3-aminomethyl-3,5,5-trimethylcyclohexan-1-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-04-06 to 2016-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-aminomethyl-3,5,5-trimethylcyclohexan-1-ol

EC Number:

239-717-0

EC Name:

3-aminomethyl-3,5,5-trimethylcyclohexan-1-ol

Cas Number:

15647-11-7

Molecular formula:

C10H21NO

IUPAC Name:

3-(aminomethyl)-3,5,5-trimethylcyclohexan-1-ol

Test material form:

other: liquid > 20°C

Details on test material:

3-aminomethyl-3,5,5-trimethylcyclohexan-1-ol manufactured by Evonik Degussa GmbH, Batch 2014-11, purity 99.25% w/w

Specific details on test material used for the study:

The test item was completely dissolved in highly purified water.

Method

Target gene:

mutated gene loci resposible for histidine auxotropy

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- obtained from Trinova Biochem according to Dr. Bruce N. AMES,

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver S9; male rats, obtained from Trinova Biochem

Test concentrations with justification for top dose:

Plate incorporation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
Preincubation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate

Vehicle / solvent:

The test item was completely dissolved in highly purified water. The vehicle highly purified water was employed as the negative control. Fresh preparations of the test item were used for the treatment in all experimental parts.

Controls

Untreated negative controls:

yes

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

Highly purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate (see tables 1 and 2). Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction.
ADMINISTRATION
- Dosing:
* Plate incorporation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
* Preincubation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
* sodium azide in highly purufied water for TA 1535 and TA 100, 10 µg/plate
* 2-nitroflurene in DMSO for TA 98, 10 µg/plate
* 9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
* 4-nitroquinoline-N-oxide in DMSO for E.coli WP2, 5 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100, TA 1535 and WP2, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, and TA 1537, 10 µg/plate
- negative control: the vehicle highly purified water was used as negative reference item (all test strains).
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

NUMBER OF REPLICATIONS: 3 per concentration and experiment

NUMBER OF CELLS EVALUATED: Overnight cultures were grown in a water bath for 15 h at 37°C in Oxoid 2 nutrient broth. The final cell density was approximately 10E8 - 10E9 cells/mL.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Rationale for test conditions:

The study was performed in compliance with:
- Regulation (EC) No. 440/2008 method B.13/14: Mutagenicity: Reverse Mutation Test Using Bacteria, adopted May 30, 2008;
- OECD Guideline for Testing of Chemicals, No. 471: Bacterial Reverse Mutation Test, adopted July 21, 1997;

Evaluation criteria:

A test item is considered to show a positive response if
- the number of revertants is significantly increased (p- in addition, a significant (pBiological relevance of the results should be considered first.
Positive results have to be reproducible and the histidine or tryptophan independence of the revertants has to be confirmed by streaking random samples on histidine or tryptophan-free agar plates.

A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
Salmonella typhimurium TA98: 20 - 60
TA100: 100 - 200
TA1535: 10 - 35
TA1537: 3 - 20
Escherichia coli WP2 uvrA: 25 - 65
The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

The results of the negative and positive control cultures have to be within the range of the historical data generated by LPT.

Statistics:

According to the OECD Guideline 471, a statistical analysis of the data is not mandatory

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with metabolic activation, cytotoxicity was noted at the top concentration of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ

CYTOTOXICITY EFFECTS:
No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in the Salmonella typhimurium strains and in the Escherichia coli test strain in the experiments without S9 mix.
In the experiments with metabolic activation, cytotoxicity (reduction of the number of revertants by more than 50%) was noted at the top concentration of 5000 µg/plate in the Escherichia coli strain WP2 uvrA [pKM101].

Any other information on results incl. tables

see attchached document

Applicant's summary and conclusion

Conclusions:

In conclusion, under the present test conditions, test item tested up to the concentration of 5000 µg/plate (cytotoxic in the Escherichia coli strain WP2 uvrA [pKM101] in the presence of metabolic activation), caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in the Escherichia coli strain WP2 uvrA [pKM101], neither in the plate incorporation test nor in the preincubation test, each carried out without and with metabolic activation.

Executive summary:

The purpose of this study was to evaluate the test substance for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and (1983).

Test item was examined in the 4 Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in one Escherichia coli strain WP2 uvrA [pKM101] in respective two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

Test item was completely dissolved in highly purified water. The vehicle highly purified water served as the negative control.

Preliminary test

Test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in the Salmonella typhimurium test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 31.6 to 5000 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in the Salmonella typhimurium strains and in the Escherichia coli test strain in the experiments without S9 mix. In the experiments with metabolic activation, cytotoxicity (reduction of the number of revertants by more than 50%) was noted at the top concentration of 5000 µg/plate in the Escherichia coli strain WP2 uvrA [pKM101].

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to the concentration of 5000 µg/plate, in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

 

In conclusion, under the present test conditions, test item tested up to the concentration of 5000 µg/plate (cytotoxic in the Escherichia coli strain WP2 uvrA [pKM101] in the presence of metabolic activation), caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in the Escherichia coli strain WP2 uvrA [pKM101], neither in the plate incorporation test nor in the preincubation test, each carried out without and with metabolic activation.