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Key value for chemical safety assessment

Additional information

In vitro studies : Ames tests

In the key study (1979), an Ames test was carried out with a mixture of 70% dioctyltin bis(2-ethylhexylmercaptoacetate) and 30% mono-octyltin tris(2-ethylhexylmercaptoacetate). This mixture was tested in strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100), with or without S9, and there are positive and negative controls. No mutagenic activity was observed in this test.

Others studies were used as supporting studies because they are less complete than the key study. All these studies used the same mixture as the key study, DOTE:MOTE, 70:30%. One of these studies gave negative results, and two old studies showed a (weak) positive response without metabolic activation.

 

In vitro studies : Mouse lymphoma assay

A GLP study guideline (OECD 473) was available. DOTE was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). DOTE was cytotoxic in both the absence and presence of S9-mix.

In the absence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. In the presence of S9-mix at 72 μg/ml the mutant frequency was significantly increased by 238 mutants per 1,000,000 clonable cells compared to the negative control. Since relatively small intervals (0.85) were used and the increase was observed at a single concentration causing more than 90% cytotoxicity compared to six concentrations causing 50-70% cytotoxicity which showed no increase in mutant frequency, it is concluded that this increase is not indicative for mutagenicity.

It is concluded that under the conditions used in this study, the test substance DOTE isnot mutagenicat the TK-locus of mouse lymphoma L5178Y cells.

In vivo studies.

Two in vivo micronucleus studies available on the mixture Dioctyltin bis(IOMA) [CAS No. 26401-97-8]:Octyltin tris(IOMA) [CAS No.26401-86-5] (80:20). Information from these studies has been read across to the registered substance on the basis Dioctyltin bis (IOMA) and dioctyltinnbis (2-EHMA) are isomers of the same compound and are structural analogues of each other. Based on the recently conducted developmental toxicity studies in two species it is considered that DOTI is likely to be more toxicoligically active and therefore use of data on this substance would be considered to be a worst case assessment of the registered substance.

Both studies followed methods similar to those which are outlined in standardised guideline OECD 474 and were assigned a reliability score of 2 in line with the criteria of Klimisch; these studies are regarded as key studies in the overall assessment of the genetic toxicity of the registered substance.

In the first of the two studies, the effect of ZK 30 434 on the incidence of micronucleated polychromatic erythrocytes from the bone marrow of mice was tested with doses of 2250, 4500, and 9000 mg/kg bodyweight administered by oral gavage in two equal doses separated by an interval of 24 hours. Negative control (1% methycellulose by oral gavage) and positive control group (14 mg/kg mitomycin C by injection) were run concurrently. After 6 hours of the second dose bone marrow smears were examined for the presence of micronuclei in 2000 polychromatic and 2000 normochromatic erythorcytes per mouse.

At all doses of ZK 30 434 both the group mean micronucleated cell counts were comparable with the concurrent control values. The ratio of normochromatic to polychromatic cells was comparable to controls for 2250 mg/kg group and significantly higher for 4500 and 9000 mg/kg groups (by Kruskal-Wallis method). Positive control produced expected increase in micronucleated cell count and ratio. It was concluded that ZK 30 434 failed to show any evidence of mutagenic potential when administered orally in the test procedure. However, evidence of bone marrow depression was observed, which is an indication that the test item reached the bone marrow.

In the second of the two studies, the effect of ZK 30 434 on the incidence of micronucleated polychromatic erythroctyes in mice was assessed; a total doseage of 4500 mg/kg bodyweight was administered by oral gavage in 2 equal doses, separated by 24 hours. A negative control group (1% methylcellulose) and positive control group (methotrexate, 40 mg/kg body weight) were run concurrently. Mice were sacrificed 12, 24, 36, and 48 hours after the second dose and bone marrow smears were examined for the presence of micronuclei in 2000 polychromatic and 200 normochromatic erythrocytes per mouse. The ratio of normochromatic to polychromatic cells was also determined in each mouse.

The group mean micronucleated cell counts in polychromatic and normochromatic erythrocytes obtained after ZK 30 434 treatment were comparable with the concurrent control after 12, 24 and 36 hours. After 48 hours the micronucleated normochromatic cell count was slightly higher than that of the concurrent control (least significant difference method). At 48 hours the micronucleated polychromatic erythrocyte count was comparable to control.

The normochromatic to polychromatic ratio at each period was higher than that for the concurrent control value. The positive control produced the expected increase in both the group mean micronucleated cell counts and also in the normochromatic to polychromatic erythrocyte ratio at 12, 24, 36 hours. After 48 hours the micronucleated normochromatic erythrocyte count was comparable with the concurrent control. There were too few polychromatic cells to count and was not possible to use this parameter in analysis.

It therefore was concluded that ZK 30 434 did not show mutagenic potential when administered orally in the test procedure. However, evidence of bone marrow depression was observed at each kill. The small increase in micronucleated normochromatic cells at the 48 hour timepoint, though significantly different from the concurrent control, cannot be considered evidence of mutagenic potential since a corresponding rise in micronucelated polychromatic cells had not been observed in an earlier time point.

Disregarded studies

Three in vivo studies with the test material Dioctyltin Dichloride (DOTC) are included in the dataset since the substance was initially considered to be a relevant substance to use for read-across purposes since findings from a gastric hydrolysis study showed that DOT(2-EHMA) was readily hydrolysed to Dichlorodioctyltilstanane (CAS no.3542-36-7) under physiological conditions (see section 7.1.1). Thus DOTC(Dichlorodioctylstannane) was considered to be an appropriate anchor compound and surrogate for the mammalian toxicology endpoints of repeated dose, in vivo genetic toxicity, reproduction and developmental effects, when it is dosed via the oral route of administration.

Read-across to the substance DOTC is no longer considered as wholly appropriate based on the results of the recent Hydrolysis study, as reported by Naßhan, H, 2014 (see section 7.1.1) which indicate the substance DOTECl is the only metabolite of DOTE which is formed in a simulated mammalian gastric environment; no dioctyltindichloride was formed under the conditions of the study. The three in vivo studies with DOTC have therefore been disregarded in the overall assessment of the registered substance with regards to genetic toxicity.


Justification for selection of genetic toxicity endpoint
Multiple studies have been provided to address the different endpoint of genetic toxicity, each addressing different types of genetic toxicity. Since all the studies showed negative results, a single study could not be selected as key over the others.

Short description of key information:
In vitro studies : Four reliable Ames tests (4 with mixture DOTE:MOTE) + Mouse lymphoma assay (with DOTE) were available.
In vivo studies : Two micronucleus tests with mixture DOT(IOMA):MOT(IOMA)) (80:20) were available.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to Directive 67/548/EEC and to regulation EC no.1272/2008 (CLP), DOTE is not classified for genetic toxicity endpoint.

Justification based on weight of evidence approach: Ambiguous results in Ames tests, negative results in the mouse lymphoma assay performed on DOTE, and negative results in in vivo micronucleus test performed with the hydrolysis product (DOTC).