Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An OECD 471compliant  Bacterial Reverse Mutation (Ames) test was conducted with Cyclohexylamine Benzoate at levels of 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 µg/plate.

There was no concentration-related or substantial test substance related increases in the number of revertant colonies observed with strains TA1535, TA1537, TA98 , TA100 or E.coli WP2 uvrA in both the absence and presence of S9 using either the plate incorporation or the pre-incubation method.

Based on these findings and on the evaluation system used, Cyclohexylamine Benzoate did not elicit evidence of bacterial mutagenicity in the Ames assay.

Under the condotions of the test, cyclohexylamine was not mutagenic.

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:
March-June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
See remark below
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
See remark below
Principles of method if other than guideline:
Deviation: The protocol requires that the performance of the test be evaluated with positive controls for each tester strain used. Due to an oversight, 2-Aminoanthracene was not added to strain TA1535 in the plate incorporation method.
As a result, strain TA1535 using the plate incorporation method was repeated along with appropriate sterility control check plates. The data collected from the initial test is maintained in the raw data. There was no impact on the study as the test was repeated for the bacteria strain.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Composition: Cyclohexyamine Benzoate - 99.7%, CAS #3129-92-8
Physical Description: Off-white crystalline powder
pH: ~7 (5% aq solution)
Stability: Test substance was expected to be stable for the duration of testing.
Expiration Date: Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (cofactor supplemented post-mitochondrial fraction)
Test concentrations with justification for top dose:
0, 1.58, 5.0, 15.8, 50, 158, 500, 1580, 5000 μg/plate

5000 μg/plate was selected as the top dose as it is the OECD standard limit dose.
Vehicle / solvent:
The test substance was found to be soluble in sterile water that was used as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: ICR 1919 Acridine, Daunomycin, 2 aminoanthracene
Details on test system and experimental conditions:
Fresh bacterial suspension cultures in nutrient broth were prepared so that they were in the late exponential phase of growth at the time of use (approximately 1 x 109 bacteria/mL). Bacterial
growth was evaluated by spectrophotometric optical density measurement.

The test substance was formulated as a solution in sterile water (0.0158, 0.05, 0.158, 0.5, 1.58, 5, 15.8 and 50 mg/mL) to provide corresponding dose levels of up to 5000 µg/plate. The solutions
were vortexed prior to use.
Evaluation criteria:
The mean revertant colony counts for each strain treated with the vehicle should lie close to or within the expected range taking into account the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012). The positive controls (with S9 where required) should produce substantial increases in revertant colony numbers with the appropriate bacterial strain.
Statistics:
Calculated means and standard deviations for all quantitative data collected.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values
(Mortelmans & Zeiger, 2000; Gatehouse, 2012). The positive control substances caused the expected substantial increases in revertant colony counts in both the absence and presence of S9
in each phase of the test confirming the sensitivity of the test and the activity of the S9 mix. Therefore, each phase of the test is considered valid.

There was no evidence of toxicity or precipitation present for any of the strains at the tested concentrations. Individual plate contamination was noted for strain TA1537 in the plate incorporation method at 158 µg/plate with S9. Contamination did not impact mutagenic evaluation.

For all strains, eight non-toxic doses were evaluated; therefore bacterial mutagenicity was adequately assessed.
Conclusions:
An OECD 471compliant Bacterial Reverse Mutation (Ames) test was conducted with Cyclohexylamine Benzoate at levels of 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 µg/plate.

There was no concentration-related or substantial test substance related increases in the number of revertant colonies observed with strains TA1535, TA1537, TA98 , TA100 or E.coli WP2 uvrA in both the absence and presence of S9 using either the plate incorporation or the pre-incubation method.

Based on these findings and on the evaluation system used, Cyclohexylamine Benzoate did not elicit evidence of bacterial mutagenicity in the Ames assay.

Under the condotions of the test, cyclohexylamine was not mutagenic.
Executive summary:

An OECD 471 Bacterial Reverse Mutation (Ames) test was conducted with Cyclohexylamine Benzoate at levels of 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 µg/plate.

The main test was conducted using the plate incorporation method in both the absence and presence of metabolic activation (chemically-induced rat liver S9 mix). The results of the test were confirmed using a similar study design but employing the pre-incubation modification of the Ames test.

There was no evidence of toxicity or precipitation present for any of the strains at the tested concentrations. Individual plate contamination was noted for strain TA1537 in the plate incorporation method at 158 µg/plate with S9. Contamination did not impact mutageni evaluation.

For all strains, eight non-toxic doses were evaluated; therefore bacterial mutagenicity was adequately assessed.

There was no concentration-related or substantial test substance related increases in the number of revertant colonies observed with strains TA1535, TA1537, TA98 , TA100 or E.coli WP2 uvrA in both the absence and presence of S9 using either the plate incorporation or the pre-incubation method.

Based on these findings and on the evaluation system used, Cyclohexylamine Benzoate did not elicit evidence of bacterial mutagenicity in the Ames assay.


Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification