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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phase of this study was performed between 08 June 2011 and 14 July 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell lines (if applicable):
Not applicable.
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle:
Vehicle: sterile distilled water

Justification for choice of solvent/vehicle:
The test item was found to be soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house. Therefore, this solvent was selected as the vehicle of choice.
Controlsopen allclose all
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Solvent controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Solvent controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Solvent controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA102
Solvent controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Solvent controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Solvent controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Solvent controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Solvent controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Solvent controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix
Negative controls:
yes
Remarks:
Spontaneous mutation rates of TA102
Solvent controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix
Details on test system and conditions:
METHODS OF APPLICATION: in agar (pre-incubation) – Experiment 1 and 2
- Pre-incubation period for bacterial strains: 10hrs
- Exposure duration: 48-72hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (in incubation with a selective agent): 25 minutes at 37 degrees C


NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
-Method: the test item must have caused a reduction in the number of spontaneous revertants (below a factor of 0.5 fold under the concurrent solvent control value) and/or the bacterial lawn should have exhibited evidence of thinning when viewed microscopically.
Evaluation criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per ml.
All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination

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).
A test item 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 judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation
Dunnetts Regression Analysis

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECFIC CONFOUNDING FACTORS:
- Colouration: A blue/black test item colouration was observed at and above 150 µg/plate. This observation did not prevent the scoring of revertant colonies.

STERILITY, VEHICLE AND POSITIVE CONTROL DATA:
- The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The S9 mix and top agar used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test

Any other information on results incl. tables

Table1          Spontaneous Mutation Rates (Concurrent Negative Controls)

 

EXPERIMENT1

 

Number of revertants(mean number of colonies per plate)

 

Base-pairsubstitution type

Frameshifttype

TA100

TA1535

TA102

TA98

TA1537

90

 

15

218

 

20

 

 

7

132

(112)

27        (21)

198

(202)

13

 

(14)

14         (12)

115

 

20

190

 

10

 

 

14

 

EXPERIMENT 2

 

 

 

Numberofrevertants(meannumberofcoloniesperplate)

Base-pairsubstitution type

Frameshifttype

TA100

TA1535

TA102

TA98

TA1537

97

 

25

207

 

8

 

 

11

 

98

(98)

22           (5)

191

(229)

15

 

(15)

4

(7)

98

 

27

289

 

21

 

 

5

 

 

19

 

14        (16)t

16

 

Table1          SpontaneousMutationRates(ConcurrentNegativeControls)

 

EXPERIMENT1

 

Numberofrevertants(mean number of colonies per plate)

 

Base-pair substitution type

Frame shift type

TA100

TA1535

TA102

TA98

TA1537

90

 

15

218

 

20

 

 

7

132

(112)

27          (21)

198

(202)

13

 

(14)

14         (12)

115

 

20

190

 

10

 

 

14

 

EXPERIMENT 2

 

 

 

Numberofrevertants(mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

97

 

25

207

 

8

 

 

11

 

98

(98)

22          (25)

191

(229)

15

 

(15)

4

(7)

98

 

27

289

 

21

 

 

5

 

 

19

 

14        (16)t

16

 

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.  

The method incorporated the Prival and Mitchell modification for azo dyes. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. The method incorporated the Prival and Mitchell modification for azo dyes.

Methods.

Salmonella Typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test item using the Ames pre-incubation method at five dose levels, in triplicate, both with and without the addition of a hamster liver homogenate metabolising system (30% liver S9 in modified co-factors). The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.oo The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver S9-mix and the uninduced hamster liver S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test item was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate.  A yellow item colouration was observed at and above 150 µg/plate. This observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the, either with or without metabolic activation.

Conclusion. The test item was considered to be non-mutagenic under the conditions of this test.