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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative Ames tests, a negative mouse lymphoma assay, a negative in vitro chromosome aberration test, negative umu-test, one reliable negative cell transformation assay (CTA) and one non-reliable positive CTA.

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

Genetic toxicity in vivo

Description of key information

One reliable negative mouse bone marrow micronucleus test (MNT) and one non-reliable equivocal MNT, a negative dominant lethal test and a non-reliable DNA fragmentation assay.

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

Additional information

1) In vitro genotoxicity:

1.1) Bacterial reverse gene mutation

- Hargitai (2015), key study, reliability: 1

A fully guideline compliant bacterial reverse mutation assay (OECD471, GLP) was performed in the tester strains Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2, including analytical verification of the concentrations applied. In a plate incorporation assay and a pre-incubation assay there was no relevant increase in the number of revertants per plate in the absence and presence of metabolic activation up to the highest requested concentration of 5 mg/plate. Thus, this reliable and scientifically valid study demonstrated the non-mutagenicity of 1,3 dioxolane in bacterial test strains.

Four supplementary bacterial mutagenicity assays were performed with 1,3-dioxolane in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA 98 and TA 100 by the plate incorporation method [1983,1980, 1979, 1976]. These studies were performed under non-GLP conditions and with deviations to the current OECD guideline 471, but well documented and thus considered to be acceptable as supplementary information for the assessment of genotoxicity in bacteria. In addition to S. typhimurium TA1535, 1537 (or TA97/TA97a), TA98 and TA100, the current OECD guideline recommends the usage of either S. typhimurium TA102 or Escherichia coli WP2 uvrA (alternatively WP2 uvrA (pKM101)) to cover also mutagens with oxidising and cross-linking properties. These two recommended strains were not included in these supporting studies.

- Kowalski et al. (1983), supporting study, reliability: 2

The publication of Kowalski et al. (1983) provides data on bacterial mutagenicity testing higher than the requested limit dose. This may not only have been a compensate for the volatility of 1,3-dioxolane, but also for a possibly low purity of the substance batch, for which no purity data were available. Reference substances for the test system were not included in the main experiments of the study, however, the sensitivity of the strains was proven by a separate spot test revealing a clear mutagenic response after treatment with positive controls. The outcome of the assay was clear non-mutagenicity and was confirmed by independent repeats in the absence and presence of a metabolic activation system.

- Jagannath (1980), supporting study, reliability: 2

Additional minor guideline deviations in the Ames test performed by Jagannath (1980) were the use of single or duplicate cultures instead of triplicates and the lack of an experimental repeat. The high substance concentration used (10 µL or 50 µL/plate) is considered to have compensated any losses due to the volatility of the substance. In the absence and presence of metabolic activation 1,3-dioxolane was found to be clearly non-mutagenic under the test conditions described.

- Bulman et al. (1979), supporting study, reliability: 2

In the study of Bulman et al. (1979) the highest applied concentration of 1,3-dioxolane (1 mg/plate) was below the requested concentration of 5 mg/plate and thus there was no compensation for volatility or a possibly low purity of the substance batch. Cytotoxicity data were not included. Up to the highest tested concentration in the absence and presence of metabolic activation there was no mutagenic response in the S. typhimurium strains. This negative outcome was not confirmed in an experimental repeat.

- Anonymous (1976), supporting study, reliability: 2

A fourth supplementary bacterial mutagenicity test used substance concentrations far beyond the requested limit concentration of the OECD guideline and compensated for the low purity and the volatility of the substance during test performance. Up to the highest concentration applied (10 mg/plate) in the absence and presence of metabolic activation, neither cytotoxicity nor mutagenicity was observed under the test conditions described. This clear non-mutagenic outcome of the assay was not confirmed by an independent repeat.

- Brusick (1975), disregarded study, reliability: 4

This Ames test is disregarded due to lack of information about material, methods and results.

There is clear evidence from one key and four supporting bacterial reverse mutation assays that 1,3 dioxolane is not mutagenic.

