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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:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
1999-12-23
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
CYANOX® 1790 Antioxidant
IUPAC nomenclature - Tris(4-tert-butyl-3-hydroxy-2,6-dimethylbenzyl) isocyanurate
Synonym - [tris(4-t-butyl-3-hydroxy-2,6-dimethyl-benzyl)-s-triazine-2,4,6-(1H,3H,5H) trione]
Lot PA0121305
Appearance - White powder, odorless
CAS No. 40601-76-1
Molecular Formula - C42H57N3O6
Molecular Weight - 699.92 g/mole
Purity 93.7 - 98.9%

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Escherichia coli WP2uvrA was obtained from Dr. C. Voogd, National Institute of Public Health and Environmental Protection, Bilthoven, the Netherlands. Frozen stocks were tested for tryptophan requirement and sensitivity to ampicillin, crystal violet and UV radiation.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were obtained from Dr. Bruce Ames (University of California, Berkeley, USA). Frozen stocks of each strain were tested for histidine requirement and sensitivity to ampicillin, crystal violet and UV radiation.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: in house
- method of preparation of S9 mix: Prepared according to Ames et al (1975) and Maron and Ames (1983) - Aroclor 1254
- concentration or volume of S9 mix and S9 in the final culture medium : 10% of S9 in S9 mix

S-9 was prepared from the livers of twelve male Wistar rats five days after they had been induced i.p. with a single dose of 500 mg/kg Aroclor 1254 in soya bean oil (20% w/v). The S-9 was assayed for sterility (0 colonies/10 microliters), protein content (32.1 g/l) and cytochrome P-450 content (29.6 micromoles/l; 0.924 micromoles/g protein).S-9 was frozen until use. On the day of use, aliquots of S-9 were thawed, mixed with a NADPH-generating system, and kept on ice until use.
Test concentrations with justification for top dose:
0, 62, 185, 556, 1667 and 5000 µg/plate (Test 1)
0, 125, 250, 500, 1000 and 2000 µg/plate (Test 2)
Vehicle / solvent:
DMSO was used as a vehicle (See details on test system and conditions).
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
ethylnitrosurea
other: 2-aminoanthracene
Details on test system and experimental conditions:
The material was tested in two separate plate incorporation assays. Test material was dissolved in DMSO at 50 mg/l for the first assay and 20 mg/l for the second. A clear solution was obtained. This solution was used to make serial dilutions by 3-fold intervals in the first assay (62, 185, 556, 1667 and 500 micrograms/plate), and 2-fold intervals in the second assay (125, 250, 500, 1000, and 2000 micrograms/plate). In both assays, the 3 highest concentrations precipitated in the top agar.

Bacteria (0.1 ml of a fully grown culture), test material, negative control (DMSO) or positive control (0.1 ml of the appropriate dilution), and 0.5 ml of sodium phosphate buffer (for tests without metabolic activation) or 0.5 ml of S-9 mix (for tests with metabolic activation) were mixed with 2 ml molten top agar (containing 0.6% agar, 0.5% NaCl and 0.05 mM L-histidine HCl/0.05 mM biotin for Salmonella and supplemented with 0.05 mM tryptophan for E coli), and the mix was poured onto minimal glucose agar plates (1.5% agar in Vogel and Bonner medium E with 2% glucose). All plates were prepared in triplicate. The plates were incubated at 37 degrees C for 3 days. Subsequently, the his+ (Salmonella) and trp+ (E. coli) revertants were counted. The background lawn of bacteria growth was examined microscopically to determine if the material caused toxicity.

A second test was to be conducted if the first test was inconclusive. The first test was considered to be inconclusive if < 5 analyzable concentrations were obtained, if a positive or equivocal response at only one concentration was observed or if positive or equivocal responses at several concentrations were observed that were not concentration-dependent.

A test material was considered to be mutagenic if a concentration-related increase or reproducible positive response was observed. A test material was not mutagenic if it produced neither a dose-related increase in the mean number of revertants nor a reproducible response at any of the time points. Both numerical significance and biological relevance were considered together in the evaluation.
Evaluation criteria:
The study was considered valid if the mean colony counts of controls were within acceptable ranges and the positive controls caused a minimum 3-fold (strains TA100 or WP2uvrA without S-9 and TA98, TA100 and TA1537 with S-9), 5-fold (strain TA1535 with and without S-9 and WP2uvrA with S-9), or 10-fold (strain TA1537 without S-9) increase in the number of revertants. In addition, plates lost through contamination or other unforeseen events had to be < = 5%.

A response was considered positive if the mean number of revertants on the test plates was 2-fold greater than that of negative controls. A response was equivocal is the mean number of revertants was increased by 2-fold in strain TA100 or slightly less than 2-fold for the other strains.
Statistics:
No data.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
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:
other: The test material was toxic at the 4th highest concentrations tested in Salmonella strain TA1537 in the first test, but not the second.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The test material was not positive in any of the strains tested in the absence and presence of S-9 mix. In test 1, the mean numbers of revertants in negative control cultures of S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA were 31, 139, 14, 11 and 34 without S-9 and 35, 136, 16, 9 and 40 with S-9 (respectively). With the test material, the numbers of revertants in negative control cultures of S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA ranged from 23-32, 103-135, 18-25, 5-9 and 26-37 without S-9 and 34-53, 111-150, 10-15, 6-11 and 21-34 with S-9 (respectively). In test 2, the mean numbers of revertants in negative control cultures of S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA were 36, 164, 19, 19 and 32 without S-9 and 64, 156, 18, 24 and 40 with S-9 (respectively). With the test material, the numbers of revertants in negative control cultures of S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA ranged from 38-45, 160-175, 17-29, 15-22 and 29-36 without S-9 and 54-66, 163-172, 14-25, 20-24 and 36-49 with S-9 (respectively).

The test was valid, as the positive controls induced at least the minimum number of revertants as specified by the protocol. The test material was toxic at the 4th highest concentrations tested in Salmonella strain TA1537 in the first test, but not the second.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
The test substance was negative for genotoxicity in all strains tested, with and without activation.
Executive summary:

The test material was examined for mutagenic activity according to OECD TG 471 and under GLP conditions in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, the tryptophan-requiring Escherichia coli strain WP2 uvrA, and a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix).

Two tests were performed with all strains in both the absence and the presence of S9-mix with different concentrations of the test substance formulation, ranging from 62 - 5000 µg/plate and 125 - 2000 µg/plate. Negative controls (DMSO) and positive controls were run simultaneously with the test substance.

The test material was not toxic to the Salmonella typhimurium strains TA 1535, TA 98, TA 100 and E. coli strain, as was evidenced by an absence in decrease in the mean number of revertant colonies with increasing concentration. S. typhimurium strain TA 1537 showed a decrease in the mean number of revertant colonies at the four highest concentrations in the first assay. However, this decrease was not observed in the second assay.

In both assays, in the absence and the presence of S9-mix and in all strains at any of the concentration of the test substance, the material did not cause a two-fold or greater reproducible increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaenous reversion rate observed in the negative control.

The positive controls gave the expected increase in the number of his+ or trp+ revertants in both the absence and the presence of the S9-mix.

It is concluded that the test material was not mutagenic under the conditions employed in this study.