Registration Dossier

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
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

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]

Appearance - White powder, odorless
CAS No. 40601-76-1
Molecular Formula - C42H57N3O6
Molecular Weight - 699.92 g/mole
Purity > 95%

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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)

Examinations

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.

Results and discussion

Test results
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).

Applicant's summary and conclusion

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.