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EC number: 947-907-6 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
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- Flash point
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- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
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- Irritation / corrosion
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- Repeated dose toxicity
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- Carcinogenicity
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- Specific investigations
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- Additional toxicological data
![](https://www.echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From September 20, 2017 to October 27, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Protein hydrolyzates, silk, reaction products with 3-chloro-2-hydroxypropyl-cocoalkyl-dimethylammonium chloride
- Molecular formula:
- Molecular formula of major active constituents: C20H43CL1N2O3 (representative molecular formula for C12 alkyl chain Quaternised alanine) C19H41CL1N2O3 (representative molecular formula for C12 alkyl chain Quaternised glycine) C20H43Cl1N2O4 (representative molecular formula of C12 alkyl chain quaternised serine)
- IUPAC Name:
- Protein hydrolyzates, silk, reaction products with 3-chloro-2-hydroxypropyl-cocoalkyl-dimethylammonium chloride
- Test material form:
- other: Paste
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
- Test concentrations with justification for top dose:
- Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (Presence and absence of S9)
Experiment 2: 0.15, 0.5, 1.5, 5, 15, 50, 150 500 μg/plate (Presence and absence of S9)
The maximum concentration was 5000 µg/plate (the maximum recommended dose level). - Vehicle / solvent:
- In solubility checks performed in–house, the test substance was noted as fully soluble in sterile distilled water at 50 mg/mL. Sterile distilled water was selected as the vehicle.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Identity: Sterile distilled water, Supplier: Aguettant, Batch number (purity): 3012486 (N/A), Expiry: 11/2018
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: Identity: 2-Aminoanthracene (2AA), CAS No.: 613-13-8, Batch number: STBB1901M9, Purity: 97.5%, Expiry date: 08 October 2019, Solvent: DMSO, Concentration: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 , 10 µg/plate for WP2uvrA
- Details on test system and experimental conditions:
- Bacteria
The five strains of bacteria used, and their mutations, are as follows:
1) Salmonella typhimurium
Strains - Genotype - Type of mutations indicated
TA1537 - his C 3076; rfa-; uvrB-: - frame shift
TA98 - his D 3052; rfa-; uvrB-; - R-factor
TA1535 - his G 46; rfa-; uvrB-: - base-pair substitution
TA100 - his G 46; rfa-; uvrB-;R-factor
2) Escherichia coli
Strain - Genotype - Type of mutations indicated
WP2uvrA - trp-; uvrA-: - base-pair substitution
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
The bacteria used in the test were obtained from:
1) University of California, Berkeley, on culture discs, on 04 August 1995.
2) British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 h. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates. - Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2) A reproducible increase at one or more concentrations.
3) Biological relevance against in-house historical control ranges.
4) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5) Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test substance activity. Results of this type will be reported as equivocal. - Statistics:
- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.
Results and discussion
Test results
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
Results
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test substance formulation was also shown to be sterile. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test substance, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 and Table 2 for Experiment 1 (plate incorporation) and Table 3 and Table 4 for Experiment 2 (pre-incubation).
Table
1:
Test Results: Experiment 1 – Without Metabolic Activation (Plate
Incorporation)
Test Period |
From: 09 October 2017 |
To: 12 October 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98† |
TA1537 |
||||||||
Solvent Control (Water) |
103 90 106 |
(100) 8.5# |
29 32 34 |
(32) 2.5 |
25 28 18 |
(24) 5.1 |
36 13 23 |
(24) 11.5 |
13 9 18 |
(13) 4.5 |
||
1.5 µg |
103 92 109 |
(101) 8.6 |
27 28 28 |
