Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro mutagenicity of the test material was dermined in bacteria and mammalian cells. An in vitro chromosomal aberration study was conducted in mammalian cells.

Link to relevant study records
Reference
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
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
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%
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).
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.
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.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo mouse micronucleus study was conducted.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982-11-29 - 1983-01-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was not conducted according to a current guideline. Otherwise it was a well conducted and documented study.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Typical procedure for micronucleus assay conducted at the time.
According to Schmid, W. The micronucleus test. Mutation Res 31: 9-15, 1975.

The test material was subjected to a micronucleus assay in mice to detect potential chromosome breaking activity according to the method of Schmid.
In the assay, 6 animals of each sex were included in each treatment group and 8 animals of each sex in the negative control group (5 animals in the treated groups and 8 animals in the control group were used for the evaluation).

Each mouse was medicated per os once daily on two consecutive days with the vehicle used for the test material (negative control), with cyclophosphamide at a concentration of 50 mg/kg bw (positive control) or with the test material at one of three dose levels (200, 1000 or 5000 mg/kg bw). The test material and the controls were administered in a standard volume of 20 ml/kg bw. This treatment schedule was selected to expose a high proportion of the cell population to the test material during two successive S-phases of the cell cycle.

24 Hours after the second treatment (48 hours after the first treatment), the mice were sacrificed and bone marrow was removed from the femora and prepared on slides for examination. One thousand polychromatic erythrocytes (PCE) as well as one thousand normochromatic erythrocytes (NCE) were scored. The ratio of PCE to NCE based on 1500 cells (PCE + NCE) counted per animal was used as a measure of the toxic efficacy of the test material.
GLP compliance:
yes
Type of assay:
micronucleus assay
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]

Appearance - White powder, odorless
CAS No. 40601-76-1
Molecular Formula - C42H57N3O6
Molecular Weight - 699.92 g/mole
Purity > 95%
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, Switzerland
- Age at study initiation: 5 weeks
- Weight at study initiation: 20-39g
- Assigned to test groups randomly: yes (using a random algorithm)
- Fasting period before study:
- Housing: housed in groups of 6 in macrolon type 3 cages with wire mesh tops and standardized granulated soft wood bedding
- Diet (e.g. ad libitum): Peletted standard Kliba 343-A mouse maintenance diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 13 days - They were clinically examined by a veterinarian during this period and did not have any symptoms of disease.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 10 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
Aqua Bidest containing 1% Tween 80
Details on exposure:
Mice were randomized into 3 treatment groups of 6 animals/sex (200, 1000 and 5000 mg/kg), one positive control group of 6 animals/sex (50 mg/kg cyclophosphamide) and one negative control group of 8 animals/sex [(Aqua Bidest with Tween 80(1%)]. For each dose of test material, a suspension was prepared by adding the material to Aqua Bidest containing 1% Tween 80. The suspension was homogenized and stirred with a magnetic stir bar while animals were being dosed. The test and control materials were administered by gavage once daily on two consecutive days in a volume of 20 ml/kg.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
Two treatments, one each on consecutive days
Post exposure period:
24 hours after the last treatment.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Six animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
One positive control group of 6 animals/sex (50 mg/kg cyclophosphamide)
Details of tissue and slide preparation:
Both femurs were removed from each mouse and freed of adherent tissue. Bone marrow cells were flushed out of the femurs using a needle and syringe containing 0.2 ml calf serum. Cells from both femurs were centrifuged at 1000 rpm for 5 min. Cells in the sediment were carefully mixed by aspiration in a siliconized Pasteur Pipette. A small drop of the suspension was smeared onto a slide and slides were air dried overnight. Two slides were prepared per animal. The following day, the slides were stained according to the Panoptic staining method of Pappenheim (as described in Queisser, Das Knochenmark, Georg Thieme Verlag, Stuttgart 1978, pg 12).