1.2) Mammalian cell gene mutation

- Cifone (1985), key study, reliability: 1

The mutagenicity of 1,3-dioxolane was further evaluated in a GLP and OECD476 (version July 1997) compliant mouse lymphoma assay in three independent experiments. The cells were exposed to the substance up to the highest recommended concentration of 5 µL/mL without consideration of its volatility. Under the test conditions described, 1,3-dioxolane treatment did not result in an increased mutation frequency in the absence and presence of metabolic activation. This study with few methodological deficiencies is reliable, scientifically valid and appropriate for the assessment of 1,3-dioxolane as non-mutagenic in mammalian cells.

1.3) Chromosome aberration

- Ivett and Myhr (1985), supporting study, reliability: 3

A chromosomal aberration assay in Chinese hamster ovary cells provides supporting non genotoxic information on 1,3-dioxolane up to the limit dose of 5 mg/mL as requested by the OECD guideline 473 (volatility was not taken into account). There were major deviations to the guideline recommendations in this GLP study. In order to guarantee for a sufficient number of cells being in first post-treatment mitosis the cells should be harvested at a time point equivalent to 1.5 of the normal cell cycle time. In this chromosomal aberration study a cell harvest time of approximately 20 hours would have been adequate to fulfill this requirement. In the assay a shortened harvest time of 10 hours was applied, being less than a normal full cell cycle. The validity of the experimental parts were proven by the positive clastogenic response of the reference substances used. The outcome of this study was clearly that the test item was non-clastogenic under the conditions described, however, there was no independent repeat to confirm this result. This study with distinct methodological deficiencies is considered not to be sufficiently reliable to fully cover the endpoint structural (numerical) chromosome aberration, but considered to provide relevant supporting information to the results of the respective in vivo assays; and thus contributing to the weight of evidence for non-genotoxicity.

1.4) Genome mutation

Cell transformation assays are not limited to the detection of potential genotoxic agents, but can also identify substances acting as potential non-genotoxic carcinogens. There are currently no OECD guidelines in place for the tests briefly summarised below. Minor deviations, such as the lack of a metabolising system in the test and the lack of an experimental repeat for data confirmation, were noted as compared to the method described in Council Regulation (EC) 440/2008 B.21.

- Matthews and Myhr (1985), supporting study, reliability: 2

In a GLP compliant cell transformation assay, Balb/c 3T3 cells were treated with 1,3 dioxolane at 5 concentrations up to a maximum concentration of 15 µL/mL for a time period of 72 hours. The chosen concentrations covered the non-toxic, moderate toxic (approx. 78%) and clearly toxic (approx. 50%) range, and showed severe toxicity at the highest applied concentration. The complete concentration range was evaluated for morphological transformation and there was no increased incidence in the mean number of transformed foci up to the top concentration under the conditions of this study. This supporting study with methodological deficiencies is considered to be reliable with restriction and to provide relevant information contributing to the weight of evidence for non genotoxicity of 1,3-dioxolane.

- Garry and Kreiger (1981), disregarded study, reliability: 3

In a non-GLP compliant study a phase I colony mode test was performed, treating murine C3H10T1/2 cells with 0.1 µg/ml up to 10 mg/ml 1,3-dioxolane in culture flasks and up to 20 mg/mL in culture dishes for 24 hours, followed by 14 days incubation before fixation and examination. In the phase II focus test cells were treated with 1 µg/ml up to 20 mg/ml in culture flasks and dishes for 24 h followed by 42 days incubation. The higher concentrations in flasks (2 – 20 mg/ml) showed contaminations, therefore information about transformation rates is lacking. In the phase I method, no transformed cells were observed using culture dishes. Thus, the single positive results in flasks (2 and 10 mg/ml), which were not dose-dependent, could not be confirmed. The phase II experimental part in culture flasks was discussed as being invalid due to the low response of the positive control. In contrast, in the phase II method a concentration-related increase in transformed foci was observed in dishes from 0.1 – 10 mg/mL, whereas at the highest applied concentration of 20 mg/mL, the number of transformed foci decreased; possibly linked to severe toxic effects. In the study report the outcome of the study is discussed as being positive due to the concentration-related increase in the phase II experiment in culture dishes. With regard to several deficiencies in study performance the positive findings should rather be discussed as being questionable. Most importantly, there are no cytotoxicity data for the phase II test, which are essential for evaluating the results, because a possible influence of test substance toxicity cannot be excluded. Additionally, there is no independent repeating experiment available, confirming the positive results. No criteria for the classification of a test substance as being positive were given in the report. In addition, there were no criteria defined for the acceptance of the reference substance showing a positive response. Finally, test substance concentrations of 5 – 20 mg/mL are far beyond the limit concentrations which are applied nowadays in the in vitro genetic toxicity assay systems. This non-GLP study with distinct methodological deficiencies is considered not to be reliable for the assessment of morphological cell transformation, and to provide a minor contribution to the weight of evidence.

