magna	7.9 × 10⁵ CFU/mL, 1.8 × 10⁶ CFU/mL, 3.4 × 10⁶ CFU/mL, 7.3 × 10⁶ CFU/mL and 2.0 × 10⁷ CFU/mL	LC₅₀ ~ 1.6 × 10⁶ CFU/mL NOEC: 7.9 × 10⁵ CFU/mL.
Freshwater aquatic invertebrate	Grass shrimp (Palaemonetes pugio)	4.0 × 10⁶ CFU/g	LC₅₀ greater than 4.0 × 10⁶ CFU/mL
Aqueous plant	Single cell green alga (Scenedesmus subspicatus)	Max dose: 5.1 × 10⁵ CFU/mL	NOEC greater than 100 mg/L LOEC greater than 100 mg/L
Non-target insect study (oral/dietary)	Honey Bee -Apil mellifera L.	Max dose: 100,000 ppm	LD₅₀ greater than 100,000 ppm
Dietary toxicity/ pathogenicity	Honey Bee -Apil mellifera L.	600, 6,000 and 60,000 ppm	LC₅₀: 5663 ppm
Non-target insect study (oral/dietary)	Green lacewing (Chrisoperla carnea)	Max dose: 60000 ppm	LC₅₀ greater than 60,000 ppm
Non-target insect study (oral/dietary)	Ladybird beetle -Hippodamia convergens	Max dose: 1.2 × 10⁹ CFU/mL (60000 ppm)	LC₅₀ greater than 60,000 ppm NOEC: 60,000 ppm (1.2 × 10⁹ CFU/g)
Toxicity and pathogenicity test (30 days)	Parasitic Hymenoptera - Nasonia vitripenis	600, 6,000 and 60,000 ppm	LC₅₀: 28,000 ppm (15 days)
Non-target insect study (oral/dietary)	Parasitic Hymenoptera - Nasonia vitripenis	Max dose: 3.2 × 10⁹ CFU/mL (60000 ppm)	LC₅₀ ~ 24,739 ppm

Footnote Appendix 11 Table A56 a

Studies done with the technical grade active ingredient (Mendelsohn and Vaituzis, 1999; U.S. EPA, 2006; U.S. EPA, 2010; PMRA-HC, 2007b).

Return to footnote Appendix 11 Table A56 a referrer

**Table A-57: Pathogenicity, toxicity and irritation testing results for *B. subtilis* strain MBI 600^{Footnote Appendix 11 Table A57 [a]}**
Study Type	Target organism	Dose concentration	Outcome
Acute oral toxicity and infectivity	CD Rats	2.0 × 10⁸ spores	Low toxicity, not infective LD₅₀ greater than 2 × 10⁸ CFU
Acute pulmonary toxicity and infectivity	CD Rats	3.3 × 10⁸ to 3.7 × 10⁸ spores	Toxic, not infective LD₅₀ greater than 3.5 × 10⁸ CFU
IV infectivity	CD Rats	10⁷ spores	No significant signs of toxicity
Acute dermal toxicity	New Zealand White Rabbits	2mL/kg body weight	Low toxicity (slight oedema) LD₅₀ greater than 2mL/kg body weight
Eye irritation	New Zealand White Rabbits	1.0 × 10⁹ CFU	Minimally irritating
Dermal irritation	New Zealand White Rabbits	2.0 × 10¹⁰ CFU	Minimally irritating
Acute Avian Oral Toxicity and Pathogenicity	Bobwhite quails (Colinus virginianus)	GUS 378 Concentrate: 4000 mg/kg BW/day	Low toxicity, not pathogenic
Acute Avian Oral Toxicity and Pathogenicity	Bobwhite quails (Colinus virginianus)	Water-soluble metabolites: 240 mg/kg BW/day	Low toxicity, not pathogenic
Acute Avian Oral Toxicity and Pathogenicity	Bobwhite quails (Colinus virginianus)	Washed spores: 3680 mg/kg BW/day	Low toxicity, not pathogenic
Plant Toxicity and Pathogenicity	Soybean seeds	10⁵ to 10⁷ viable spores/seed	Not pathogenic
Freshwater Fish Toxicity and Infectivity	Carp	2.0 × 10⁶ CFU/mL, 2.0 × 10⁷ CFU/mL, 2.0 × 10⁸ CFU/mL	No treatment-related toxicity or pathogenicity

