4-Nitrophenyl 2-(methylsulphonyl)ethanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria was evaluated based on OECD 471.

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

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2014-03-24 to 2014-05-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: PNP-0020313
Purity: 99.5%

Target gene:

histidine locus of Salmonella strains, tryptophan operon of E. coli tester strain

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation system
Type and composition of metabolic activation system:
- source of S9: Lot No. PB/βNF S9 02 March 2014 was used in this study. The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4.
- method of preparation of S9 mix: The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test with following recipe:
S9: 5.0 mL
1. 65 M KCl/0.4 M MgCl2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL
- concentration or volume of S9 mix and S9 in the final culture medium:
Experiment 1: 0.5 mL of S9-mix was added to molten trace amino-acid supplemented media (final volume 2.0 mL).
Experiment 2: 0.5 mL of S9-mix was added to the tube together with 0.1 mL of the appropriate bacterial strain culture, and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control and incubated at 37 ℃ for 20 mins prior to addition of 2 mL of molten amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.
- quality controls of S9: S9-mix used in both experiments was shown to be sterile

Test concentrations with justification for top dose:

Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, the maximum dose level of the test item was selected as the maximum recommended dose level of 5000 μg/plate.
Experiment 2 - Pre-Incubation Method: 5, 15, 50, 150, 500, 1500 μg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains at and above 1500μg/plate in both presence and absence of metabolic activation (S9-mix) in the first mutation test (plate incorporation method). Consequently, the toxic limit of the test item was selected as the maximum dose level in the second mutation test (pre-incubation method).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was fully soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA): For TA100, TA1535, TA1537 and WP 2uvrA with S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: in triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar: plate incorporation (Experiment 1); pre-incubation (Experiment 2)

TREATMENT AND HARVEST SCHEDULE:
- Pre-incubation period: Experiment 2: 20 minutes
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: viewed microscopically for evidence of thinning (toxicity)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- OTHER: All of the plates were scored for the presence of revertant colonies using an automated colony counting system.

Evaluation criteria:

Any, one, or all of the following can be used to determine the overall positive 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 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 judgment about test item activity. Results of this type will be reported as equivocal.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Ames test:
- Signs of toxicity: The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains at and above 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix) in the first mutation test (plate incorporation method). Results from the second mutation showed weakened lawns to all of the bacterial strains at 1500 μg/plate in both the presence and absence of S9-mix.
- Genotoxicity: There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the presence of S9-mix at 150 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle control.
- Precipitate: A yellow test item induced coloration was noted at and above 500 μg/plate, this observation did not affect the scoring of revertant colonies. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- Positive/negative control results:
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

Conclusions:

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

Executive summary:

The purpose of the study was to evaluate test item for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria based on OECD 471.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvr A were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). 

The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains at and above 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix) in the first mutation test (plate incorporation method). Results from the second mutation showed weakened lawns to all of the bacterial strains at 1500 μg/plate in both the presence and absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the presence of S9-mix at 150 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle control.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Ames test, OECD 471, negative

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the test substance should not be classified as germ cell mutagens.