REAKTIV-ORANGE DYPR 1410

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:

13 April 1999 - 29 April 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to EU, OCED & US EPA test guidance in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

Name or number of compound (I.N.N, or U.SAN): Reaktiv-Orange DYPR 1410
Molecular weight: 515.53 g/mol
Batch number: DYPR 1410
Certificate of analysis: no. NB 208 dated December 15th, 1998, Aventis Research & Technologies, Dr. B. Ciommer
Content: 73 % (w/w) + 2 %
Appearance: orange powder
Date of production: August 17th, 1998
Date of receipt: August 26th, 1998
Date of expiry: August 2003
Storage: conditions darkness at approximately 5°C in a refrigerator
Solubility: dissolved in cell culture medium
Stability in the solvent: not necessary because the test substance was dissolved directly into the test system (MEM)
Concentration of stock solution: 5 mg/ml

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species of animals: mouse
Strain of animals: HsdWin:NMRI
Origin (supplier) of animals: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
Animal identification: fur marking with KMnO4 and cage numbering

Body weight at start of study

Male animals
mean= 37,0 g
min= 33 g
max= 44 g
n= 15

Female animals:
mean = 25,7 g
min= 23 g
max= 28 g
n= 15

Age at the start of study; male/female animals approximately 8 weeks
Animal maintenance: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate
Room temperature: 22 ± 3°C
Relative humidity: 50 ± 20 %
Lighting times: 12 hours daily
Acclimatization: 5 days under study conditions
Food: rat/mice diet ssniff® R/M-H (V 1534), ad libitum ssniff GmbH, Postbox 2039, 59480 Soest
Water: tap water in plastic bottles, ad libitum

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

Deionized water

Details on exposure:

Test groups
In a preliminary dose range finding study, oral administration of 2000 mg Reakttv Orange DYPR 1410 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.

Group 1; 5 males, 5 females. Dose(mg/kg bwt): 0 Dose Concentration (% w/v): 0, Volume (ml/kg bwt): 10, killing time: 24 hours

Group 2; 5 males, 5 females. Dose(mg/kg bwt): 2000 Dose Concentration (% w/v): 20, Volume (ml/kg bwt): 10, killing time: 24 hours

Group 3; 5 males, 5 females. Dose(mg/kg bwt): 50 , Dose Concentration (% w/v): 0.5, Volume (ml/kg bwt): 10, killing time: 24 hours

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 Endoxan which was administered once orally by gavage at a dose of 50 mg per kg body weight.

Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Twice

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2000 mg/kg
Basis:

No. of animals per sex per dose:

Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

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

Examinations

Tissues and cell types examined:

polychromatic erythrocytes

Details of tissue and slide preparation:

Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. 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 farmed. 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-Grunwald'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®

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 micronucleafed 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.

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 evalua¬tion 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-relationship one-sided Wilcoxon tests were performed starting with the highest dose group. These test were performed with a multiple level of significance of 5%

Statistics:

A one-sided Wilcoxon-Test was evaluated to check the validity of the study

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mice were treated twice with 2000 mg Reaktiv Orange DYPR 1410 per kg body weight to study the induction of micronuclei in bone marrow cells.
All animals survived after treatment. No signs of toxicity were observed, but feces and urine were red discolored up to 24 hours after second application.

The dissection of the animals revealed no test substance related macroscopic findings.

The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.

The incidence of micronucleated polychromatic erythrocytes in the dose group of Reaktiv Orange DYPR 1410 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.

Any other information on results incl. tables

The present study was conducted in compliance withOECD Guideline For Testing Of Chemicals, 474 Genetic Toxicology, Mammalian Erythrocyte Micronucleus Test, Adopted 21^st, July 1997 and U.S. EPA: OPPTS 870.5395 Health Effects Test Guidelines; Mammalian Erythrocyte Micronucleus Test, August 1998 and EEC Directive 92/69, L 383 A, Annex B. 12., p. 154 -156

 

The micronucleus test was carried out with Reaktiv Orange DYPR 1410. 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 (see preliminary study). According to the test procedure the animals were killed 24 hours after 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 withReaktiv Orange DYPR 1410and 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 ratioof polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.    

 

Under the conditions of the present study the results indicate that Reaktiv Orange DYPR 1410 is not mutagenic in the micronucleus test.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The results lead to the conclusion that Reaktiv Orange DYPR 1410 did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test.