2,7-NAPHTHALENEDISULFONIC ACID 5-[[4-CHLORO-6-(ETHYLPHENYLAMINO)-1,3,5-TRIAZIN-2-YL]AMINO]-3-[[5-[(2,3-DIBROMO-1-OXOPROPYL)AMINO]-2-SULFOPHENYL]AZO]-4-HYDROXY-,SODIUM SALT

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40508/A
- Batch No.: Roe TV IP
- Purity: approx. 60%
- Stability: Stable
- Expiration date: December 31, 1997

Test animals

Species:

mouse

Strain:

Tif:MAGf

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Farm of Ciba-Geigy, Sisseln, Switzerland
- Fasting period before study: 12 hours
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: Tolerability test: Males: 33-34g, Females: 23-27g, Micronucleus test: Males: 24-32g, Females 20-26g
- Housing: Individually or in groups of two.
- Diet (e.g. ad libitum): Pelleted, certified standard diet (NAFAG No. 890 Tox) was administered ad libitum.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 - 23.0
- Humidity (%): 48.0 - 72.0
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Carboxymethylcellulose (CMC), 0.5% in water
- Application volume: 20 mL/kg bw

Frequency of treatment:

Single treatment

Post exposure period:

- High dose and negative control group: 16, 24 and 48 hours
- Intermediate, low and positive control group: 24 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
625, 1250, 2500 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide, 64 mg/kg bw (ENDOXAN), dissolved in bidistilled water
- Intraperitoneal, Application volumes: 10 mL/kg bw

Examinations

Tissues and cell types examined:

Normochromatic and polychromatic erythrocytes.

Details of tissue and slide preparation:

- Preparation of bone marrow and preparation of slides: The animals were sacrificed by CO2 gas. Bone marrow was harvested in fetal calf serum from the shafts of both femurs, centrifuged and resuspended in fetal calf serum. Thereof smears were made. They were air-dried and then stained with May-Grünwald/Giemsa solution. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.
- Criteria for scoring micronuclei: Micronuclei are uniform, darkly stained, more or less round bodies in the cytoplasm of erythrocytes. Inclusions which are reflective, improperly shaped or stained, or which are not in the focal plain of the cell are judged to be artifacts and are not scored as micronuclei. Cells containing more than one micronucleus are only counted once. Prior to analysis the slides were coded. The slides of five animals/sex/dose, showing good differentiation between mature and polychromatic erythrocytes, were scored by a laboratory technician. The incidence of micronucleated polychromatic erythrocytes (MNPCE) among at least 1000 polychromatic erythrocytes (PCE), and the ratio of PCE to normochromatic erythrocytes (NCE) among a total of 1000 erythrocytes was determined for each slide.

Evaluation criteria:

- Assay evaluation criteria: The results of the experiments were evaluated with respect to the mean number of PCEs with micronuclei. The groups compared differed by treatment, sampling time and sex of the animals. Since there was no significant difference between animals of either sex, the data from females and males were pooled for evaluation.
- Criteria for a negative effect: The test substance is considered to be inactive in this test system if there is no statistically significant difference (Chi Squared < 3.84) between the mean number of micronucleated PCEs in the groups treated with the test substance and that of the respective negative control and the former does not exceed the range accepted for the negative control (< 0.20 %).
- Criteria for a positive effect: The test substance is considered to be active in this test system if at any group treated with the test substance the mean number of micronucleated PCEs exceeds the value of 0.20% and if there is a statistically significant difference (Chi Squared = 3.84) of the number of micronucleated PCEs in comparison with the negative control.
- Exceptions: If the positive effect occurs in a minority of the treated animals only and if their number of micronucleated normochromatic erythrocytes (NCEs) is also enhanced in comparison with the respective negative control, the effect on PCEs is not attributed to the treatment. If the limits of the criteria for a positive or for a negative response are reached, the Study Director will additionally interpret the results based on previous experience with this test system.

Statistics:

The significance of differences was assessed by the Chi-Squared-Contingency-Test (F=l, p<0.05).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

only at the 5000 mg/kg bw dose group in the tolerability test.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Tolerability test: In the first step of the tolerability test the dose of 2000 mg/kg bw FAT 40508/A was administered to each one male and one female animal. Both animals survived the treatment and showed slight loss of body weight on day one after treatment. In the second step of the tolerability test each one male and one female animal was dosed with 5000 mg/kg (in deviation to the dose limit given in the protocol). Both animals survived the treatment and showed slight loss of body weight on day one after treatment. In the third step of the tolerability test (confirmatory experiment) each one male and one female received 5000 mg/kg bw. Both animals survived the treatment and showed diarrhea a few hours after treatment and on the following day. Coloured urine and feaces were observed with animals dosed with 5000 mg/kg bw. Based on these results, the dose of 5000 mg/kg bw was chosen as the highest dose to be administered in the micronucleus test.
- Micronucleus test: Initially, the assay was started with 5000 mg/kg bw as the high dose. Since 50% of the animals receiving this dose died within the treatment period, the assay was continued with 2500 mg/kg bw as the high dose and residual animals from the 5000 mg/kg bw dose groups were excluded from the study. The test was performed with the dose of 2500 mg/kg bw at three sampling times (16, 24 and 48 hours) and with the doses of 625 and 1250 mg/kg bw at the sampling time of 24 hours. In all three dosage groups none of the animals died or showed signs of toxicity. At all sampling times (16, 24 and 48 hours) there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the respective doses of FAT 40508/A as compared with the negative control animals. At the 16 hours sampling time in the group treated with 2500 mg/kg bw of the test substance the mean percentage of micronucleated PCEs was 0.05 compared to a negative control value of 0.06. At the 24 hours sampling time in the groups treated with the test substance the mean percentage of micronucleated PCEs was 0.05 (625 mg/kg bw), 0.05 (1250 mg/kg bw) and 0.09 (2500 mg/kg bw) respectively. The respective negative control value for this group was 0.04%. At the 48 hours sampling time in the group treated with 2500 mg/kg bw of the test substance the mean percentage of micronucleated PCEs was 0.10 compared to a negative control value of 0.05. In the positive control (24 hours) the percentage of micronucleated cells within polychromatic erythrocytes was clearly increased. The mean percentage of micronucleated PCEs was 1.02. In comparison with the negative control (0.04) this value is highly significant (p<0.05).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test substance.

Executive summary:

In a GLP-compliant micronucleus test, performed according to OECD guideline 474, Tif: MAGf mice (5/sex/treatment group) were treated by oral gavage with the test substance (625, 1250, 2500 mg/kg bw). From the high dose group and from the negative control group, animals were sacrificed 16, 24 and 48 hours thereafter. From the intermediate and the low dose group and from the positive control group animals were sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei. The high dose applied did not lead to visible symptoms of toxicity. In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective negative control group. It was therefore concluded that under the given experimental conditions no evidence for clastogenic or aneugenic effects were obtained in mice treated with the test substance.