Cyclohexanamine, 4,4’-methylenebis[N-(2,2-dimethyl-3-(4-morpholinyl)propylidene)]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a Bacterial Reverse Mutation Assay according to OECD guideline 471, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used with and without metabolic activation. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

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:

2021-03-25 to 2021-06-08

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:

corrected 26 June, 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use

Version / remarks:

November 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S.typhimurium: his;
E.coli: trp

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: defective for DNA excision repair (uvrA mutation)

Remarks:

genotype: trpE

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: increased cell wall permeability (rfa mutation)

Remarks:

defective in excision repair (uvrB mutation); genotypes: hisG46, hisC3076, hisD3052, hisG46

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) provided by Trinova Biochem GmbH
-Preparation of S9: Performed according to Ames et al. (1975)
- Volume of S9 mix and S9 in the final culture medium: 500 µL S9 mix (containing 10% S9) in a final culture volume of 2750 µL.
- Quality controls of S9: Yes

Reference:
B.N. Ames, J. Mccann and E. Yamasaki (1975): Methods for Detecting Carcinogens and Mutagens with the Salmonella / Mammalian - Microsome Mutagenicity Test. Mutation Research, 31: 347-364, 1975.

Test concentrations with justification for top dose:

Selection of the concentrations was done on the basis of a solubility test and a
concentration range finding test:

5000; 1600; 500; 160, 50 and 16 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle and for percentage of solvent in the final culture medium: based on the results of the solubility test

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone (test item), DMSO (NPD, 9AA, 2AA), water (SAZ)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other:

Remarks:

Without S9:
NPD: 4 μg/plate (TA 98)
SAZ: 2 μg/plate (TA 100, TA 1535)
9AA: 50 µg (TA 1537)
MMS: 2 µL (E.coli WP2 uvrA)

With S9:
2AA: 2 μg (all TA strains), 50 μg (E.coli WP2 uvrA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two (a plate incorporation test (experiment I, initial mutation test) and in a pre-incubation test (experiment II, confirmatory mutation test))

METHOD OF TREATMENT/ EXPOSURE:
- Cell density (overnight culture): 2.25 x 10^9 CFU/mL (E.coli); approx. 1.15 x 10^9 CFU/mL (S.typhimurium)
- The experiment I (initial mutation test) of the main study the plate incorporation method was used. In the experiment II (confirmatory mutation test) the pre-incubation method was applied and the concentrations examined were the same as investigated in the experiment I.

- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 10^9cells/mL; the viable cell number of the cultures was determined by plating experiments and manual counting.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The cytotoxicity was indicated by decreased revertant colony counts (revertants below the historical control data ranges and/or corresponding vehicle control data ranges) and/or affected background lawn development (slightly reduced background lawn).

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mean revertant numbers

CONDITIONS FOR THE VALIDITY OF THE TEST
The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 10^9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analysable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).
A dose level is considered toxic if
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the vehicle historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.

Evaluation criteria:

The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined (counted manually, evaluated by unaided eye), the mean values, standard deviations and the mutation rates were calculated.
Mutation Rate = (Mean number of revertants at the test item (or control*) treatments)/(Mean number of revertants of vehicle control)
* : untreated, vehicle or positive control
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

According to the guidelines, the biological relevance of the results was the
criterion for the interpretation of results, a statistical evaluation of the results
was not regarded as necessary.

Key result

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

Five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a preincubation test (experiment II, confirmatory mutation test). Each assay was conducted with and without metabolic activation (±S9). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. No substantial increases in revertant colony numbers were observed in any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments; however, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.In the initial mutation test inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. However, in the confirmatory mutation test (pre-incubation test) clear inhibitory effect of the test item on bacterial growth was observed in the all Salmonella typhimurium strains, at 5000 µg/plate, in the absence and presence of exogenous metabolic activation (±S9). The cytotoxicity was indicated by decreased revertant colony counts (revertants below the historical control data ranges and/or corresponding vehicle control data ranges) and/or affected background lawn development (slightly reduced background lawn). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study.

 

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro, Ames

Five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a preincubation test (experiment II, confirmatory mutation test). Each assay was conducted with and without metabolic activation (±S9). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. No substantial increases in revertant colony numbers were observed in any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments; however, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.In the initial mutation test inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. However, in the confirmatory mutation test (pre-incubation test) clear inhibitory effect of the test item on bacterial growth was observed in the all Salmonella typhimurium strains, at 5000 µg/plate, in the absence and presence of exogenous metabolic activation (±S9). The cytotoxicity was indicated by decreased revertant colony counts (revertants below the historical control data ranges and/or corresponding vehicle control data ranges) and/or affected background lawn development (slightly reduced background lawn). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study.

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is not classified and labelled for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.