1.5) DNA damage / repair

- Nakamura et al. (1987), disregarded study, reliability: 4

In this publication 1,3-dioxolane was considered to be non-genotoxic in the umu-test system, according to the methods of Oda et al. (1986). Due to low amount of details, the study is regarded as non-assignable.

- Jaros-Kaminska et al. (1985), disregarded study, reliability: 3

The changes in hyperchromicity and melting temperature of DNA incubated with 1,3-dioxolane were determined with and without metabolic activation. This is not a recognized or established test system to detect mutagenicity. Reported results were not interpretable and no reliable conclusion was possible.

2) In vivo genotoxicity:

2.1) Chromosome aberration

- Putman and Melhorn (1989), key study, reliability: 1

A GLP compliant mouse bone marrow micronucleus assay was performed in male and female animals at dose levels in a range of 525 to 2100 mg/kg bw with only minor deviations to the OECD guideline 474. The highest dose applied by intraperitoneal injections was slightly higher than the limit dose requested by the guideline. Cytotoxicity of 1,3-dioxolane was observed in the highest dose applied indicated as a reduction in the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), thus demonstrating bone marrow exposure. The evaluation of 1000 PCE at sampling times of 24, 48 and 72 hours revealed no treatment-related increases in the number of micronucleated cells. The current OECD guideline (latest revision Sept 2014) requests the evaluation of 4000 cells for micronucleus formation, however, since the criteria for a valid test were clearly fulfilled for the solvent and positive controls it was concluded that the outcome of the test was non-mutagenicity of 1,3-dioxolane. This key GLP study with few deviations to guideline is reliable and scientifically valid and demonstrates that 1,3-dioxolane is neither clastogenic nor aneugenic in vivo.

- Przybojewska et al. (1984), disregarded study, reliability: 3

In a mouse micronucleus study published by Przybojewska et al. (1984) male mice were treated twice (24 hours apart) with 1,3-dioxolane and bone marrow cells were prepared 6 hours after the last injection. The treatment resulted in a dose-related increase in PCE with micronuclei in a concentration range of 750 to 6000 mg/kg bw, showing statistical significance in a range of 1500 to 6000 mg/kg bw when compared to the concurrent vehicle control and thus the substance was considered to be mutagenic by the authors under the conditions of the test performance. This non-GLP study, however, is less reliable as compared to the key study since it did not follow the recommendations of the OECD guideline 474, resulting in distinct methodological deficiencies. The number of animals used for testing was below 5 mice per dose group, no data on toxicity (e.g. mortality, clinical signs, PCE/NCE ratio) were given for the main experiment and the sampling time should have been 18 - 24 hours after the last substance application. The lack of cytotoxicity data represents a major deficiency as positive results at highly toxic doses should be interpreted with caution. There are no data on the purity of the test substance batch used for the assay and thus it cannot be excluded that any impurity might be responsible for the observed effects. In addition, the two highest applied dose levels are far above the limit dose requested by the guideline. This non-GLP supporting study with clear methodological deficiencies and limited scientific validity provides equivocal evidence for the induction of chromosome aberrations by 1,3 dioxolane, which is overruled by the clearly negative result of the GLP and guideline compliant, reliable and scientifically valid key study by Putman and Melhorn (1989). The key study was conducted with a defined batch of high purity and its result is supported by the negative outcome of an in vitro chromosomal aberration assay by Ivett and Myhr (1985).

