Yellow PE 3260

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:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

1

Reference substance name:

bis[2-hydroxy-N,N-bis(2-hydroxyethyl)ethanaminium] 7-{[4-(4-{[2-(cyanoamino)-4-hydroxy-6-oxo-1,6-dihydropyrimidin-5-yl]diazenyl}benzamido)-3-methoxyphenyl]diazenyl}naphthalene-1,3-disulfonate

Cas Number:

115811-45-5

Molecular formula:

C29H19N9O10S2.2C6H16NO3

IUPAC Name:

bis[2-hydroxy-N,N-bis(2-hydroxyethyl)ethanaminium] 7-{[4-(4-{[2-(cyanoamino)-4-hydroxy-6-oxo-1,6-dihydropyrimidin-5-yl]diazenyl}benzamido)-3-methoxyphenyl]diazenyl}naphthalene-1,3-disulfonate

Test material form:

solid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 Füllingsdorf
- Age at study initiation: 9-13 weeks
- Weight at study initiation: males mean value 31.3 ± 3.4 g, females mean value 24.3 ± 1.7 g
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon Type I cages with wire mesh top and granulated soft wood bedding
- Fasting period before study: approximately 18 hours
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 7 °C
- Humidity (%): 15-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle used: Deionised water
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test article was dissolved in deionised water.

Duration of treatment / exposure:

Single administration.

Frequency of treatment:

Single oral administration of test item and controls, respectively.

Post exposure period:

Sampling of the bone marrow was done 24 (all dose groups) and 48 hours (only high dose group) after treatment, respectively.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

670 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males and 6 females per dose/control group

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control substance: cyclophosphamide
- Route of administration: orally
- Doses / concentrations: 40 mg/kg bw at 10 mL/kg bw in deionised water

Examinations

Tissues and cell types examined:

femural bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w., the substance dissolved in deionised water. As no severe toxic reactions were observed within 48 after exposure, 2000 mg/kg bw was estimated to be suitable as high dose concentration.

DETAILS OF SLIDE PREPARATION:
The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald /Giemsa. Cover slips were mounted with Eukitt. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

Significant increase can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

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:

RESULTS OF RANGE-FINDING STUDY
- Dose range: limit test with 2000 mg/kg bw
- Solubility: well soluble at the tested concentration
- Clinical signs of toxicity in test animals: reduction of spontaneous activity in 4 of 4 animals (fully reversible within 48h), eyelid closure in 2 of 4 animals (fully reversible within 24h), apathy in 2 of 4 animals (fully reversible within 24h),

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 as compared to the mean value of NCEs of the vehicle control. However, the enhancement of the mean value was caused mainly by the results of two males (62 = 2160 NCEs per 1000 PCEs; 63 = 1377 NCEs per 1000 PCEs). Therefore, it cannot be concluded that the test article induced cytotoxic effects. Mean data are given in Table 1.
- Statistical evaluation: In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with the substance were in the range of the vehicle control group. Cyclophosphamide administered was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:

The substance is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed according to the First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted May 26, 1983, „Micronucleus Test" and the method laid down in EEC Directive 92/69, L 383, Annexe V, B 12, dated December 29, 1992 to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was dissolved in deionised water. Deionised water was used as vehicle control. The volume administered orally was 20 ml/kg bw 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670 and 2000 mg/kg bw

48 h preparation interval: 2000 mg/kg bw

The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions.

After treatment with the test article at preparation interval 48 h the mean number of normochromatic erythrocytes was increased as compared to the mean value of NCEs of the vehicle control. However, the enhancement of the mean value was caused mainly by the results of two males (62 = 2160 NCEs per 1000 PCEs; 63 = 1377 NCEs per 1000 PCEs). Therefore, it cannot be concluded that the test article induced cytotoxic effects.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, the substance is considered to be non-mutagenic in this micronucleus assay.