[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro gene mutation study was conducted in accordance with the OECD Testing Guideline 471. The registered substance was not mutagenic under the conditions of the test, with and without metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 August 2020 - 15 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 21 July 1997, Corrected 26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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:

Rat liver S9-mix induced by Aroclor 1254 (500 mg/kg body weight)

Test concentrations with justification for top dose:

First experiment:
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512 and 1000 µg/plate (tested in TA100 and WP2uvrA, both with and without S9-mix)
Direct plate assay: 5.4, 17, 52, 164, 512 and 1000 µg/plate (tested in TA1535, TA1537 and TA98, both with and without S9-mix)

Second experiment:
Pre-incubation assay: 5.4, 17, 52, 164, 512 and 1000 µg/plate ( tested in TA1535, TA1537, TA98 TA100 and WP2uvrA, with and without S9-mix)

Vehicle / solvent:

- Vehicle used: ethanol

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

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

PLATING
- During plating, the test item solutions were kept in a water bath of 45 +/- 2°C; test item concentrations were used within 4 h after preparation.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 ± 2 minutes
- Exposure duration/duration of treatment: 48 ± 4 h

ENVIRONMENTAL CONDITIONS
- Incubation temperature (37.3 - 44.6°C)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two
times the concurrent control, and the total number of revertants in tester strains TA1535,
TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two
times the concurrent control, or the total number of revertants in tester strains TA1535,
TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of
the tester strains, the positive response should be reproducible in at least one follow up
experiment.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the additional experiment with tester strain TA1537, cytotoxicity, as evidenced by a decrease in the number of revertants, was observed at a dose level of 512 µg/plate in the presence of S9-mix.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First Experiment: Direct Plate Assay
Solvent control: Because several solvent control plates demonstrated a number of revertants below the historical negative control range in tester strain TA1537 in the absence and presence of
S9-mix, two repeat experiments with TA1537 (one in the absence of S9-mix, another in the presence of S9-mix) were performed to confirm initial observations. These repeat experiments are reported as part of the first mutation experiment.

Toxicity: In strain TA100 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no doserelationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies. In the additional experiment with tester strain TA1537, cytotoxicity, as evidenced by a decrease in the number of revertants, was observed at a dose level of 512 µg/plate in the presence of S9-mix.

Mutagenicity: In the direct plate test, no relevant increase in the number of revertants was observed upon treatment with the test item under all conditions tested. In tester strain TA1537, the three-fold increase observed at the highest tested concentration was considered to be not to be biologically relevant due to the low control value. Moreover, this increase was within the historical control data ranges of the solvent control and was not confirmed in the additional and second experiments.

Second experiment: Pre-incubation assay
Toxicity: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

ACCEPTIBILITY:

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

 

Conclusions:

In an AMES test, performed according to OECD guideline 471 and in accordance with GLP principles, Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD guideline 471 and in accordance with GLP principles. In the dose-range finding study, the test item was initially tested up to concentrations of 1000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose levels of 512 µg/plate and upwards. The test item precipitated heavily on some of the plates at a concentration of 1000 µg/plate; the number of revertants and bacterial background lawn of this dose level could not be determined.
In the first mutation experiment, the test item was tested up to concentrations of
1000 µg/plate in the strains TA1535, TA1537 and TA98. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
Since in the first experiment in tester strain TA1537, some values of the individual solvent control plates were below the historical data range an additional experiment was performed.
In the additional experiment, the test item was tested up to concentrations of 1000 µg/plate in strain TA1537 in the absence and presence of S9-mix. The test item was tested up to or beyond a precipitating dose level. In the absence of S9-mix, the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in
the number of revertants was observed. In the presence of S9-mix, cytotoxicity, as evidenced by a decrease in the number of revertants, was observed at a dose level of 512 µg/plate in the presence of S9-mix. In addition, the test item precipitated heavily on the plates at a concentration of 1000 µg/plate; the number of revertants and bacterial background lawn at this dose level could not be determined.
In the second mutation experiment, the test item was tested up to concentrations of 1000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item was tested up to or beyond a precipitating dose level. The test item precipitated heavily on some of the plates at the concentration of 1000 µg/plate; the number of revertants and bacterial background lawn of this dose level could not be determined. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The mean negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro gene mutation in bacteria
An AMES test was performed according to OECD guideline 471 and in accordance with GLP principles. In the dose-range finding study, the test item was initially tested up to concentrations of 1000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose levels of 512 µg/plate and upwards. The test item precipitated heavily on some of the plates at a concentration of 1000 µg/plate; the number of revertants and bacterial background lawn of this dose level could not be determined.
In the first mutation experiment, the test item was tested up to concentrations of
1000 µg/plate in the strains TA1535, TA1537 and TA98. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
Since in the first experiment in tester strain TA1537, some values of the individual solvent control plates were below the historical data range an additional experiment was performed.
In the additional experiment, the test item was tested up to concentrations of 1000 µg/plate in strain TA1537 in the absence and presence of S9-mix. The test item was tested up to or beyond a precipitating dose level. In the absence of S9-mix, the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in
the number of revertants was observed. In the presence of S9-mix, cytotoxicity, as evidenced by a decrease in the number of revertants, was observed at a dose level of 512 µg/plate in the presence of S9-mix. In addition, the test item precipitated heavily on the plates at a concentration of 1000 µg/plate; the number of revertants and bacterial background lawn at this dose level could not be determined.
In the second mutation experiment, the test item was tested up to concentrations of 1000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item was tested up to or beyond a precipitating dose level. The test item precipitated heavily on some of the plates at the concentration of 1000 µg/plate; the number of revertants and bacterial background lawn of this dose level could not be determined. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
The mean negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
In conclusion, based on the results of this study it is concluded that Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Based on the outcome of in vitro testing, the registered substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008.