- Baranski et al. (1984), supporting study, reliability: 2

In a non-GLP dominant lethal study by Baranski et al. (1984) similar to OECD guideline 478 male rats were repeatedly dosed with 1,3-dioxolane by gavage (0.58 or 1.16 g/kg bw/d for 8 weeks) or exposed to the substance by whole body inhalation (2.5 mg/L, 5h/d for 12 months) and then mated with untreated females. All female animals were subject to examination of reproductive parameters. The male animals were examined for organ weight and histopathology of testicles. Minor deviations that were not considered to have affected the study were the missing data on substance batch and purity and the lack of a positive control. The lower number of animals was compensated with a longer evaluation period providing sufficient pregnant rats. Two dose levels were applied instead of three for oral exposure, however, toxic effects were observed at these levels and toxicity was also found for the concentration applied by inhalation. There was no evidence of a dominant lethal effect and the study is considered as being valid.

2.2) DNA damage / repair

- Jaros-Kaminska et al. (1985), disregarded study, reliability: 3

The interaction of 1,3-dioxolane with DNA in vivo (in hepatocytes, 4 hours after intraperitoneal injection in rats) was examined by Jaros-Kaminska et al. (1985) in an explorative non-GLP study. In hepatocyte DNA isolated from treated rats a statistical significant increase in single strand breaks was observed without showing any dose dependency. The calculated DNA fragmentation index was far below the values obtained for the reference substances. Interaction with DNA in vivo was observed, however, this is not an accepted method for the testing of genotoxicity. This non-GLP study conducted with a test item batch of unknown purity (purity not reported) is not considered to provide adequate information for a reliable assessment of the endpoint DNA fragmentation.

3) Overall conclusion:

There are sufficient data available from reliable and scientifically valid in vitro and in vivo studies to conclude that, based on the available weight of evidence,1,3-dioxolane does not exhibit a genotoxic potential. 1,3-Dioxolane was shown to be non-mutagenic in bacteria and mammalian cells. There was no indication for 1,3-dioxolane-related cell transformation based on key study results. 1,3-Dioxolane did not induce chromosome aberration according to the outcome of the key in vivo bone marrow micronucleus assay, as supported by the lack of clastogenic activity noted in an in vitro chromosome aberration assay. Supplementary in vivo data indicated that exposure to 1,3-dioxolane was not associated with a dominant lethal effect. The assessment of 1,3-dioxolane as being non-genotoxic both in vitro and in vivo is supported by the results of the 2-year carcinogenicity key study in rats (Busey, 1985) where no relevant increases in any tumor incidence of the animals were reported. In addition, no indication for a carcinogenic potential was found in the repeated dose oral and inhalation toxicity key studies performed by Hoberman (1991) and Landry et al. (1990).


Justification for selection of genetic toxicity endpoint
A variety of in vitro and in vivo genotoxicity assays consistently concluded that 1,3-dioxolane did not exhibit genotoxic potential.

Short description of key information:
- overall conclusion: non-genotoxic
- in vitro data: negative Ames tests, a negative mouse lymphoma assay, a negative in vitro chromosome aberration test, negative umu-test, one reliable negative cell transformation assay (CTA) and one non-reliable positive CTA
- in vivo data: one reliable negative mouse bone marrow micronucleus test (MNT) and one non-reliable equivocal MNT, a negative dominant lethal test and a non-reliable DNA fragmentation assay

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Regarding the variety of in vitro and in vivo genotoxicity assays, it could be concluded that 1,3-dioxolane does not exhibit genotoxic potential. Therefore, a classification for mutagenicity according to Regulation (EC) No 1272/2008 is not warranted.