PIGMENTADDITIV RL

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: Guideline study (OECD TG 474) according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Test animals

Species:

mouse

Strain:

other: NMRI, strain NMRKf (SPF71)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Kastengrund , SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 30.1 g (26 g-34 g), females: 23.8 g (20 g-27 g)
- Housing: fully air-conditioned rooms in Macrolon cages (type 3), on softwood granulate in groups of 5 animals
- Diet: rat/mice diet Altromin 1324, ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/-10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 25%
- Amount of vehicle: 10 mL/kg bw (controls); 2 x 10 mL/kg bw (dose groups)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 6250 mg of test compound were weight in a breaker, mixed with sesame oil, washed out in a 25 mL flask and topped up to the calibration mark. A suspension was formed.

Duration of treatment / exposure:

The animals were killed 24, 48 or 72 h after administration (test compound and negative control) and 24 h after administration (positive control).

Frequency of treatment:

The test article was admistered in two equal parts within two hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females per time point

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide-Endoxan (Charge 107480) (50 mg/kg bw) was used as standard mutagenic substance. It was put in solution using water as vehicle. A 2% stock solution was prepared. Animals treated with the positive control via gavage were sacrificed 24 h after exposure.

Examinations

Tissues and cell types examined:

bone marrow of femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Oral administration of 5000 mg test substance per kg bw did not lead to a partial lethality in male and female mice (6 animals). It was considered the maximal applicable dose and was selected as dose level for the main study.

DETAILS OF SLIDE PREPARATION:
The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. After centrifugation and resuspension of the cells the smears were prepared: One drop of the mixed sedimet was smeared on a cleaned slide and dried for 24 h. The slides were stained with May-Grünwald-Giemsa.

METHOD OF ANALYSIS:
Microscopic examination of the slides was performed. For each animal 1000 polychromatic erythrocytes were examined. The number od cells with micronuclei was recorded. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. At the same time the ratio of polychromatophile erythrocytes to normochromatophile erythrocytes was established.

Evaluation criteria:

A possible statistically significant increase as compared to the controls and the possible deviation from the normal range of the negative control groups were used as evaluation criteria to discriminate between positive and negative results. Measurement parameters used: incidence of micronuc lea ted pol ychromatic erythrocytes, number of normochromatic erythrocytes with micro nuclei, the ratio of polychromat ic erythrocytes to normocytes

Statistics:

Wilcoxon (paired, two sides)

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Faeces were blue coloured

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- no significant increase of the number of polychromatic erythrocytes bearing micronuclei in test groups at any time point
- the number of normochromatic erythrocytes with micronuclei did not differ significantly from the values of the simultaneous control animals at any time point
- no marked decrease in the polychromatic erythrocytes / normochromatic erythrocytes ratio, i.e. no toxic effects in test and control group
- positve control was valid

Any other information on results incl. tables

No signs of toxicity have been observed in the pretest and main study.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of this study, the test item did not lead to a substantial increase of micronucleated polychromatic erythrocytes.

Executive summary:

The possible genotoxicity of the test item was examined in a micronucleus test in vivo according to OECD guideline 474 and GLP. The test compound was dosed orally per gavage at 0 and 5000 mg/kg bw to mice, based upon the results of a dose range finding assay (vehicle: sesame oil). The test compound was given in two equal parts within two hours. In accordance with the test procedure the animals (5 males and 5 females per dose group and examination time) were killed 24, 48 or 72 hours after administration. Cyclophosphamide (Endoxan) was used as positive control substance at a dose of 50 mg/kg bw. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound and was statistically not different from control values. The positive control induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females, indicating the sensitivity of the test system. Under the conditions of this study, the test item did not induce micronuclei in mouse bone marrow after oral exposure to 5000 mg/kg bw.