(28) 0.6 |
27 26 18 |
(24) 4.9 |
25 36 24 |
(28) 6.7 |
21 10 15 |
(15) 5.5 |
||
5 µg |
101 87 99 |
(96) 7.6 |
33 31 25 |
(30) 4.2 |
29 24 30 |
(28) 3.2 |
19 19 29 |
(22) 5.8 |
12 14 18 |
(15) 3.1 |
||
15 µg |
96 98 92 |
(95) 3.1 |
29 29 26 |
(28) 1.7 |
29 21 34 |
(28) 6.6 |
29 19 16 |
(21) 6.8 |
12 20 16 |
(16) 4.0 |
||
50 µg |
75 94 83 |
(84) 9.5 |
32 29 30 |
(30) 1.5 |
26 24 33 |
(28) 4.7 |
15 22 16 |
(18) 3.8 |
15 16 11 |
(14) 2.6 |
||
150 µg |
8 S 10 S 18 S |
(12) 5.3 |
15 S 12 S 17 S |
(15) 2.5 |
24 17 19 |
(20) 3.6 |
11 S 5 S 5 S |
(7) 3.5 |
4 S 4 S 9 S |
(6) 2.9 |
||
500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
8 S 20 S 20 S |
(16) 6.9 |
0 T 0 T 0 T |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
||
1500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
466 611 578 |
(552) 76.0 |
1141 1211 1265 |
(1206) 62.2 |
757 858 930 |
(848) 86.9 |
206 193 227 |
(209) 17.2 |
435 497 498 |
(477) 36.1 |
Table
2: Test
Results: Experiment 1 – With Metabolic Activation(Plate
Incorporation)
Test Period |
From: 09 October 2017 |
To: 12 October 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98† |
TA1537 |
||||||||
Solvent Control (Water) |
106 87 89 |
(94) 10.4# |
36 39 39 |
(38) 1.7 |
34 35 35 |
(35) 0.6 |
32 31 33 |
(32) 1.0 |
15 10 14 |
(13) 2.6 |
||
1.5 µg |
69 81 76 |
(75) 6.0 |
42 45 37 |
(41) 4.0 |
38 36 41 |
(38) 2.5 |
33 23 28 |
(28) 5.0 |
9 19 15 |
(14) 5.0 |
||
5 µg |
72 107 95 |
(91) 17.8 |
38 39 38 |
(38) 0.6 |
22 30 42 |
(31) 10.1 |
24 29 25 |
(26) 2.6 |
12 23 15 |
(17) 5.7 |
||
15 µg |
104 68 120 |
(97) 26.6 |
43 39 41 |
(41) 2.0 |
23 37 39 |
(33) 8.7 |
35 38 25 |
(33) 6.8 |
18 14 14 |
(15) 2.3 |
||
50 µg |
104 95 87 |
(95) 8.5 |
40 41 41 |
(41) 0.6 |
32 35 37 |
(35) 2.5 |
35 18 31 |
(28) 8.9 |
6 12 18 |
(12) 6.0 |
||
150 µg |
73 S 52 S 63 S |
(63) 10.5 |
38 44 43 |
(42) 3.2 |
34 28 24 |
(29) 5.0 |
23 23 23 |
(23) 0.0 |
9 12 16 |
(12) 3.5 |
||
500 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
27 S 20 S 28 S |
(25) 4.4 |
0 V 0 V 0 V |
(0) 0.0 |
3 S 3 S 9 S |
(5) 3.5 |
||
1500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
2047 2156 2207 |
(2137) 81.7 |
385 377 326 |
(363) 32.0 |
601 512 560 |
(558) 44.5 |
248 285 260 |
(264) 18.9 |
465 475 437 |
(459) 19.7 |
Table 3: Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)
Test Period |
From: 24 October 2017 |
To: 27 October 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Water) |
83 99 83 |
(88) 9.2# |
14 8 11 |
(11) 3.0 |
21 22 37 |
(27) 9.0 |
22 17 18 |
(19) 2.6 |
6 12 12 |
(10) 3.5 |
||
0.15 µg |
96 89 106 |
(97) 8.5 |
12 18 11 |
(14) 3.8 |
17 20 36 |
(24) 10.2 |
15 22 18 |
(18) 3.5 |
9 9 20 |
(13) 6.4 |
||
0.5 µg |
106 103 105 |
(105) 1.5 |
20 10 11 |
(14) 5.5 |
19 25 17 |
(20) 4.2 |
18 25 14 |
(19) 5.6 |
13 17 8 |
(13) 4.5 |
||
1.5 µg |
106 98 91 |
(98) 7.5 |
26 19 13 |
(19) 6.5 |
25 20 22 |
(22) 2.5 |
29 19 21 |
(23) 5.3 |
16 17 12 |
(15) 2.6 |
||
5 µg |
94 97 104 |
(98) 5.1 |
11 12 11 |
(11) 0.6 |
23 23 24 |
(23) 0.6 |
15 13 13 |
(14) 1.2 |
18 14 6 |
(13) 6.1 |
||
15 µg |
114 90 110 |
(105) 12.9 |
19 9 21 |
(16) 6.4 |
18 31 22 |
(24) 6.7 |
17 24 26 |
(22) 4.7 |
11 20 14 |
(15) 4.6 |
||
50 µg |
76 S 89 S 91 S |
(85) 8.1 |
6 S 18 S 13 S |
(12) 6.0 |
31 16 18 |
(22) 8.1 |
15 S 20 S 10 S |
(15) 5.0 |
12 S 4 S 9 S |
(8) 4.0 |
||
150 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
21 S 12 S 21 S |
(18) 5.2 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
||
500 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
786 849 829 |
(821) 32.2 |
1071 1118 1220 |
(1136) 76.2 |
833 777 715 |
(775) 59.0 |
208 233 200 |
(214) 17.2 |
171 209 185 |
(188) 19.2 |
Table
4: Test
Results: Experiment 2 – With Metabolic Activation(Pre-Incubation)
Test Period |
From: 24 October 2017 |
To: 27 October 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (Water) |
83 94 96 |
(91) 7.0# |
13 18 8 |
(13) 5.0 |
33 33 39 |
(35) 3.5 |
21 17 26 |
(21) 4.5 |
13 14 15 |
(14) 1.0 |
||
0.15 µg |
93 97 101 |
(97) 4.0 |
10 8 13 |
(10) 2.5 |
38 33 34 |
(35) 2.6 |
27 19 26 |
(24) 4.4 |
18 15 19 |
(17) 2.1 |
||
0.5 µg |
98 93 99 |
(97) 3.2 |
13 11 12 |
(12) 1.0 |
42 36 38 |
(39) 3.1 |
37 12 20 |
(23) 12.8 |
25 14 8 |
(16) 8.6 |
||
1.5 µg |
114 103 101 |
(106) 7.0 |
15 12 13 |
(13) 1.5 |
32 39 28 |
(33) 5.6 |
23 14 27 |
(21) 6.7 |
19 15 16 |
(17) 2.1 |
||
5 µg |
88 73 103 |
(88) 15.0 |
13 8 9 |
(10) 2.6 |
31 22 37 |
(30) 7.5 |
36 22 25 |
(28) 7.4 |
20 6 17 |
(14) 7.4 |
||
15 µg |
99 108 96 |
(101) 6.2 |
8 10 7 |
(8) 1.5 |
26 21 31 |
(26) 5.0 |
19 34 25 |
(26) 7.5 |
21 16 14 |
(17) 3.6 |
||
50 µg |
116 93 82 |
(97) 17.3 |
15 9 9 |
