reaction products of 1 mole of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs and 1 mole of cyclohexylamine

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:

10 February 2017 - 14 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Experiments without S9 mix
The selected dose levels were as follows:
- 4.1, 12.3, 37, 111.1, 333.3 and 1000 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the first experiment and for the TA 1537, TA 98 and TA 100 strains in the second experiment,
- 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 102 strain in the first and second experiments,
- 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1535 strain in the second experiment,
- 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1535 strain in the third experiment.

Experiments with S9 mix
The selected dose levels were as follows:
- 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains in the first experiment and for the TA 1535, TA 1537, TA 98, and TA 102 strains in the second experiment,
- 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the TA 100 strain in the second experiment,
- 3.4, 10.3, 30.9, 92.6, 277.8, 833.3 and 2500 µg/plate for the TA 1537 strain in the third experiment.

Using a test item concentration of 50 mg/mL in the vehicle and a treatment volume of 100 µL/plate, the highest recommended dose level of 5000 µg/plate was achievable. Thus, the dose levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle used: water for injections
- Justification for choice: According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control plates was water for injections.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);
The preliminary test, all experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second and third experiments with S9 mix were performed according to the pre-incubation method.

DURATION
- Preincubation period: 60 minutes
- Incubation duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: observation of a decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

NUMBER OF REPLICATIONS: ??????

Rationale for test conditions:

Not applicable.

Evaluation criteria:

In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose levels,
- nor any evidence of a dose-response relationship is noted.

When inconclusive results were observed, an additional confirmatory experiment was specified by an amendment to the study plan (phrase à conserver ??????).

Statistics:

no

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING STUDY:
No precipitate was observed in the Petri plates when scoring the revertants at any dose levels, either with or without S9 mix.

A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose levels = 500 µg/plate in the TA 98 and TA 100 strains without S9 mix and = 2500 µg/plate in the TA 102 strain without S9 mix as well as in the TA 98 and TA 100 strains with S9 mix.
No noteworthy toxicity was noted at any dose levels in the TA 102 strain with S9 mix.

RESULTS OF CYTOTOXICITY and GENOTOXICITY:
Experiments without S9 mix:
A moderate to strong toxicity was noted at dose levels = 111.1 µg/plate in the TA 1537 strain, = 333.3 µg/plate in the TA 100 strain, at 1000 µg/plate in the TA 98 strain, and at 5000 µg/plate in the TA 102 strain.
No noteworthy toxicity was noted in the TA 1535 strain in the first experiment whereas a toxicity was noted at 5000 µg/plate in the second experiment then at dose levels = 185.2 µg/plate in the third experiment (microcolonies was a marker of strong toxicity preventing the plates reading), though performed under the same experimental conditions.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in any experiments.

These results in the absence of S9 mix met the criteria of a negative response.

Experiments with S9 mix:
Using the direct plate incorporation method (i.e. first experiment), a moderate to strong toxicity was noted at the highest tested dose level of 5000 µg/plate in the TA 1535 and TA 98 strains, and at dose levels = 2500 µg/plate in the TA 1537 and TA 100 strains. No noteworthy toxicity was noted in the TA 102 strain.
Using the pre-incubation method (i.e. second and third experiments), a moderate to strong toxicity was noted at the highest tested dose level of 5000 µg/plate in the TA 1535 and TA 102 strains, at dose levels = 2500 µg/plate in the TA 98 strain, = 833.3 µg/plate in the TA 1537 strain and = 625 µg/plate in the TA 100 strain (microcolonies was a marker of strong toxicity preventing the plates reading).

An increase in the number of revertants was noted at 2500 µg/plate in the TA 1537 strain in the second experiment (pre-incubation method). This increase exceeded the positive threshold of 3-fold the vehicle control value (6.3-fold the vehicle control value) and the corresponding mean number of revertants was above the vehicle control historical range (52.7 versus [5-18] for historical data). However, this increase occurred only at a second highly toxic dose level (strong thinning of the bacterial lawn) and was not reproduced in the third experiment performed under the same experimental conditions. Moreover, microcolonies were observed in the plates at 2500 µg/plate in the third experiment suggesting that the increase noted in the second experiment was also due to high toxicity rather than genotoxicity. As a consequence, the increase observed in the second experiment was considered to be non-biologically relevant.

