2,2'-[6-(4-methoxyphenyl)-1,3,5-triazine-2,4-diyl]bis{5-[(2-ethylhexyl)oxy]phenol}

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-03-25 - 2003-05-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): FAT 70' 884/B
- Physical state: Solid, light yellow powder
- Analytical purity: 98.6%
- Lot/batch No.: 06484CL2
- Storage condition of test material: At room temperature, protected from light.
- Expiry date: 30 November 2007.

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, l´Arbresle, France
- Age at study initiation: animals were approximately 6 weeks old
- Housing: animals were housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust (SICSA, Alfortville, France)
- Diet: All animals had free access to A04 C pelleted maintenance diet (SAFE, Villemoisson-sur-Orge,France).
- Water: drinking water filtered by a FG Millipore membrane (0.22 micron), ad libitum
- Acclimation period: at least 5 days before the day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 30 to 70 %
- Air changes: at least 12 cycles/hour of filtered non-recycled fresh air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (07:00 - 19:00)

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Corn oil

Details on exposure:

• route: intraperitoneal,
• frequency: two treatments separated by 24 hours,
• volume: 10 mL/kg bw

The quantity of each item administered to each animal was adjusted according to the most recently recorded body weight.

Duration of treatment / exposure:

two treatments separated by 24 hours

Frequency of treatment:

two treatments separated by 24 hours

Post exposure period:

Sacrifice: 24 hours after the last administration

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (8 treated with 2000 mg/kg bw/d, but only 5 assessed vor MN)
3 (Pretest)

Control animals:

yes, concurrent vehicle

Positive control(s):

- yes: cyclophosphamide
- Route of administration: oral, single treatment
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In order to select the top dose-level for the cytogenetic study, 2000 mg/kg/day were administered twice, to three males and three females. The interval between each administration was 24 hours. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no toxic effects were observed, the top dose-level selected for the main test was 2000 mg/kg/day.
The two other selected dose-levels were 1000 and 500 mg/kg/day.


DETAILS OF SLIDE PREPARATION:
At the time of sacrifice, all animals were killed by CO2 inhalation in excess. The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer was unaware of the treatment group of the slide under evaluation ("blind" scoring).


METHOD OF ANALYSIS:
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normo chromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data or other considerations of biological relevance was also taken into account in the evaluation of data obtained.

Statistics:

When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value (Lovell et al., 1989), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the x2 value (Lovell et al., 1989).
When there was significant within-group heterogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969).
The student "t" test was used for the PE/NE ratio comparison.
Probability values of p <=0.05 was considered as significant.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary test: no toxic effects were observed.

Main Test:
No clinical signs and no mortality were observed in the animals ofboth sexes given 500, 1000 or 2000 mg/kg/day.

For both males and females, the mean values of MPE as weil as the PE/NE ratio in the groups treated with the test item, were equivalent to those ofthe vehicle group.

Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Applicant's summary and conclusion

Executive summary:

The objective of this study was to evaluate the potential ofthe test item FAT 70'884/B to induce structural or numerical damage in bone marrow cells of mice.

A preliminary toxicity test was performed to define the dose-levels tobe used for the cytogenetic study.

In the main study, three groups of five male and five female Swiss mice were given intraperitoneal administrations of FAT 70'884/B at dose-levels of 500, 1000 or 2000 mg/kg/day, over a 2-day period. One group of five males and five females received the vehicle (com oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test item, were equivalent to those of the vehicle group. Cyclophosphamide induced a highly significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

In conclusion, the test item FAT 70'884/B did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, with a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg/day under the chosen testing conditions.