Slides from each animal (with the exception of 1 animal/sex from each treatment group) were blindly evaluated for the presence of micronuclei. One thousand polychromatic erythrocytes (PCE) and one thousand normochromatic erythrocytes (NCE) from each slide were screened under a microscope (at 1000x). The ratio of polychromatic to normochromatic erythrocytes based on 150 PCE and NCE per slide was calculated. The ratio of PCE/NCE was calculated and used as an index of toxicity of the test material.
Evaluation criteria:
The test material was considered positive if the statistical T value (one-sided) was significant.
Statistics:
Homogeneity of test results was confirmed using the Poisson Heterogenicity test. The results of the positive control tests were not included in the analysis since they were so much higher than those of the test material. The 95% confidence limits for the L parameter of the Poisson distributions were taken from Geigy tables. Since results did not appear to be dependent on dose of the material, all doses of the material per sex were analyzed globally (compared to the negative control) using regression. The test material was considered positive if the T value (one-sided) was significant.
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
One male mouse treated with 5000 mg/kg died after the second application of test material. There was no significant difference between the numbers of micronucleated polychromatic (PCE) or normochromatic erythrocytes (NCE) in treated animals versus vehicle-treated controls. The average numbers of PCE and NCE in control animals were 1.8 and 0.95, respectively. In the three groups of treated animals, the average numbers of PCE and NCE ranged from 1.2-2.0 and 0.6-1.4, respectively. There was no effect of treatment on the ratio of PCE to NCE (ranged from 0.2-2.2 in control and 0.8-2.1 in treated). The test was valid, as there was a significant increase in the number of micronucleated erythrocytes in the positive control group (23-46 micronucleated PCE/animal and 6-14 micronucleated NCE/animal in positive controls vs. 0-3 micronucleated PCE and 0-2 micronucleated NCE/animal in the negative control).
Conclusions:
Interpretation of results: negative
The test material was negative for mutagenicity in both males and females under the condition of the study.
Executive summary:

The test material was subjected to a micronucleus assay in mice to detect potential chromosome breaking activity according to the method of Schmid.

In the assay, 6 animals of each sex were included in each treatment group and 8 animals of each sex in the negative control group (5 animals in the treated groups and 8 animals in the control group were used for the evaluation).

Each mouse was medicated per gavage once daily on two consecutive days with the vehicle used for the test material (negative control), with cyclophosphamide at a concentration of 50 mg/kg bw (positive control) or with the test material at one of three dose levels (200, 1000 or 5000 mg/kg bw). The test material and the controls were administered in a standard volume of 20 ml/kg bw. This treatment schedule was selected to expose a high proportion of the cell population to the test material during two successive S-phases of the cell cycle.

24 Hours after the second treatment (48 hours after the first treatment), the mice were sacrificed and bone marrow was removed from the femora and prepared on slides for examination. One thousand polychromatic erythrocytes (PCE) as well as one thousand normochromatic erythrocytes (NCE) were scored. The ratio of PCE to NCE based on 1500 cells (PCE + NCE) counted per animal was used as a measure of the toxic efficacy of the test material.

After treatment of the mice with the test material, no substance-related increase of micronucleated erythrocytes was observed at any dose level tested in comparison with the negative control group. No toxic effects of the test material were observed. As expected, the positive controlg roup showed a significant increase in the number of micronucleated erythrocytes in comparison with the negative control group.

In conclusion, it can be stated that during this mouse micronucleus assay with the test material no chromosome-breaking activity or damage to the mitotic apparatus could be detected under the experimental conditions reported and thus no evidence of any potential chromosome mutagenic activity was demonstrable.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

in vitro

In an OECD Guideline 471 study, the test material was negative for mutagenicity inS. typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100 and inE. colistrain WP2 uvr A either with or without metabolic activation. In an OECD Guideline 476 study in mouse lymphoma L5178Y cells, the test material was negative for mutagenicity. In an OECD Guideline 473 study with cultured Chinese hamster ovary cells (CHO K-1 line), the test substance did not produce chromosomal effects either with or without metabolic activation.

in vivo

The test substance was negative for mutagenicity in a mouse micronucleus study conducted with NMRI KFM mice following oral administration at dose levels as high as 5000 mg/kg bw.


Justification for selection of genetic toxicity endpoint
Well conducted in vitro and in vivo genetic toxicity studies

Justification for classification or non-classification

Based on the universally negative results obtained in adequate in vitro and in vivo test for mutagenicity, the test material is not classifed.