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Key value for chemical safety assessment

Additional information

No relevant human data currently available.

In a well-conducted study, Chlorparaffin 56 (a C12 chlorinated paraffin; 57% chlorination) was assessed for its ability to induce gene mutations in the Ames test. Seven concentrations (ranging from 4 -5000 ug/mL) of the test substance were assessed in a plate incorporation assay with Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 and Eschericha coli WP2uvrA, both with and without a rat microsomal activation fraction (S9). No increase in mutant frequency was evident with the test substance, either with or without S9, compared to the untreated controls. The positive control substances gave the expected increases in the numbers of revertant colonies. Therefore, Chlorparaffin 56 showed no mutagenic potential in an Ames test for bacterial mutagenicity when tested up to 5 mg/plate in the absence or presence of S9 (Muller, 1988).

Negative results were also obtained in an Ames test using S. typhimurium strains TA97, TA 98, TA 100 and TA 1535, when a C12 chlorinated paraffin (60% chlorination) was tested at up to 3333 ug/plate, in the presence and absence of Aroclor-induced rat or hamster liver S9. This study employed a 20 minute preincubation period (NTP, 1986). [However, cytotoxicity was not observed and precipitation was not reported; therefore it is possible that the maximum concentration tested could have been increased further (up to 5000 ug/plate).]

 

Similarly, a C10-13 chlorinated paraffin (50% chlorination) did not produce an increase in revertants in S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1538, in the absence or presence of Aroclor-induced rat liver S9, when tested up to 2500 ug/plate (ICI, 1989; cited in Birtley et al. 1980 and EU, 2000). [As above, cytotoxicity was not observed and precipitation was not reported and hence the maximum concentration tested could have been increased.]

 

Cereclor 56L (a C10-13 chlorinated paraffin; 56% chlorination) at unspecified concentrations was negative in an Ames assay with five strains of S. typhimurium both in the presence and absence of a mammalian metabolic activation fraction (Elliot, 1989). Negative results were also claimed in another Ames test (ICI; 1977; cited in the EU, 2000), but the results of this study and the Elliot (1989) study provide very little experimental details, therefore there reliability cannot be assessed.

 

A C10-13 chlorinated paraffin (50% chlorination; containing 1% epoxy stabiliser) was assessed for its mutagenic potential in the Ames test with S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 and E. coli WP2uvrA. These bacterial strains were incubated with up to 10 mg/plate of the test substance in the presence or absence of a rat liver S9. Dose-related increases in mutant frequency occurred with S9 in TA100 and without S9 in TA100 and TA98. However, the increase in TA100 with S9 was just less than 2 -fold, and in TA98 without S9 only just reached 2 -fold. Furthermore, the presence of the epoxy stabiliser made it uncertain which substance was responsible for the increase (Hoechst AG, 1986; cited in EU, 2000). Overall it is not possible to draw firm conclusions from this study.

 

Chlorparaffin 56 flϋssig (a C10-13 chlorinated paraffin; 56% chlorination) was assessed for its mutagenic potential in mammalian cells using the HPRT assay. After a preliminary study to determine cytotoxicity of the test substance, it was tested at concentrations of up to 75 or 30 ug/mL with or without a rat liver metabolic activation system (S9), respectively, in cultures of Chinese hamster lung fibroblast (V79) cells. Mutants were selected by their resistance to 6-thioguanine. Two independent experiments were conducted. [According to an expert review (EU, 2000) on SCCPs, the study was well-conducted and carried out to modern protocols.] The test substance did not induce an increase in mutant frequency; cytotoxicity was evident at levels of 50 or 20 ug/mL with or without S9, respectively. In contrast positive control substances (not specified in the available abstract of the report) performed as expected, increasing the mutant frequency, thus demonstrating the validity of the assay. In conclusion, in a gene mutation (HPRT) study in Chinese hamster V79 cells, Chlorparaffin 56 flϋssig did not induce a significant, reproducible increase in mutant frequency at doses of up to 75 or 30 ug/mL with or without S9, respectively (Muller, 1987).

