Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 1. According to the Guidelines for the Ministry of Labour, Japan 2. Some study data are in appendices, which were not available for review 3. No GLP certificate

Data source

Mutagenicity Test Data of Existing Chemical Substances
Japan Chemical Industry Ecology - Toxicology & Information Cetnter
Bibliographic source:
Edited and Published by: Japan Chemical Industry Ecology-Toxicology & Information Center, Japan (JETOC)

Materials and methods

Test guidelineopen allclose all
according to guideline
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Ministry of Labour, Japan (1979, 1985, 1988)
equivalent or similar to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Performed by the following methods: Ames et al. (1975), Maron and Ames (1983), and Matsushima et al. (1980)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- The study examined several compounds. The first choice of solvent was water, followed by dimethyl sulfoxide (DMSO) second, and acetone third (depending on solubility). The solvent was not specifically stated for methylamine.


Target gene:
Species / strain
Species / strain / cell type:
S. typhimurium, other: S.typhimurium strains TA98, TA100, TA102, TA104, TA1535, TA1537, TA1538 and E.coli WP2uvrA and WP2uvrA/pKM101
Additional strain / cell type characteristics:
other: Salmonella were histidine deficient; E. coli were L-tryptophan deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 from rat liver induced with sodium phenobarbital and 5,6-benzofravone
Test concentrations with justification for top dose:
0, 0.05, 0.1, 0.5, 1, 5, 10, 50%
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
sodium azide
Further substances: BLM: bleomycin, PA: pyruvic aldehyde
Details on test system and experimental conditions:
- Male Sprague-Dawley rats (7 weeks old, 200g) were used for the preparation of liver S9 fractions
- Sodium phenobarbital and 5,6-benzofravone were used to induce the rat metabolic activation system by i.p. injection
- S9 was prepared from rat liver samples, it was frozen and stored at -80 deg C

- The test substance was dissolved in 0.05 or 0.1 mL of the solvent and supplemented with 0.5 mL of S9 mix (with metabolic activation) or 0.1M phosphate buffer pH 7.4 (without metabolic activation) and 0.1 mL of tester strains which had been cultured in nutrient broth
- The mixture was incubated for 20 min. at 37 deg C, then rapidly mixed with agar containing 0.05 umol/mL of L-histidine and biotin for the Salmonella test
- In the E.coli test, 0.05 umol/mL of L-tryptophan was used instead of L-histidine and biotin
- All plates were incubated for 48 hours at 37 deg C and the number of revertant colonies were scored
Evaluation criteria:
- Two-hold rule criteria was used for data evaluation.
- The chemicals are considered to be mutagenic when a dose-related increase in revertant colonies is observed and the number of revertant colonies per plate with the test substance is more than twice that of the negative control (solvent control) and when a reproducibility of test result is observed
- Mutagenic potency was calculated by the following equation and maximum potency was expressed as a specific activity on the data sheet: mutagenic potency (induced revertants / mg test substance) = (number of induced revertants on the dose X - number of revertant on the solvent control) / mg of test chemical on the dose X

Results and discussion

Test results
Metabolic activation:
with and without
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Table 2

Tester Strain

Solvent Control

Positive Control

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix


138 +/- 27

139 +/- 29

728 +/- 196

1011 +/- 210


14 +/- 5

13 +/- 4

300 +/- 81

255 +/- 70


20 +/- 8

26 +/- 7

413 +/- 82

404 +/- 118


16 +/- 3

23 +/- 5

376 +/- 79 (2NF)

556 +/- 216

343 +/- 34 (4NQO)


8 +/- 2

11 +/- 4

497 +/- 254

196 +/- 70


260 +/- 49

317 +/- 52

758 +/- 175

1676 +/- 562


269 +/- 40

332 +/- 48

1973 +/- 755

1196 +/- 252


29 +/- 11

34 +/- 11

273 +/- 126

879 +/- 177


141 +/- 43

198 +/- 49

2080 +/- 884

928 +/- 250

Control values (mean +/- standard deviation) for solvent controls and positive controls

Applicant's summary and conclusion

Interpretation of results:
- negative with metabolic activation
- negative without metabolic activation

Methylamine is negative in the Ames Test.
Executive summary:

Methylamine was assayed for mutation (according to OECD 471, Klimisch 2 - reliable with restrictions) in seven histidine requiring strains (TA98, TA100, TA102, TA104, TA1535, TA1537 and TA1538) of Salmonella typhimurium and in two strains of Escherichia Coli (WP2uvrA and WP2uvrA/pKM101) both in the absence and presence of metabolic activation by an sodium phenobarbital and 5,6-benzofravone induced rat liver post-mitochondrial fraction (S-9).

An initial toxicity range-finder experiment was carried out. No toxicity was observed at 2000 µg/plate, therefore that concentration was chosen as the top dose for the mutation experiments.

In the mutation experiment each strain (0.1 mL) was treated with the test substance (test substance was dissolved in 0.05 or 0.1 mL of the solvent and supplemented with 0.5 mL of S9 mix (with metabolic activation) or 0.1M phosphate buffer pH 7.4 (without metabolic activation).

The mixture was incubated for 20 min. at 37 deg C, then rapidly mixed with agar containing 0.05 µmol/mL of L-histidine and biotin for the Salmonella test. In the E.coli test, 0.05 µmol/mL of L-tryptophan was used instead of L-histidine and biotin. Incubations for 48 h were carried out and the number of colonies were scored. Negative (solvent) and positive control treatments were included for all strains. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. So the study met the acceptance criteria and is considered to be valid.

Treatment of the strains in the absence of SS-9 and in the presence of S-9 did not rise the numbers of revertant colonies significantly. So methylamine did not induce revertants in all bacterial strains tested both with and without metabolic activation.

It is concluded that the results fully satisfy the requirements for a non-mutagenic response, the compound being considered non-mutagenic in this assay.