Direct Yellow 169

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP- and Guideline-conform study, but number of analysed erythrocytes are 1000 only (2000 are required)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

number of cells counted: 1000

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

At the time of study conduct, no test guideline was issued. Number of cells counted (=1000) was based on current knowledge.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): FAT 40'403/A
- Physical state: solid
- Analytical purity: main product 85.6 %
- Impurities (identity and concentrations): unknown coloured by-products 6.8 %; water 7.5%
- Composition of test material, percentage of components: main product 85.6 %; unknown coloured by-products 6.8 %; water 7.5%
- Purity test date: 1991-10-18
- Lot/batch No.: EN. SCR 905 / TV
- Expiration date of the lot/batch: May, 1995
- Stability under test conditions: Pure: years; In vehicle: aqua dest > 48 hours, DMSO > 48 hours, DMF > 48 hours
- Storage condition of test material: room temperature, light protected

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf; CH-4414 Füllinsdorf/Basel
- Age at study initiation: minimiim 10 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours before treatment
- Housing:single
- Diet (e.g. ad libitum): pelleted standard diet (ALTROMIN, D-49 37 Lage/Lippe)
- Water (e.g. ad libitum):tap water, ad libitum (Gemeindewerke, D-6101 Rossdorf)
- Acclimation period:5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The vehicle was chosen to its nontoxicity for the animals.
- Amount of vehicle (if gavage or dermal): All animals received a single standard dose volume of 20 ml/kg body weight orally.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
no data

Duration of treatment / exposure:

once

Frequency of treatment:

single dose

Post exposure period:

24-72h

Doses / concentrations

Remarks:

Doses / Concentrations:
5000 mg/kg b.w.
Basis:
actual ingested

No. of animals per sex per dose:

Six

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: orally, singly
- Doses / concentrations:40 mg/kg b.w. (10 ml/kg b.w.)

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The volume to be administered should be compatible with physiological space available. The maximum tolerated dose level was determined to be the dose
that caused toxic reactions without having major effects on survival within 7 2 hours.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 5 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-Griinwald (MERCK, D-6100 Darmstadt,) / Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.


METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes, with lOOx oil immersion objectives. 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 scune sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points. A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at anyone of the test points is considered non-mutagenic in this system. This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT FOR TOXICITY
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w., FAT 40'403/A dissolved in aqua dest. The volume administered was 20 ml/kg b.w.. None of the treated animals expressed toxic reactions. Higher dosing was not attainable: a) Appropriate suspensions (homogeneity, viscosity) could be obtained only up to 250 mg/ml. b) Application volumes higher than 20 ml/kg b.w. were not justifiable for the rodents used.

MAIN STUDY
The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that FAT 40'403/A had no cytotoxic properties. In comparison to the corresponding negative controls there was no significant, enhancement in the frequency of the detected micronuclei at any preparation interval after application of the tdst
article. The mean values of micronuclei observed after treatment with FAT 40'403/A were in the same range as compared to the negative control groups. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus frequency.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
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, FAT 40'403/A is considered to be non-mutagenic in this
micronucleus assay.

Executive summary:

This study was performed to investigate the potential of FAT 40'403/A to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was dissolved in aqua dest.. This solvent was used as negative control. The volume administered orally was 20 ml/kg b.w. . 24 h, 48 h and 72 h after a single application 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 level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w.. In a pre-experiment this dose level was estimated to be the maximum attainable dose.

After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative

controls thus indicating no cytotoxic effects. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency

.