REAKTIV OLIV F00-149

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

The test item has been tested in vitro in a reverse gene mutation assay in bacteria (OECD 471), a mammalian cell cytogenetics assay (OECD 473), a mammalian cell gene mutation assay (OECD 476) and in vivo in a mammalian cell gene mutation assay (OECD 474). Based on a weight of evidence assessment the test item is not considered genotoxic.

Link to relevant study records

Reference

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:

2002-02-27 to 2002-04-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
- Age at study initiation: 6 weeks
- Weight at study initiation: mean male: 190.6 g; mean female: mean= 149.9 g
- Housing: five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air-conditioned room (room number 011)
- Diet (e.g. ad libitum): rat/mice diet ssniff R/M-H (V 1534), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12h light/dark

Route of administration:

oral: gavage

Vehicle:

deionized water

Duration of treatment / exposure:

24 h

Frequency of treatment:

two doses separated by an interval of 24 hours (except positive control with only one dose)

Remarks:

Doses / Concentrations:
2000 mg/kg
Basis:
no data

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control was with Endoxan, which was administered once orally at a dose of 40 mg per kg body weight.

Tissues and cell types examined:

bone marrow: 2000 polychromatic erythrocytes were counted for each animal

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the results of the acute oral toxicity-study PTOl-0361 the dose of 2000 mg/kg bw was selected as the limit dose

DETAILS OF SLIDE PREPARATION:
bone marrow smaples were fentrifuged with fetal bovine serum, sediment was smeared and stained as follows:
Staining procedure
-5 minutes in methanol
-5 minutes in May-Griinwald' 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

Evaluation criteria:

Criteria for a positive response:
A substance is considered as positive if there is a dose-related increase in the number of micronucleated polychromatic erythrocytes or a significant increase in at least one dose group compared with the concurrent negative control group which is above the range of the historical control data. A test substance producing no significant or dose-related increase in the number of micronucleated polychromatic erythrocytes is considered non-clastogenic in this system.

Statistics:

Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5% is adopted for all tests.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was a slight increase in micronucleated polychromatic erythrocytes in male animals, but was within the historical control range of the negative control groups and was concluded to have no biological relevance.

Conclusions:

Interpretation of results (migrated information): negative
Reactive Olive F00-0149 did not cause a substantial increase of micronucleated polychromatic erythrocytes and is therefore not considered clastogenic in the micronucleus test in vivo.

Executive summary:

In a Sprague-Dawley rat bone marrow micronucleus assay, 15 rats/sex were treated orally with two doses, 24 hours apart, of Reactive Olive at 2000 mg/kg bw. Bone marrow cells were harvested 24h post-treatment. The vehicle was deionized water. 

There were no signs of substance-related toxicity during the study. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after treatment.

This study is classified asacceptable and satisfies the requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537 of S. typhimurium and E. colli WP2 uvrA were exposed to Reactive Olive F00-0149 in water at concentrations of 50, 160, 500, 1600, and 5000 ug/plate in the presence and absence of metabolic activation. There was evidence of a significant dose-response increase in the number of revertant colonies in the presence of metabolic activation with strain TA 98 at the highest dose and a slight increase in the number of reverant colonies with strain TA 1535 at the highest dose. In this study, the test item is mutagenic in bacteria in the presence of metabolic activation using the standard Ames Test procedure (plate incorporation test).

Additionally, in a mammalian cell cytogenetics assay (chromosome aberration), Chinese hamster lung fibroblasts (V79) were exposed to Reactive Olive F00-0149 in test medium at concentrations of 1000, 2500, 3750,5000 µg/mL, with and without metabolic activation for 3h and at concentrations of 250, 500, 1000, 1500, 2000 µg/mL without metabolic activation for 20h. Results show significantly enhanced aberration rates at the 3h treatment time with S9-mix at 2500 and 5000 ug/mL. Further, there was an increase in polyploidy cells at the highest dose with and without S9-mix. There was also a slight increase in the number of aberrations compared to historical controls without S9-mix at 2500 ug/mL. This experiment indicated there was evidence of chromosome aberration induced over background in the absence and presence of metabolic activation. 

In a mammalian cell gene mutation assay, Chinese hamster lung fibroblast (V79) cells cultured in vitro were exposed to Reactive Olive F00-0149 in culture medium at concentrations 10, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL in the presence and absence of mammalian metabolic activation S9. The test item did not induce gene mutations, i.e. was not mutagenic, either in the presence or in the absence of metabolic activation under the conditions described in this report.

Finally, in a Sprague-Dawley rat bone marrow micronucleus assay, 15 rats/sex were treated orally with two doses, 24 hours apart, of Reactive Olive F00-0149 at 2000 mg/kg bw in deionized water. Bone marrow cells were harvested 24h post-treatment. This test concluded no signs of substance-related toxicity during the study as well as no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after treatment. The test item is therefore not considered clastogenic in the micronucleus test in vivo.

In summary, two in vitro experiments (Ames test and in vitro CA) indicated genetic toxicity induced by Reactive Olive F00-0149. The effects have been invalidated by a higher tier gene mutation assay in mammalian cells (HPRT) and an in vivo micronucleus test in rat. Based on the weight of evidence, the test item is not considered genotoxic in an appropriate testing battery.

Justification for selection of genetic toxicity endpoint
In vivo genetic toxicity study according to OECD 474 for in vivo cytogenicity. Additionally, several in vitro studies are available for genetic toxicity of Reactive Olive F00-0149

Justification for classification or non-classification

The test item is not considered genotoxic based on a weight of evidence assessment of the results of an appropriate testing battery.