Methylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

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

Reference

Title:

Mutagenicity Test Data of Existing Chemical Substances

Author:

Japan Chemical Industry Ecology - Toxicology & Information Cetnter

Year:

1997

Bibliographic source:

Edited and Published by: Japan Chemical Industry Ecology-Toxicology & Information Center, Japan (JETOC)

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Ministry of Labour, Japan (1979, 1985, 1988)

Test guideline 2

Qualifier:

equivalent or similar to guideline

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

Test material information

Constituent 1

Reference substance name:

Methylamine

EC Number:

200-820-0

EC Name:

Methylamine

Cas Number:

74-89-5

Molecular formula:

CH5N

IUPAC Name:

methanamine

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.

Method

Target gene:

his-

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: The Salmonella strains were histidine deficient.

Species / strain 2

Species / strain / cell type:

S. typhimurium, other: TA104

Additional strain / cell type characteristics:

other: The Salmonella strain was histidine deficient

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: The Salmonella strain was histidine deficient.

Species / strain 4

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: The E. coli strain was L-tryptophan deficient.

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: The E coli. strain was 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%

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other:

Remarks:

Further substances: BLM: bleomycin, PA: pyruvic aldehyde

Details on test system and experimental conditions:

PREPARATION OF S9 FRACTION
- 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

PREINCUBATION METHOD
- 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:

DATA EVALUATION
- 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

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 3

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 4

Species / strain:

S. typhimurium, other: 104

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 6

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 7

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 8

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 9

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

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
TA100	138 +/- 27	139 +/- 29	728 +/- 196	1011 +/- 210
TA1535	14 +/- 5	13 +/- 4	300 +/- 81	255 +/- 70
TA98	20 +/- 8	26 +/- 7	413 +/- 82	404 +/- 118
TA1538	16 +/- 3	23 +/- 5	376 +/- 79 (2NF)	556 +/- 216
			343 +/- 34 (4NQO)
TA1537	8 +/- 2	11 +/- 4	497 +/- 254	196 +/- 70
TA102	260 +/- 49	317 +/- 52	758 +/- 175	1676 +/- 562
TA104	269 +/- 40	332 +/- 48	1973 +/- 755	1196 +/- 252
WP2uvrA	29 +/- 11	34 +/- 11	273 +/- 126	879 +/- 177
WP2uvrA/pKM101	141 +/- 43	198 +/- 49	2080 +/- 884	928 +/- 250

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

Applicant's summary and conclusion

Conclusions:

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.