(11) 3.5 |
31 25 22 |
(26) 4.6 |
32 25 31 |
(29) 3.8 |
17 19 16 |
(17) 1.5 |
||
150 µg |
106 S 104 S 111 S |
(107) 3.6 |
7 S 18 S 10 S |
(12) 5.7 |
40 32 19 |
(30) 10.6 |
22 32 19 |
(24) 6.8 |
15 20 15 |
(17) 2.9 |
||
500 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
60 S 34 S 41 S |
(45) 13.5 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1234 1172 1126 |
(1177) 54.2 |
222 237 255 |
(238) 16.5 |
259 202 186 |
(216) 38.4 |
117 114 149 |
(127) 19.4 |
329 288 299 |
(305) 21.2 |
†: Experimental procedure repeated at a later date due to high colony counts in the original test
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
S: Sparse bacterial background lawn
T: Toxic, no bacterial background lawn
V: Very weak bacterial background lawn
#: Standard deviation
The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) the test substance induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9‑mix) initially from 150 µg/plate. Consequently, the toxic limit of the test substance was selected as the maximum dose level in the second mutation test. Results from the second mutation test showed that the test substance induced a stronger toxic response employing the pre‑incubation modification with weakened bacterial background lawns initially noted in the absence of S9‑mix from 50 µg/plate (all Salmonella strains) and 150 µg/plate (WP2uvrA). In the presence S9 -mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100 and TA1535) and 500 µg/plate (TA98, TA1537 and WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9 -mix and experimental methodology. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9 -mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9 -mix) in Experiment 2 (pre‑incubation method). Minor statistical values were noted in Experiment 2 (TA100 at 0.5 and 15 µg/plate in the absence of S9 -mix), however these responses were within the in-house historical vehicle/untreated control values and were, therefore considered of no biological relevance. Based on the study results, the test substance was considered to be non-mutagenic with and without metabolic activation.
Applicant's summary and conclusion
- Conclusions:
- Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation
- Executive summary:
A study was conducted to determine the genotoxic potential of the test substance, 'Cocodimonium hydroxypropyl hydrolysed silk' (active: 51%), using Ames test method, according to OECD Guideline 471, EU Method B13/14 and the USA, EPA OCSPP harmonized guideline, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using both the Ames plate incorporation (Experiment 1) and pre-incubation (Experiment 2) methods at eight dose levels (i.e., 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation method) was predetermined and ranged from 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended for Experiment 2, following the results of Experiment 1 and ranged from 0.15 to 500 µg/plate . There were eight test substance concentrations (i.e., 0.15, 0.5, 1.5, 5, 15, 50, 150 500 μg/plate), which were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the toxic limit of the test substance following the change in test methodology. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the first mutation test (Experiment 1), the test substance induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9‑mix) initially from 150 µg/plate. Consequently, the toxic limit of the test substance was selected as the maximum dose level in the second mutation test (Experiment 2). Results from the second mutation test showed that the test substance induced a stronger toxic response employing the pre‑incubation modification with weakened bacterial background lawns initially noted in the absence of S9‑mix from 50 µg/plate (all Salmonella strains) and 150 µg/plate (WP2uvrA). In the presence S9 -mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100 and TA1535) and 500 µg/plate (TA98, TA1537 and WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9 -mix and experimental methodology. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9 -mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9 -mix) in Experiment 2 (pre‑incubation method). Minor statistical values were noted in Experiment 2 (TA100 at 0.5 and 15 µg/plate in the absence of S9 -mix), however these responses were within the in-house historical vehicle/untreated control values and were, therefore considered of no biological relevance. Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (Envigo, 2017).
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