2,4,6-trimethylbenzene-1,3-diamine

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:

2016-03-17 to 2016-04-14

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:

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

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

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:

2,4,6-trimethylbenzene-1,3-diamine

EC Number:

221-456-9

EC Name:

2,4,6-trimethylbenzene-1,3-diamine

Cas Number:

3102-70-3

Molecular formula:

C9H14N2

IUPAC Name:

2,4,6-trimethylbenzene-1,3-diamine

Test material form:

other: crystalline powder

Details on test material:

2,4,6-trimethylbenzene-1,3-diamine supplied by Evonik Fibres GmbH (Lenzing, Austria), batch 20150703 (Dragon), purity 99.82 % w/w

Specific details on test material used for the study:

The test item was not soluble in any of the solvents generally recommended: highly purified water or dimethylsulfoxide (DMSO). However, the test item was completely dissolved in ethanol, a solvent acceptable for this test system. The vehicle ethanol served as the negative control.

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 TA 102

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 ethanol, a solvent acceptable for this test system. The vehicle ethanol served as the negative control.

Controls

Untreated negative controls:

yes

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

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/plate were tested. No signs of cytotoxicity were observed 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 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
* Mitomycin C in highly purifies water for TA 102, 10 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100 and TA 1535, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, TA 102 and 1537, 10 µg/plate
- negative control: the vehicle DMSO 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: approximately 10E8 viable cells in the late exponential or early stationary phase

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 solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation
coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Acceptance Criteria
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 102

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

Test results 2

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

The results point to a base-pair substitution.

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

Additional information on results:

GENTOXIC EFFECTS:
- With and without metabolic activation: In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation a pronounced concentration-related mutagenic effect (significant at p No mutagenic effect was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test.

CYTOTOXICITY EFFECTS:
No signs of cytotoxicity were observed 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 all test strains.

Any other information on results incl. tables

see attchached document

Applicant's summary and conclusion

Conclusions:

In conclusion, under the present test conditions, the test item caused a pronounced concentration-related mutagenic effect in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at concentrations of 3160 and/or 5000 µg test item/plate in test strain TA 100.
No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
The results point to a base-pair substitution.

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).

The potential of the test item to induce gene mutations was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in 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.

The test item was not soluble in any of the solvents generally recommended: highly purified water or dimethylsulfoxide (DMSO). However, the test item was completely dissolved in ethanol, a solvent acceptable for this test system.The vehicle ethanol 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 test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. No signs of cytotoxicity were observed 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 observed 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 all test strains.

Mutagenicity

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation a pronounced concentration-related mutagenic effect (significant at p </= 0.05) was observed in test strain TA 100.

All criteria for a positive response (a concentration (log value)-related effect and a 2-fold increase in revertant colony numbers compared with control counts, significant at p </= 0.05) were met in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at a concentration of 5000 µg test item/plate and, in addition, in the experiments without S9 mix at the concentration of 3160 µg/plate in test strain TA 100.

No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the 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, the test item caused a pronounced concentration-related mutagenic effect in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at concentrations of 3160 and/or 5000 µg test item/plate in test strain TA 100. No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. The results point to a base-pair substitution.