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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1998-09-07 to 1998-11-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Isolan Rot S-RL

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann Gartenstr. 27; D-33178 Borchen
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: M=38.3 g; F=30.5 g
- Assigned to test groups randomly: yes, randomization schemes 98.0818 and 98.0819
- Fasting period before study: -
- Housing: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate
- Diet (e.g. ad libitum): rat/mice diet ssniff RIM-H (V 1534), ad libitum ssniff GmbH, Postbox 2039, 59480 Soest
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 3 degrees C
- Humidity (%): 50± 20%
- Light/dark cycles: 12 hours daily

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the days of administration the test substance was suspended in deionized water at the appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.
The test substance preparation was administered twice at an interval of 24 hours orally by gavage
Duration of treatment / exposure:
The test substance was administered twice at an interval of 24 hours orally by gavage to the test animals at a dose of 2000 mg per kg body weight; animals were killed 24 hours after dosing
Frequency of treatment:
twice at an interval of 24 hours
Post exposure period:
24 h
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide: 50 mg per kg body weight. orally per gavage

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a preliminary dose range finding study, oral administration of 2000 mg Acid Red 414 per kg body weight did not cause any toxic effects in male and female mice over an observation period of 7 days. This dose level was therefore the regularly limit dose, selected as the highest dose for the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test substance was administered twice at an interval of 24 hours orally by gavage to the test animals at a dose of 2000 mg per kg body weight. The vehicle, deionized water, was administered in the same way to the negative control groups. The study included a concurrent positive control using Cyclophosphamide, which was administered once orally by gavage at a dose of 50 mg per kg body weight.
All animals were killed by carbon dioxide asphyxiation 24 hours after dosing and bone marrow samples were taken from both femora.

DETAILS OF SLIDE PREPARATION:
For each animal, about 3 ml fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Staining was performed as follows:
- 5 minutes in methanol
- 5 minutes in May-Grünwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan

METHOD OF ANALYSIS:
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
If the validity of the study had been shown the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone-dose-relation-ship one-sided Wilcoxon tests were performed starting with the highest dose group. These test were performed with a multiple level of significance of 5%.

OTHER:
Evaluation criteria:
Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
If the validity of the study had been shown the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone-dose-relation-ship one-sided Wilcoxon tests were performed starting with the highest dose group. These test were performed with a multiple level of significance of 5%.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
but reddish urine was observed
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All animals survived after treatment. Red discolored feces and reddish urine but no signs of toxicity were observed during the duration of the whole study.
The dissection of the animals revealed red coloured contents of the gastro-intestinal tract as macroscopic finding.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The incidence of micronucleated polychromatic erythrocytes in the test substance treated group was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20% of the control values.
Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:
Acid Red 414 did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not genotoxic in the micronucleus test
Executive summary:

The Mammalian Erythrocyte Micronucleus Test according to OECD Test Guideline No 474 was carried out with Acid Red 414 in male and female NMRI mice. The test compound was suspended in deionized water and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay. According to the test procedure the animals were killed 24 hours after test substance administration.

Cyclophosphamide was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Acid Red 414 and was not less than 20% of the control value.

Cyclophosphamide induced a marked statistically significant increase in the number of poly-chromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that Acid Red 414 is not mutagenic in the micronucleus test.