Footnote Appendix 11 Table A57 a

Studies done with the technical grade active ingredient and not the end-use product containing the micro-organism, (PMRA-HC, 2007a)

Return to footnote Appendix 11 Table A57 a referrer

**Table A-58: Pathogenicity and toxicity testing results for *B. subtilis* ATCC 6051A and ATCC 55405^{Footnote Appendix 11 Table A58 [a]}**
Study Type	Target organism	Dose concentration	Outcome
Pathogenicity/toxicity testing	Red fescue(Festuca rubra)	10⁵ CFU/g soil dry weight	Shoot length significantly affected (p = 0.03)^{Footnote Appendix 11 Table A58 [b]}
Pathogenicity/toxicity testing	Springtail(Folsomia candida)	10³ CFU^{Footnote Appendix 11 Table A58[c]}/g soil dry weight ; 10⁴ CFU^{Footnote Appendix 11 Table A58[d]}/g soil dry weight	Significant reduction (p less than 0.01) in juvenile production^[c] No juvenile production (statistical analysis could not be performed)^[d] Adult survival not affected by either strain
Pathogenicity/toxicity testing	Earth worm(Eisenia andrei)	10⁵ CFU^[c]/g soil dry weight; 10⁴ CFU^[d]/g soil dry weight	No adverse effects reported

Footnote Appendix 11 Table A58 a

Data generated by Environment Canada's Biological Methods Division

Return to footnote Appendix 11 Table A58 a referrer

Footnote Appendix 11 Table A58 b

The survival, growth and reproduction of test organisms were significantly inhibited in the field-collect soil relative to the artificial soil

Return to footnote Appendix 11 Table A58 b referrer

Footnote Appendix 11 Table A58 c

B. subtilis ATCC 55405

Return to footnote Appendix 11 Table A58 c referrer

Footnote Appendix 11 Table A58 d

B. subtilis ATCC 6051A

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Appendix 12: Virulence and pathogenicity testing of the DSL B. licheniformis/subtilis strains

Murine exposure data generated by Health Canada's Healthy Environments and Consumer Safety Branch. Female BALB/c mice were exposed to 10⁶ CFU/25μL of bacteria (vegetative cells or spores) via an endotracheal nebulizer for pulmonary exposure. Animals were necropsied at 24 hours and 1 week for vegetative cells and spores exposures respectively.

Clearance following endotracheal exposure

**Table A-59: Enumeration of vegetative cells (CFU/mg) of DSL *B. licheniformis/subtilis* group strains following endotracheal exposure**
Strain^{Footnote Appendix 12 Table A59[a]}	Lung	Trachea	Esophagus
B. amyloliquefaciens 13563-0	468.5	124.1	16.1
B. atrophaeus 18250-7	270.7	96.6	45.0
B. licheniformis ATCC 12713	323.1	31.4	14.6
B. subtilis subsp. inaquosorum ATCC 55406	228.4	1.5	4.8
Bacillus species 16970-5	249.2	17.6	30.2
Bacillus species 2 18118-1	235.0	51.8	17.8
Bacillus species 4 18121-4	310.7	47.3	44.3

Footnote Appendix 12 Table A59 a

No data available for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051^T and Bacillusspecies 7 18129-3

Return to footnote Appendix 12 Table A59 a referrer

Figure A-1: Enumeration of vegetative cells (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure

Figure A-1 (See long description below)

Long description of the figure A-1

Figure A-1: Enumeration of vegetative cells (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure

Bar graph visualizes information presented in Table A-48.

