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Key value for chemical safety assessment

Additional information

In vitro data

Bacterial reverse mutation

The potential of the test material to cause gene mutation in bacterial strains was determined in a GLP study which was conducted in accordance with the standardised guidelines OECD 471. Five strains of Salmonella typhimurium (TA1535, TA1537, TA98,TA100 and TA102) were treated in the presence and absence of at rat liver derived metabolic activation system (S9 mix), in two separate experiments.

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S9, at concentrations of test material of 5, 15.81, 50, 158.1, 500, 1581 and 5000 mg/plate, plus negative (vehicle) and positive controls. Following these treatments, no evidence of toxicity was observed.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S9. The maximum test concentration of 5000 mg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 156.3 – 5000 mg/plate, in order to examine more closely those concentrations of test material approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity was observed at 5000 mg/plate in all strains in the presence of S9 only.

The test material was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed. Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for negative control treatments, and were significantly elevated by positive control treatments.

Following test materialtreatments of all the test strains in the absence and presence of S9, no increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1 % level using Dunnett’s test.

It was therefore concluded that the test material did not induce mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study.

Gene mutation in mammalian cells

The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in a study which was conducted to a methodology which was similar to that outlines in the standardised guideline OECD 476. L51788Y TK+/-mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived auxillary metabolic system (S9 mix) in two independent experiments. Mutant colonies were scored for all cultures in each experiment. The test material was tested up to a maximum concentration of 5000 µg/mL in the presence and absence of metabolic activation. This concentration is approximately equivalent to the limit concentration for this assay.

Under the conditions of the study precipitation of the test material was observed at 1000 µg/mL, but testing was continued up to 5000 µg/mL. In the absence of S9 mix there was no indication of a mutagenic response in two experiments. In the presence of S9 mix, one experiment similarly failed to show any mutagenic effect, while significant increases in mutant fraction occurred in a second experiment. The Lowest Observed Effect Dose in this experiment was 2000 µg/mL, higher than the precipitation concentration. Significant toxicity was observed in all experiments. It was therefore concluded that the test material was not mutagenic in this system.

In vivo data

Chromosome aberration (Micronucleus)

The potential genotoxicity and clastogenicity of the test material to the bone marrow cells of male mice in vivo was assessed in a study conducted to a methodology which was similar to that outlined in the standardised guideline with OECD 474. Following a preliminary toxicity test, the top dose of 2000 mg/kg was selected for the micronucleus test (the limit dose recommended by the guideline). Under the conditions of the study the test material was found to be non-genotoxic and non-clastogenic.

Chromosome aberration (Dominant Lethal Test)

The potential of the test material to exert dominant lethal mutations and antifertility effects was investigated in a non-GLP study which was conducted to a methodology that was similar to that which is outlined in the standardised guideline OECD 478. During the study groups of 10 male mice received a single intraperitoneal injection of test material at dose levels of 0.5, 1.0, 5.0 and 10.0 mL/kg. Two groups of mice received distilled water at the highest dose volume (10.0 mL/kg), and served as controls. Immediately after injection, two virgin female mice were caged with each male mouse. During the 8 week period the females were replaced weekly with two virgin females. Thus, each weekly group was composed of 20 female mice. The onset of gestation was established by the presence of vaginal plug and was designated as Day 0. The pregnant mice were sacrificed with an overdose of ether in Day 15 ± 2 of gestation. The uterine horns and ovaries were exposed and the number of corpora lutea, total number of implantations, preimplantation losses, early and late fetal deaths, and viable fetuses were recorded. The antifertility effect in the dominant lethal test was considered to be a reduction in the incidence of pregnancies, while the dominant lethal mutation was determined directly from the increased number of early fetal deaths in individual mice, and indirectly from the reduced number of total implantations.

Under the conditions of the study the test material produced dose-dependent antifertility and mutagenic effects, as indicated by reduced percentage of pregnancies and increased numbers of early fetal deaths, respectively. the highest dose of test material produced a distinct reduction in incidence of pregnancies, especially during the first 3 - 4 weeks; this effect was less evident with the lower doses. There was a reduction in the number of implantations and live fetuses/pregnancy for one or more dose levels. Mutational effects, expressed by an increase in early fetal deaths/pregnancy, occurred mainly during the postmeiotic stage of spermatogenesis in mice. Adverse effects upon the premeiotic stage were also observed. Thus, early fetal deaths and semisterility constitute some of the genetic and reproductive effects elicited from intraperitoneal injection of the test material to male mice subsequently mated to untreated females.

It should be noted however that the result from this assay is confounded by the route of administration and the nature of the test material. The test material was effectively an insoluble hydrocarbon. Such materials (e.g. mineral oils, squalene) are often used as adjuvants to provoke immune responses in in vivo assays via the intraperitoneal route, and in addition are known to invoke autoimmune responses in mice (the primary test species for immunological research). As such the apparent positive result in this study is disregarded.


Justification for selection of genetic toxicity endpoint
As multiple studies are presented to address genetic toxicity, not one single study was selected as the key study as they represent different types of genetic toxicity and are therefore not comparable.

Short description of key information:
IN VITRO DATA
Reverse mutation in bacteria: Negative (S. typhimurium strains TA 100, TA 98, TA 1535, TA 1537 and TA 102 with and without metabolic activation), OECD 471, McGarry 2013

Gene mutation in mammalian cells: Negative, L5178Y TK+/-, McGregor et al. 1988

IN VIVO DATA
Chromosome aberration (micronucleus test): Negative up to 2000 mg/kg bw (limit concentration), Shelby et al. 1993

Chromosome aberration (dominant lethal test): Ambigous (males), negative (females), ICR mice, Singh et al. 1975

Endpoint Conclusion:

Justification for classification or non-classification

With respect to mutagenicity, no classification is proposed as no relevant mutagenicity or genotoxicity was observed in adequately conducted studies. The male dominat lethal assay produced a positive result however the result from this assay is confounded by the route of administration and the nature of the test material. The test material was effectively an insoluble hydrocarbon. Such materials (e.g. mineral oils, squalene) are often used as adjuvants to provoke immune responses in in vivo assays via the intraperitoneal route, and in addition are known to invoke autoimmune responses in mice (the primary test species for immunological research).