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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods

Data source

Reference
Reference Type:
publication
Title:
Mutagenicity test data of existing chemical substances
Author:
Chemical Substance Investigation Division, Industrial Safety and Health Department, Labour Standatds Bureau, Ministry of Labour, Japan
Year:
1996
Bibliographic source:
Japan Chemical Industry Ecology-Toxicology & Information Center, Japan (JETOC), January 1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
comparable to OECD 471
Principles of method if other than guideline:
The mutagenicity assay was carried out according to the guidelines of Ministry of Labour, Japan (1979, 1985 and 1988) and performed by the following methods of 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:
Adipic acid
EC Number:
204-673-3
EC Name:
Adipic acid
Cas Number:
124-04-9
Molecular formula:
C6H10O4
IUPAC Name:
adipic acid
Details on test material:
Test substance: 99% pure
Specific details on test material used for the study:
Miti No.: (2)-858
purity: guaranteed reagent
source: Tokyo Kasei Kogyo Co. LTD, Tokyo, Japan
lot No.: AL01

Method

Target gene:
histidine gene for Salmonella strains and tryptophane gene for E. coli strain
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : male Sprague Dawley rats (weight about 200 g, seven weeks old) pretreated with sodium phenobarbital (3 i.p. injections at 30 or 60 mg/kg) and 5.6-benofravone (one i.p. injection at 80 mg/kg) before sacrifice. The S9 mix contained 4mM NADPH, 5mM G-6-P, 8 mM MgCl2, 33 mM KCl, 100 mM sodium phosphate buffer and 10% S9. The S9 mix was freshly prepared before the test.
Test concentrations with justification for top dose:
100, 200, 500, 1000, 2000, 5000, and 10000 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide and bleomycin for TA100, and TA102 without S9 mix; 2-aminoanthracene (0.5 to 10 µg/plate for all strains with S9)
Details on test system and experimental conditions:
The preincubation procedure in the Salmonella/mammalian microsome test was performed as described by Matsushima et al., 1980. The test compound dissolved in 0.05 or 1 ml of solvent was supplemented with 0.5 ml of S9 mix or 0.1 M phosphate buffer and 0.1 ml of the tester strains. developed in 1975 by Ames et al.
The mixtures of S9 and test chemcials were incubated at 37°C for 20 minutes, and then added to 2 mL of molten top agar (45°C). The contents of each tube were mixed and poured onto a minimal glucose agar plate immediately. The plates were incubated at 37°C for 48 hours, and then the number of revertant colonies on each plate was scored with an automated colony counter.
Evaluation criteria:
Two-hold rule criteria was used for data evaluation (Ames et al., 1975). The chemicals are ocnsidered to be mutagenic when a dose-related increase in revertant colony count 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 the test result is observed.
Mutagenic potency was calculated by the following equation and maximum value of mutagenic 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 contro) / mg of test chemical on the dose X.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Adipic acid gave no evidence of mutagenicity in any of the
bacterial strains used. Positive controls gave the expected results.

Applicant's summary and conclusion

Executive summary:

Adipic acid was not mutagenic in an Ames test performed by the Ministry of Labour (Japan, 1996) similar to OECD TG 471 in bacteria such as Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli WP2 up to concentrations of 10 mg/plate with or without metabolic activation. Solvent and positive controls were functional in all experiments.