**Table A-60: Enumeration of spores (CFU/mg) of DSL *B. licheniformis/subtilis* group strains following endotracheal exposure**
Strain^{Footnote Appendix 12 Table A60[a]}	Lung	Trachea	Esophagus
B. amyloliquefaciens 13563-0	132.2	29.5	20.7
B. atrophaeus 18250-7	54.7	7.0	2.6
B. licheniformis ATCC 12713	6.9	1.3	1.6
B. subtilis ATCC 6051A	0.0	0.0	0.0
B. subtilis ATCC 55405	52.1	85.1	20.0
B. subtilis subsp. subtilis ATCC 6051^T	7.0	0.0	0.0
B. subtilis subsp. inaquosorum ATCC 55406	10.6	1.4	0.3
Bacillus species 2 18118-1	54.8	8.5	0.4
Bacillus species 4 18121-4	28.4	15.0	4.1
Bacillus species 7 18129-3	7.0	0.0	0.0

Footnote Appendix 12 Table A60 a

No data available for Bacillus species 16970-5

Return to footnote Appendix 12 Table A60 a referrer

Figure A-2: Enumeration of spores (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure

Figure A-2 (See long description below)

Long description of the figure A-2

Figure A-2: Enumeration of spores (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure

Bar graph visualizes information presented in Table A-49.

Pulmonary Cytokines

**Table A-61: Pulmonary cytokine expression (pg/mL) from vegetative cell exposures**
DSL Strain^{Footnote Appendix 12 Table A61[a]}	IL-1 beta	IL-6	MCP-1	IL-12 (p70)	KC	TNF-alpha
Control (saline)	73.58 ± 13.26	2.02 ± 0.61	1501.94 ± 288.19	18.07 ± 2.26	7.32 ± 1.16	728.57 ± 107.77
B. amyloliquefaciens 13563-0	141.39 ± 60.31	1.46 ± 1.11	1597.70 ± 177.87	13.05 ± 5.77	62.46 ± 16.28	575.77 ± 58.43
Bacillus atrophaeus 18250-7	1619.34 ± 564.47	4.20 ± 0.73	4446.72 ± 1536.15	16.49 ±9.96	452.83 ± 160.76	718.82 ± 135.08
B. licheniformis ATCC 12713	1818.28 ± 573.73	5.80 ± 1.94	6032.81 ± 2094.65	19.08 ± 7.44	387.79 ± 146.88	705.84 ± 228.92
B. subtilis subsp. inaquosorum ATCC 55406	262.22 ± 44.63	1.10 ± 0.49	1624.45 ± 242.78	12.23 ± 4.65	34.61 ± 19.19	591.58 ± 87.85
Bacillus species 16970-5	101.13 ± 5.94	1.10 ± 0.53	1651.50 ± 319.11	16.38 ± 5.78	26.29 ± 18.31	667.01 ± 133.49
Bacillus species 2 18118-1	1444.876 ± 778.68	4.53 ± 2.24	5554.10 ± 2162.64	13.95 ± 2.00	660.90 ± 74.56	647.20 ± 205.74
Bacillus species 4 18121-4	7545.602 ± 1988.01	53.43 ± 21.70	20278.06 ± 7401.54	24.40 ± 7.21	2082.50 ± 501.70	983.90 ± 172.32

Footnote Appendix 12 Table A61 a

No data available for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051and Bacillus species 7 18129-3