No in vitro cytogenetic studies on SCCPs in mammalian cells are available. In accordance with column 2 of REACH Annex VIII, an in vitro cytogenicity study in mammalian cells or an in vitro micronucleus study, do not usually need to be conducted if adequate data from an in vivo cytogenicity test are available. In this instance, two reliable in vivo mammalian cytogenicity tests (Muller, 1989; IRDC, 1982) have been performed with SCCPs. Both these in vivo bone marrow studies demonstrate that C10-13 chlorinated paraffins (58% chlorination) are not clastogenic at up to 5 g/kg bw to this sensitive target tissue. The available toxicokinetic data demonstrate that SCCPs undergo significant absorption following oral administration, with one study (Darnerud et al. 1982; see IUCLID Chapter 7.1.1 for further details) shows distribution to the bone marrow of three C12 chlorinated paraffins (17, 56 and 69% chlorination) following oral or intravenous administration to mice; indicating the reliability of these negative genotoxicity study results.

In a study conducted to a protocol equivalent to OECD Guideline 478, a C10-13 chlorinated paraffin (58% chlorination) was tested for its ability to induce dominant lethal mutations in the sperm of male Charles River COBS CD rats. Groups of 15 sexually mature male rats were treated with 0, 250, 750 or 2000 mg/kg bw/day (in corn oil) by gavage for 5 consecutive days followed 2 days later with the sequential mating with groups of 2 untreated virgin females to provide a total of 20 mated females per male. This was to cover all the various germ cell stages. The dams were sacrificed 15 days after pairing and the uteri examined for foetal deaths and viable embryos. No increase in foetal deaths or decrease in viable embryos were evident in the treated groups compared to the untreated controls. In contrast the positive control (cyclophosphamide) produced a dominant lethal mutation affecting the post-meiotic stage of spermatogenesis. Under the conditions of this study, a C10-13 chlorinated paraffin (58% chlorination) showed no evidence of germ cell mutagenicity in a dominant lethal assay in rats (IRDC, 1983).

Although not mutagenicity assays, the results of two cell transformation assays, using BHK21/C13 cells, have been summarised here for convenience. In the first, cells were treated, in the presence of Aroclor-induced rat liver metabolic activation, with up to toxic concentrations of a C10-13, 50% chlorinated paraffin (cited in Birtley et al.1980 and EU, 2000). There was no evidence of an increase in cell transformation frequency. The test was not conducted in the absence of metabolic activation mix. In contrast, increases in transformation frequency were obtained in the presence and absence of Aroclor-induced rat liver activation mix when cells were treated with Chlorowax 500C (a C12 chlorinated paraffin; 58% chlorination) (ICI, 1982; cited in EU, 2000). Large increases (5 to1000-fold) in the transformation frequency were obtained at both cytotoxic and nontoxic concentrations. The relationship between this effect and neoplastic activity of chlorinated paraffinsin vivois not clear.


Short description of key information:
No human data is currently available. Various C10-13 chlorinated paraffins (50-60% chlorination) have shown no evidence of mutagenic activity to bacteria (S. typhimurium and E. coli) in a number of Ames tests when tested at up to 5 mg/plate in the presence or absence of a mammalian liver activation system.

In a gene mutation (HPRT) study in Chinese hamster lung (V79) cells, a C10-13 chlorinated paraffin (56% chlorination) did not induce a significant, reproducible increase in mutant frequency at doses of up to 75 or 30 ug/mL with or without S9, respectively (Muller, 1987).

No standard in vitro cytogenetic studies are available, but two well-conducted in vivo studies (Muller, 1989; IRDC, 1982) suggest that C10-13 chlorinated paraffins (58% chlorination) do not produce mutagenicity in somatic (bone marrow). In addition, a C10-13 chlorinated paraffins (58% chlorination) showed no evidence of germ cell mutagenicity in a dominant lethal assay in rats (IRDC, 1983).

Overall, the data available and a consideration of the generally unreactive nature of these substances indicate that SCCPs (as a group) are not mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Under EU CLP and DSD regulations, SCCPs would not be classified as mutagenic based on the information described above.