Reaction mass of Chromate(1-), [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-, hydrogen, compd. with N-cyclohexylcyclohexanamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1) and hydrogen bis[N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthyl]acetamidato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Oct. 1994 to 09 Dec. 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26 May 1983

Deviations:

yes

Remarks:

The negative control was administered at a volume of 10 ml/kg body weight by gavage

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Dec. 1992

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

19 June 1987

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

05 Dec. 1986

GLP compliance:

yes

Type of assay:

other: micronucleus assay, mouse

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

12237-23-9

Specific details on test material used for the study:

- Test material: TK 10358 (ORASOL BLACK CN)
- Batch No.: 72079449
- Purity: Current quality
- Physical appearance: Black-bluish powder
- Expiry date: January 31,2000
- Stability (of test material itself): Stable (see Expiry date)
- Material submitted by (sponsor): CIBA-GEIGY Limited, Pigments Division, Basle, Switzerland.

Test animals

Species:

mouse

Strain:

other: MAGf (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: reared at the Animal Farm of CIBA-GEIGY, Sisseln, Switzerland
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: females 24-31 g; males 30-36 g
- Assigned to test groups randomly: yes
- Housing: 2 per cage
- Diet: standard diet NAFAG No. 890 ad libitum up to 12 hours before dosing.
- Water: ad libitum
- Acclimation period: at least four days prior to being used in the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 44-76
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Carboxymethylcellulose (CMC), 0.5% in water

Details on exposure:

- The test substance was dosed by gavage at volumes of 20 ml/kg b.w. (5000 mg/kg) and 10 ml/kg b.w. (all other doses).

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Single treatment

Post exposure period:

24h for 200 and 400 mg/kg bw/day; 24 and 48h for 800 mg/kg bw/day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

at the sampling time of 24h

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

at the sampling time of 24h

Doses / concentrations 3

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

at the sampling time of 24h and 48h

No. of animals per sex per dose:

5 females and 5 males

Control animals:

yes, concurrent vehicle

other: with the positive control cyclophosphamide (64 mg/kg).

Positive control(s):

- Cyclophosphamide, 64 mg/kg (ENDOXAN)
- Dissolved in bidistilled water
- Administred orally

Examinations

Tissues and cell types examined:

Bone marrow cells were used. At least 1000 PCEs scored per animal.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: TOLERABILITY (RANGE FINDING) TEST
Prior to the mutagenicity test the maximum tolerated dose (MTD) of the test substance to be applied was determined in a tolerability test. The MTD is defined as the highest dose which does not induce severe toxicity or lethality. The experiments were performed stepwise according to the fixed dose procedure described by Mackay and Elliot (Lit. 12). The highest dose tested was 5000 mg/kg according to acute oral LD50 data in the rat (3250 mg/kg), provided by the sponsor. In each step one male and one female animal were treated. The observation period was 3 days. The dose which was finally selected as the MTD was confirmed by treating an additional pair of mice. Body weight and signs of toxicity were recorded hourly for the first few hours after application and once the following days.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- The negative control was administered at a volume of 10 ml/kg body weight by gavage (in deviation to protocol).
- The positive control was dosed at a volume of 10 ml/kg b.w. by gavage.

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-Grunwald/Giemsa solution. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA
- The result of the experiment should not be influenced by a significant technical error or a recognised artifact.
- The high dose of the test substance applied should be the maximum tolerated dose causing no death in a group of four animals in the range finding-test. In the absence of lethality in the tolerability test, the high dose should be up to a maximum of 5000 mg/kg body weight or the highest applicable dose due to the limited solubility of the test substance.
- The quality of the slides must allow a clear differentiation between PCEs and NCEs.
- The mean number of micronucleated PCEs in the negative control groups should not exceed the value of 0.20%.
- The positive control should fulfill the criteria for an active substance.

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.

Statistics:

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

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The high dose applied was toxic as manifested in the tolerability test by various symptoms.

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 5000 mg/kg test substance was administered to one male and one female animal. Both animals showed several symptoms of toxicity on the day after treatment and were found dead on the following day. In the second step one male and one female were dosed with 2000 mg/kg. Again, symptoms were observed with both animals on the day after treatment. The animals were found dead three days after treatment. In the third step a pair of animals received 800 mg/kg. Both animals survived the treatment. The female showed no signs of toxicity while the male showed transient loss of body weight. In the fourth step of the tolerability test (confirmatory experiment) one male and one female were dosed with 800 mg/kg. Both animals survived the treatment and showed several signs of toxicity. Based on these results the dose of 800 mg/kg was chosen as the highest dose to be administered in the micronucleus test.

Any other information on results incl. tables

Blue skin and blue feces were observed with all animals dosed with the 800 mg/kg of the test material on the day of sacrifice. No signs of toxicity were seen. (In the mutagenicity test, registration of symptoms was restricted to the time of test substance application and to the time immediately before bone marrow preparation).

At all three doses and at both sampling times (24 and 48 hours) there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the test material as compared with the respective negative control animals.

At the 24 hours sampling time in the groups treated with the test substance the mean percentage of micronucleated PCEs was 0.04 (200 mg/kg), 0.06 (400 mg/kg) and 0.06 (800 mg/kg) respectively. The respective negative control value for this group was 0.04%.

At the 48 hours sampling time in the group treated with 800 mg/kg of the test substance the mean percentage of micronucleated PCEs was 0.07 compared to a negative control value of 0.07.

In the positive control (24 hours) the percentage of micronucleated cells within polychromatic erythrocytes was clearly increased. The mean percentage of micronucleated PCEs was 1.25. In comparison with the negative control (0.04) this value is highly significant (p<0.05).