Return to footnote Appendix 12 Table A61 a referrer

**Table A-62: Pulmonary cytokine expression (pg/mL) from spore exposures**
DSL Strain^{Footnote Appendix 12 Table A62[a]}	IL-beta	IL-6	MCP-1	IL-12 (p70)	KC	TNF-alpha
Control (saline)	90.86 ± 13.65	2.29 ± 1.27	837.12 ± 147.20	12.62 ± 8.05	9.13 ± 2.46	484.79 ± 160.58
B. amyloliquefaciens 13563-0	98.07 ± 11.21	2.87 ± 1.75	921.78 ± 187.96	9.82 ± 7.62	11.89 ± 4.39	476.05 ± 167.15
Bacillus atrophaeus 18250-7	120.36 ± 52.91	2.53 ± 1.60	750.55 ± 146.43	14.23 ± 8.82	9.48 ± 2.70	521.90 ± 110.22
Bacillus species 2 18118-1	104.37 ± 9.54	2.13 ± 1.78	843.20 ± 101.71	13.86 ± 6.73	10.0 ± 3.74	526.06 ± 115.86
Bacillus species 4 18121-4	90.31 ± 19.54	3.06 ± 1.47	884.30 ± 105.05	17.10 ± 6.72	7.17 ± 0.74	374.76 ± 91.07

Footnote Appendix 12 Table A62 a

No data available for B. licheniformisATCC 12713, B. subtilis ATCC 6051A, B. subtilisATCC 55405, B. subtilis subsp. inaquosorum ATCC 55406, Bacillus species 16970-5 and Bacillusspecies 7 18129-3

Return to footnote Appendix 12 Table A62 a referrer

Acute phase response

**Table A-63: Serum Amyloid A (SAA)^{Footnote Appendix 12 Table A63 [a]} Levels (µg/mL) in serum samples obtained from BALB/c mice treated with vegetative cells or spores of DSL strains**
Strain^{Footnote Appendix 12 Table A63[b]}	Vegetative cells	Spores
Control (saline)	13.80 ± 3.52	14.08 ± 0.63
B. amyloliquefaciens 13563-0	17.87 ± 2.73	16.60 ± 2.09
Bacillus atrophaeus 18250-7	20.73 ± 1.21	21.56 ± 14.0
B. licheniformis ATCC 12713	39.54 ± 28.54	No data
B. subtilis subsp. inaquosorum ATCC 55406	17.76 ± 4.34	No data
Bacillus species 16970-5	13.72 ± 2.66	13.88 ± 3.10
Bacillus species 2 18118-1	23.35 ± 3.29	16.04 ± 2.38
Bacillus species 4 18121-4	17.68 ± 7.89	No data

Footnote Appendix 12 Table A63 a

Serum amyloid A, an indicator of systemic effects, was measured using ELISA

Return to footnote Appendix 12 Table A63 a referrer

Footnote Appendix 12 Table A63 b

No data available for B. subtilis ATCC 6051A, B. subtilis ATCC 55405, B. subtilis subsp. subtilis ATCC 6051 and Bacillus species 7 18129-3

Return to footnote Appendix 12 Table A63 b referrer

Figure A-3: Serum Amyloid A (SAA) Levels (µg/mL) in serum samples obtained from BALB/c mice treated with vegetative cells of DSL strains

Figure a-3 (See long description below)

Long description of the figure A-3

Figure A-3: Serum Amyloid A (SAA) Levels (µg/mL) in serum samples obtained from BALB/c mice treated with vegetative cells of DSL strains

Bar graph visualizes information presented on vegetative cells in Table A-52.

Figure A-4: Serum Amyloid A (SAA) Levels (µg/mL) in serum samples obtained from BALB/c mice treated with spores of DSL strains

Figure A-4 (See long description below)

Long description of the figure A-4

Figure A-4: Serum Amyloid A (SAA) Levels (µg/mL) in serum samples obtained from BALB/c mice treated with spores of DSL strains

Bar graph visualizes information on spores presented in Table A-52.