The test item did not induce any other noteworthy increase in the number of revertants, in any other strains or test conditions.

The overall results in the presence of S9 mix were considered to meet the criteria of a negative response.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

 

Methods

A preliminary toxicity test was performed to define the dose levels of the test item, formulated in water for injections, to be used for the mutagenicity experiments. The test item was then tested in three independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Treatments were performed according to the direct plate incorporation method except for the second and third experiments with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least six dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

Results

Since the test item was found to be freely soluble in the final treatment medium but toxic in the preliminary test, the selection of the highest dose level for the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose levels for each strain and test condition. The study was therefore considered to be valid.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose levels, either with or without S9 mix.

 

Experiments without S9 mix

The selected dose levels were as follows:

.         4.1, 12.3, 37, 111.1, 333.3 and 1000 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the first experiment and for the TA 1537, TA 98 and TA 100 strains in the second experiment,

.         156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 102 strain in the first and second experiments,

.         20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1535 strain in the second experiment,

.         6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1535 strain in the third experiment.

 

A moderate to strong toxicity was noted at dose levels >= 111.1 µg/plate in the TA 1537 strain, >= 333.3 µg/plate in the TA 100 strain, at 1000 µg/plate in the TA 98 strain, and at 5000 µg/plate in the TA 102 strain.

No noteworthy toxicity was noted in the TA 1535 strain in the first experiment whereas a toxicity was noted at 5000 µg/plate in the second experiment then at dose levels >= 185.2 µg/plate in the third experiment (microcolonies was a marker of strong toxicity preventing the plates reading), though performed under the same experimental conditions.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in any experiments.

 

These results in the absence of S9 mix met the criteria of a negative response.

 

Experiments with S9 mix

The selected dose levels were as follows:

.         156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains in the first experiment and for the TA 1535, TA 1537, TA 98, and TA 102 strains in the second experiment,

.         78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the TA 100 strain in the second experiment,

.         3.4, 10.3, 30.9, 92.6, 277.8, 833.3 and 2500 µg/plate for the TA 1537 strain in the third experiment.

 

Using the direct plate incorporation method (i.e. first experiment), a moderate to strong toxicity was noted at the highest tested dose level of 5000 µg/plate in the TA 1535 and TA 98 strains, and at dose levels >= 2500 µg/plate in the TA 1537 and TA 100 strains. No noteworthy toxicity was noted in the TA 102 strain.

Using the pre-incubation method (i.e. second and third experiments), a moderate to strong toxicity was noted at the highest tested dose level of 5000 µg/plate in the TA 1535 and TA 102 strains, at dose levels >= 2500 µg/plate in the TA 98 strain, >= 833.3 µg/plate in the TA 1537 strain and >= 625 µg/plate in the TA 100 strain (microcolonies was a marker of strong toxicity preventing the plates reading).

 

An increase in the number of revertants was noted at 2500 µg/plate in the TA 1537 strain in the second experiment (pre-incubation method). This increase exceeded the positive threshold of 3-fold the vehicle control value (6.3-fold the vehicle control value) and the corresponding mean number of revertants was above the vehicle control historical range (52.7 versus [5-18] for historical data). However, this increase occurred only at a second highly toxic dose level (strong thinning of the bacterial lawn) and was not reproduced in the third experiment performed under the same experimental conditions. Moreover, microcolonies were observed in the plates at 2500 µg/plate in the third experiment suggesting that the increase noted in the second experiment was also due to high toxicity rather than genotoxicity. As a consequence, the increase observed in the second experiment was considered to be non-biologically relevant.

 

The test item did not induce any other noteworthy increase in the number of revertants, in any other strains or test conditions.

 

The overall results in the presence of S9 mix were considered to meet the criteria of a negative response.

 

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.