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Additional information

From OECD SIDS 2006:

With the exception of the micronucleus assay in vivo there are no studies available which have been performed according to the OECD test guidelines but there are valid studies which give sufficient information to evaluate this endpoint.

In vitro Studies

There are three Ames-tests (two plate incorporation assays and one suspension test) each of them of borderline validity. However, in combination the tests provide enough detailed information for evaluation of this endpoint. In a plate incorporation assay with Salmonella typhimurium TA 100 1,4-dichlorobut-2-ene (76.8 % trans- and 21.6 % cis-isomer) proved to be mutagenic at 12.5 µg/mL and above in presence of S9-mix from mouse liver (both with and without addition of cofactors for a NADPH generating system) and from human liver samples (Barbin et al., 1978; Bartsch, 1976; Bartsch et al., 1979; Bartsch et al., 1980). In another plate incorporation assay with Salmonella typhimurium TA 1535, TA 1537, and TA 1538 1,4-dichlorobut-2-ene (720 µg/mL; no data on isomer composition) showed mutagenic activity only towards strain TA 1535 with and without addition of S9 mix from liver, lung, and testes of rats, mice, and monkeys, respectively; a concentration of 1440 µg/mL led to 50% survival of bacteria of strain TA 1537. The positive controls (non-activation: ethylmethane sulfonate, 2-nitrosofluorene, quinacrine mustard; with activation: dimethylnitrosamine, 2-acetylaminofluorene) were functional (Litton Bionetics data, 1974). In the suspension test without addition of S9 mix 1,4-dichlorobut-2-ene (720 µg/mL and 343 µg/mL) was highly mutagenic to strain TA 1535 and weakly mutagenic to strain TA 1538. In presence of S9 mix from liver, lung, or testes of mice 1,4-dichlorobut-2-ene (720 µg/mL and 343 µg/mL) was mutagenic to strain TA 1535 only; a concentration of 1440 µg/mL led to 50 % survival of bacteria of strain TA 1537. The positive controls (non-activation: ethylmethane sulfonate, 2-nitrosofluorene, quinacrine mustard; with activation: dimethylnitrosamine, 2-acetylaminofluorene) were functional (Litton Bionetics data, 1974). In a yeast mitotic gene conversion test (suspension test) 1,4-dichlorobut-2-ene (720 and 1440 µg/mL) was mutagenic to Saccharomyces cerevisiae strain D4 without and with metabolic activation (S9 mix from liver, lung or testes of mice); 1440 µg/mL were cytotoxic. The positive controls (non-activation: ethylmethane sulfonate; with activation: dimethylnitrosamine) were functional (Litton Bionetics data, 1974). In a further yeast forward mutation assay (suspension test) with limited documentation 1,4-dichlorobut-2-ene (3.75 - 12 500 µg/mL) showed mutagenic activity in Schizosaccharomyces pombe (no further details given) (Loprieno, Barale, and Rossi, 1979; Barale, Presciuttini, and Rossi, 1979). In a mammalian cell mutagenicity test (HPRT-assay) 1,4-dichlorobut-2-ene (= 2 µg/mL) showed clear mutagenic activity in Chinese hamster ovary cells both without and with metabolic activation by rat liver S9 mix. At the highest concentrations tested (4 and 5.5 µg/mL, respectively) the cell survival amounted to 30 - 40 %. The positive controls (non-activation: ethylmethane sulfonate; with activation: dimethylbenzanthracene) were functional (DuPont, 1980).

In vivo Studies

In a micronucleus assay which has been performed according to OECD TG 474 groups of 5 rats per sex and treatment group were exposed nose-only against 1,4-dichlorobut-2-ene (65 % trans- and 35 % cis-isomer) concentrations of 0; 0.1; 1; 10 ppm (0; 0.52; 5.2; 52 mg/m3) for 6 hours/day on 5 days/week for 2 weeks. Bone marrow smears were prepared on the day of the last exposure. At least 1000 polychromatic erythrocytes (PCE) per animal were evaluated for the presence of micronuclei. Males and females of the 10 ppm group showed a significant decrease in bw gain (88 % and 100 % reduction, respectively) after 10 days of exposure. There was no statistically significant increase in micronucleated PCEs and no significant depression in the proportion of PCEs among total erythrocytes observed in any 1,4-dichlorobut-2-ene - treated group. The positive control (cyclophosphamide) was functional (DuPont, 1995). In a non-guideline study with limited documentation rats were exposed to 1,4-dichlorobut-2-ene vapour concentrations of 0; 1.7 and 7.9 mg/m³ for 4 hours/day, 5 days/week for 1 day, 30 days and 120 days, respectively and sacrificed after the last treatment. Part of the animals exposed for 120 days were kept for a recovery period of 45 days without further exposure prior to sacrifice. 1,4-dichlorobut-2-ene led to a significantly increased rate of chromosomal aberrations of chromatid type in rat bone marrow both at the low (after 30 and 120 days treatment) and the high exposure concentration (at all time points). At the end of the recovery period the aberration rate had returned to control level in the low exposure group only (Nalbandyan and Gizhlaryan, 1985). 1,4-dichlorobut-2-ene was mutagenic in a recessive lethal assay with Drosophila melanogaster leading to a significant increase in lethals compared to the control at a concentration of 4 mM (500 µg/mL) in the drinking water (Vogel, 1976; Vogel, 1979).


Short description of key information:
1,4-Dichlorobut-2-ene is mutagenic to bacteria and yeasts as well as to mammalian cells in vitro. In vivo a negative result was obtained in a micronucleus assay performed according to OECD TG 474 with inhalation exposure of rats although the highest concentration tested (52 mg/m³) led to systemically toxic effects. In another non-guideline study with limited documentation 1,4-dichlorobut-2-ene showed a clastogenic activity after inhalational exposure of rats. Overall 1,4-dichlorobut-2-ene is mutagenic in vitro and there are some indications for a possible clastogenic activity in vivo.

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

On the basis of the above exposed, it is proposed to classify 1,4-dichlorobut-2-ene as Muta. Cat. 3, R68; and Muta. Cat. 2, H431 according respectively to DSD and CLP criteria.