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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Animals treated with up to 5000 mg/kg bw/d (2 applications, one daily) did not show any clinical sign. All animals survived until the study termination. The number of micronuclei containing polychromatic erythrocytes and normocytes were not influenced by the treatment with Hostanox SE 10. The ratio polychromatic erythrocytes/normocytes did not differ to the ratio obtained from the negative control animals. Cyclophosphamide (positive control) showed a significant increase of induced mirconucleus frequency. In conclusion, it can be stated that during the study the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
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:
24 May - 3 June 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Scientifically acceptable; Guideline conform; GLP study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
The animals were treated twice at interval of 24hours at dose levels of 50, 500 and 5000 mg/kg bw. Bone marrow cells were collected 6 hours after the second treatment.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Pharma Forschung Toxikologie, Hattersheim, Germany
- Age at study initiation:7 to 12 weeks
- Weight at study initiation: 27 - 38 g (males), 22 - 31 g (females)
- Housing: groups of five animals in Makrolon type-3 cages with wire mesh tops and softwood bedding
- Diet (e.g. ad libitum): pelleted Altromin diet 1324 ad libitum
- Water (e.g. ad libitum): Community tap-water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 27°C
- Humidity (%): 42 - 65%
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sesame oil
- Amount of vehicle (if gavage or dermal): 25 mg/ kg bw
Details on exposure:
A pre study revealed that 5000 mg/kg bw/d was the maximum tolerated dose.

Treatment:
Five males and five females were assigned to each test group. Animals were treated orally twice (one application per day) with either three doses of the test substance, the positive control (Cyclophosphamide) or the vehicle control (deinised water). Six hours after the last application the bone marrow of the animals was prepared.

Preparation of the animals:
The animals were killed and the femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1000 rpm for 5 min and the supernatant was discarded. A small drop of re-suspended cell pellet was spread on a slide. The smear was air-dried and stained with May-Grünwald/Giemsa.

Bone marrow smears were scored microscopically for micronucleated polychrmoatic erythrocytes. To analyse target organ cytotoxicity the ratio between polychrmoatic and normochromatic erythrocytes was determined. 2000 polychromatic erythrocytes per animal were analysed for appearance of micronuclei. The count of micronuclei containing normocytes was determined.
Duration of treatment / exposure:
The animals were treated twice at interval of 24hours.
Frequency of treatment:
twice
Post exposure period:
6 hours after second treatment
Remarks:
Doses / Concentrations:
50, 500, 5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide, 100 mg/kg bw
Tissues and cell types examined:
2000 immature erythrocytes per animal are examined for the incidence of micronucleated immature erythrocytes.
1000 mature erythrocytes per animal are examined for the incidence of micronucleated mature erythrocytes.
The proportion of immature among total (immature + mature) erythrocytes was determined.
Details of tissue and slide preparation:
Staining Methods: May-Gruenwald
Statistics:
Performed by computer programm "Diamant" (Code: SG-PA Nr. 4453) programmed by the Department of Praktische Methamatik of the Hoechst AG, Frankfurt, Germany.
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:
For all treated animals the incidences of micronucleated immature erythrocytes were comparable to control values. Also the incidence of micronucleated mature erythrocytes were not changed. The ratios of immature erythrocytes to total erythrocytes were not affected by the treatment.

Animals treated with up to 5000 mg/kg bw/d (2 applications, one daily) did not show any clinical sign. All animals survived until the study termination.

The number of micronuclei containing polychromatic erythrocytes and normocytes were not influenced by the treatment with Hostanox SE 10. The ratio polychromatic erythrocytes/normocytes did not differ to the ratio obtained from the negative control animals.

Cyclophosphamide (positive control) showed a significant increase of induced mirconucleus frequency.

In conclusion, it can be stated that during the study the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Conclusions:
Interpretation of results (migrated information): negative
The oral application of Hostanox SE 10 to mice did not induce increased micronucleated polychromatic erythrocytes in bone marrow.
Executive summary:

The test substance was investigated for its micronuclei formation potential in bone marrow in mice. Each 5 males and 5 females were treated by gavage at daily doses of 0, 50, 500 and 5000 mg/kg bw for two days. No treatment related clinical effect was observed. The ratios of polychromatic erythrocytes to normochromatic erythrocyte were not affected by the treatment. No increase of micronuclei formation was observed for treated animals.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Mutagenic activity of Dioctadecyl disulphide was investigated in one bacterial reverse mutation assay (Ames test; test strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 andE. coliWP2 uvr A), in three in vitro gene mutation studies in mammalian cells, one in vitro cytogenicity study (CHO cells) and in one in vivo Micronucleus Test in mice. Negative results were obtained in all tests with and without metabolic activation.

 

Dioctadecyl disulphide showed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The study was performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvr A. Test concentrations up to 1000 µg/plate were tested in the experiment (precipitation reported at 100 µg/plate and higher). The test compound proved to be not mutagenic to the bacterial strains.

Dioctadecyl disulphide was tested for mutagenicity using mammalian cells L5178Y TK+/- with and without metabolic activation in the dose range of 75-1000 µg/ml. The highest dose level was well above the solubility limit of the test substance in the test medium and obvious cell toxicity was not observed. No mutagenic activity was found.

Dioctadecyl disulphide was investigated for its UDS potential using A549 human cells with and without metabolic activation in the dose range of 0.1-30 µg/ml, the highest concentration corresponding to the solubility limit in the test medium. No substantial increase of DNA synthesis was observed for the treated cells.

Furthermore, the test substance was tested for mutagenicity using mammalian cells BHK21/C13 with and without metabolic activation in the dose range of 1- 20µg/ml. Due to the insolubility of the test substance an application of higher dose level than 20µg/ml was not possible. Neither significant cytoxicity nor mutagenicity was observed.

Dioctadecyl disulphide was tested for the SCE induction potential using CHO cells with and without metabolic activation in the dose range of 1-100 µg/ml. Due to the insolubility of the test substance the use of higher concentrations was not possible. A slight reduction in cell grow was observed at concentrations of 33.3 µg/ml and higher without metabolic activation. No further cell toxicity was observed and no significant increase of SCE was observed.

Animals treated with up to 5000 mg/kg bw/d (2 applications, one daily) did not show any clinical sign. All animals survived until the study termination.
The number of micronuclei containing polychromatic erythrocytes and normocytes were not influenced by the treatment with Hostanox SE 10. The ratio polychromatic erythrocytes/normocytes did not differ to the ratio obtained from the negative control animals.
Cyclophosphamide (positive control) showed a significant increase of induced mirconucleus frequency.
In conclusion, it can be stated that during the study the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.


Justification for selection of genetic toxicity endpoint
This in vivo study is selected as key study representing the toxicological endpoint "Genetic toxicity" since it was performed using mammalians and examines a very sensitive genotoxic mechanism. The study was performed according to the current OECD Guideline 474 and GLP.

Justification for classification or non-classification

In conclusion,Dioctadecyl disulphideis not mutagenic in the bacterial reverse mutation assay, the in vitro gene mutation studies, as well as in the in vitro sister chromatid assay and the in vivo micronucleus test in the presence and absence of metabolic activation up to the tested concentrations.

Dioctadecyl disulphide does not have to be not classified for mutagenicity since this substance did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation in concentrations up to 1000 µg/plate (precipitation at 100 µg/plate and higher), in the in vitro gene mutation assays, the sister chromatid exchange assay or in the in vivo micronucleus test in concentrations up to 5000 mg/kg bw/d.