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Appendix 13: Food poisoning outbreaks

Table A-64: Food poisoning outbreaks involving *B. licheniformis*
Place	Year	# of Cases^{Footnote Appendix 13 Table A64[a]}	Symptoms	Cause	Death(s)
Veterans Administration Hospital, Denver, CO^{Footnote Appendix 13 Table A64 [b]}	1959	161	Gastroenteritis including abdominal cramps, diarrhoea and vomiting	Cooked turkey meat that was held at room temperature overnight	1
Australia^{Footnote Appendix 13 Table A64[c]}	1976	49	Abdominal pain, diarrhoea and vomiting	Meals-on-wheels co-contaminated with Clostridium perfringens and B. cereus	1
Prison in Ohio, USA^{Footnote Appendix 13 Table A64[d]}	1995	165	No data	Turkey and gravy were implicated	No data
Kindergarten in Split, Croatia^{Footnote Appendix 13 Table A64 [e]}	2000	12	Nausea, headache and vomiting	Contaminated milk powder that was prepared two hours prior to consumption and not boiled. Co-contamination with B. subtilis	0

Footnote Appendix 13 Table A64 a

Case refers to an individual person diagnosed with food poisoning

Return to footnote Appendix 13 Table A64 a referrer

Footnote Appendix 13 Table A64 b

(Tong et al. 1962), though the authors implicate B. subtilis as the causative agent, the food poisoning was likely caused by B. licheniformis as the onset and symptoms are more in line with the description by Lund (1990) and biochemical testing results appear to be closer to B. licheniformis (e.g. growth in salt and anaerobic growth).

Return to footnote Appendix 13 Table A64 b referrer

Footnote Appendix 13 Table A64 c

(Jephcott et al. 1977)

Return to footnote Appendix 13 Table A64 c referrer

Footnote Appendix 13 Table A64 d

(CDC 1995)

Return to footnote Appendix 13 Table A64 d referrer

Footnote Appendix 13 Table A64 e

(Pavic et al. 2005), contamination of food as the result of toxin-producing isolated of B. licheniformisand B. subtilis was proven via vacuolation assay and MTT cell culture test.

Return to footnote Appendix 13 Table A64 e referrer

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Date modified:: 2015-07-31

Appendices of the Final Screening Assessment

Environment CanadaHealth CanadaAugust 2015

Appendices

Appendix 1: Colony Morphologies of Domestic Substances List (DSL) B. licheniformis/subtilis group members

Appendix 2: Characteristics of DSL B. licheniformis/subtilis group members – 16S ribosomal RNA gene sequence analysis

Appendix 3: Characteristics of DSL B. licheniformis/subtilis group members – Fatty acids methyl ester (FAME) analysis

Appendix 4: Cellular content of select fatty acids

Appendix 5: List of some mobile elements and associated traits identified in certain isolates of the B. subtilis complex

Appendix 6: Virulence genes

Appendix 7: Virulence and Pathogenicity Testing of DSL B. licheniformis/subtilis Strains: Hemolytic Activity

Appendix 8: Virulence and pathogenicity testing of DSL B. licheniformis/subtilis strains: Catalase production

Appendix 9: Antimicrobial Compound, Other Metabolites and Toxins Produced by certain isolates of the B. subtilis Complex

Appendix 10: Virulence and pathogenicity testing of DSL B. licheniformis/subtilis strains: Cytotoxicity

Appendix 11: Pathogenicity, toxicity and irritation testing results for strains of the B. subtilis complex on terrestrial and aquatic vertebrates, invertebrates and plants

Appendix 12: Virulence and pathogenicity testing of the DSL B. licheniformis/subtilis strains

Clearance following endotracheal exposure

Figure A-1: Enumeration of vegetative cells (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure

Figure A-2: Enumeration of spores (CFU/mg) of DSL B. licheniformis/subtilis group strains following endotracheal exposure

Pulmonary Cytokines

Acute phase response

Figure A-3: Serum Amyloid A (SAA) Levels (µg/mL) in serum samples obtained from BALB/c mice treated with vegetative cells of DSL strains

Figure A-4: Serum Amyloid A (SAA) Levels (µg/mL) in serum samples obtained from BALB/c mice treated with spores of DSL strains

Appendix 13: Food poisoning outbreaks

Environment Canada
Health Canada
August 2015