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Toxicological information

Genetic toxicity: in vitro

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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:
16 June to 19 July 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP and guideline-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
TMPDE. The trade name of the substance was NEOALLYL T-20, lot no. 30341, received on 7 June 1993. The substance was a mixture of Trimethylolpropane Diallyl Ether (87.4%), Trimethylolpropane Monoallyl Ether (6.9%), and Trimethyolpropane Triallyl Ether (5.7%). The purity was given as 100%. The substance was described as a colourless liquid, and was stored in a metal canister at 4°C in the dark.

Method

Target gene:
Histidine and tryptophan auxotrophic bacterial strains were used
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix obtained from the British Industrial Biological Research Association, prepared from the livers of Aroclor 1254 induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Preliminary study: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate.
Mutation study experiment 1: 0, 8.0, 40, 200, 1000 and 5000 µg/plate.
Mutation study experiment 2: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate.
Vehicle / solvent:
The test substance was accurately weighed and dissolved in dimethyl sulphoxide and appropriate dilutions made. The test material preparations were prepared according to the proportion of the major component of the mixture, the dose levels therefore corresponded to the concentration of trimethylolpropane diallyl ether. Test concentrations were used within 90 minutes of preparation.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Migrated to IUCLID6: without S9; WP2uvrA-, TA100 and TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: without S9; TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: without S9; TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, with S9; Ta1535 ad WP2uvrA-
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: with S9; TA100, TA1537 and TA98
Details on test system and experimental conditions:
Overnight sub cultures of the appropriate stock cultures were prepared in nutrient broth (Oxoid Ltd.) and incuated at 37°C for 10 hours.
Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodiumn chloride. For the Salmonella strains, 5 ml of 1.0 mM histidine/1.0 mM biotin solution was added to each 100 ml of top agar, and for E. coli 5 ml of 1.0 mM tryptophan was added to each 100 ml of top agar. Base agar plates were prepared using 1.2% Oxoid Agar Technical No. 3 with Vogel-Bonner Medium E and 20 mg/ml D-glucose.

Preliminary study: a preliminary test was carried out to determine the toxicity of the test substance. 0.1 ml bacterial suspension (TA100 or WP2uvrA-), 0.1 ml test solution and 0.5 ml phosphate buffer were added to 2 ml molten, trace histidine/tryptophan supplemented media and overlayed onto sterile plates of Vogel-Bonner minimal agar ( 30 ml/plate). Five doses of the test material and a solvent control (DMSO) were tested in duplicate. After 48 hours incubation at 37°C the plates were scored for revertant colonies and examined for a thinning of the background lawn.

Mutation study Experiment 1: Five concentrations of the test substance were assayed in triplicate against each tester strain using the direct plate incorporation method. 0.1 ml aliquots of bacterial suspension were dispensed into sets of sterile test tubes containing 2.0 ml of molten trace histidine/tryptohphan supplemented top agar at 45°C. These sets comprised two test tubes for each bacterial tester strain. 0.1 ml test substance or negative control was also added followed by either 0.5 ml S9 mix or 0.5 ml of pH 7.4 buffer. The contents of each tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates. Positive controls were plated in an identical manner. The plates were then incubated at 37°C for 48 hours and the number of revertant colonies counted was counted using an automated colony counter.
Mutation study Experiment 2: The second experiment was performed according to the methods used in the first experiment, using fresh bacterial cultures, test material and control solutions (in triplicate).
Evaluation criteria:
Toxicity was evaluated by a thinning of the background lawn.
Mutagenicity was evaluated by the number of revertant colonies. A substance was considered positive if it induced a dose-related doubling of the spontaneous reversion rate in one or more strains in both experiments.
Statistics:
Statsitical evaluation was not required.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary study: the mean numbers of revertant colonies for the toxicity assay were not affected by the test substance tested up to a concentration of 5000 µg/plate, therefore the test substance was considered to be non-toxic to strains TA100 and WP2uvrA-.

Main study: the results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory. No toxicity was exhibited to any of the strains of bacteria used.
No significant increases in the number of revertant colonies of bacteria were recorded for any of the strains of bacteria used, at any dose level, either with or without metabolic activation. The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No significant increases in the number of revertant colonies of bacteria were recorded for any of the strains of bacteria used, at any dose level, either with or without metabolic activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test substance was found to be non-mutagenic under the conditions of this test.
Executive summary:

The mutagenic potential of TMPDE (NEOALLYL T-20) was determined in the Ames test, according to OECD method 471. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA- were treated with the test substance by the plate incorporation method at five dose levels (up to a maximum concentration of 5000 µg/ml). Test concentrations were plated in triplicate for each strain, both with and without exogenous metabolic activation (S9 mix). The test substance was found to be non-toxic to TA100 and WP2uvrA, when tested up to a concentration of 5000 µg/ml in a preliminary experiment with and without metabolic activation. The mutation assay was repeated in an independent experiment using fresh bacterial cultures and test substance preparations. The solvent (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increased in the numbers of revertant colonies, both with and without metabolic activation. No significant increase in the numbers of revertant colonies was recorded for any strain with any dose of test substance, either with or without metabolic activation. The test substance was found to be non-mutagenic under the conditions of this test.