Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

In vitro:

In vitro bacterial cells - Gene mutations

The key study for gene mutations in bacterial cells is an Ames reverse mutation test (K1). The test substance was subjected to the Ames test according to the procedure described in the original paper by Ames et al. (1975). Test concentrations were 0.1, 1.0, 10.0, and 500.0 µg/plate. S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were used. Through the use of positive controls, the bacterial strains were shown to be active. Increased numbers of revertants were seen in TA100 strains in replicate assays. This activity was observed only in the presence of S9 activating enzymes. All other strains were negative, both with and without metabolic activation.

Two supporting (K2) Ames studies (one performed with Santowax R and one with a mixture of quaterphenyls and higher polyphenyls) - in which the same strains and concentrations as in the key study were used - showed no mutagenic activity in any of the strains, both with and without metabolic activation. Additionally in these tests a Saccharomyces cerevisiae D4 strain and a S. typhimurium TA 1538 strain were subjected to the same test conditions, and showed no gene mutation effect both with and without metabolic activation.

Based on the overall results from these three tests the test substance was not shown to be genotoxic in the majority of the tests. Without metabolic activation, no genotoxicity was observed in any of the tests, with metabolic activation genotoxicity was observed in one strain (TA100) in one of the tests. Therefore, no definitive conclusion on gentotoxicity can be drawn based on the results of these Ames tests alone.

In vitro mammalian cells – Gene mutations

The key study for gene mutations in mammalian cells is a GLP (OECD guideline 476) , K1 study addressing forward gene mutation effects on Chinese Hamster Ovary (CHO) cell lines targeting the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. Tested concentrations were 5, 25, 50, 75 and 100 µg/ml, both with and without metabolic activation. The cytotoxicity of the test substance was tested at 8 doses ranging from 0.3 to 1000 µg/ml of media, which was well above the solubility limit of the test substance. Cell lines treated with test substance did not show increased mean mutation frequencies both with and without metabolic activation. The test substance as a consequence is not considered to introduce gene mutations in mammalian cells according to the described study protocol.

The supporting study (K2) evaluated the same endpoints as the key study, but used a substance with a slightly different composition (Santowax R). This supporting study completely supported the findings of the key study, no gene mutations in mammalian cells were observed.

In vitro mammalian cells – Chromosome aberration

A GLP in vitro chromosome aberrations test was performed according to the OECD 473 guideline. Chinese hamster ovary cells were exposed to 37.5, 75.0 or 150.0 µg/ml of test substance for 24 hours in the absence of metabolic activation or 2 hours with metabolic activation. Cytotoxicity was evaluated by exposing cells in culture in a range-finder test with concentrations ranging from 9.4 to 150.0 µg/ml. The doses chosen for the cytogenetic assay were based upon the findings from this cytotoxicity test. The cells were microscopically evaluated for mitotic indices and for chromosomal aberrations. The chromosome aberrations test showed no significant differences between the solvent control and the cells treated with test substance. As a consequence, the outcome of the test is negative and the test substance can be considered to have no clastogenic effects in Chinese hamster ovary cells.

In vitro mammalian cells - DNA damage

DNA damage is studied via the in vitro Unscheduled DNA-synthesis test (UDS). Two K2 tests were conducted according to GLP guidelines and similar to OECD guideline 482. One was performed with Santowax R and one with Therminol 75. Hepatocytes from male Fischer 344 rats were treated with the test substance. The outcome of the UDS tests were negative for both substances since the test substance was unable to induce DNA-damage in rat hepatocytes.

Conclusion in vitro results:

Based on all available in vitro tests, the substance does not appear to be genotoxic.


In vivo:

One K1 in vivo micronucleus test is available. The test is conducted according to GLP and equivalent to OECD Guideline 475. Groups of 6 animals per sex per dose per time of sacrifice received a single intraperitoneal dose of test substance at 500, 2500 and 5000 mg/kg bw. Groups of rats were sacrificed 6, 12 and 24 hours after treatment. At 6, 12 and 24 hour post-treatment, no significant differences in mitotic index, % of chromosomally aberrant cells and frequency of chromosomal aberrations per cell were observed between the group treated with the test substance and the negative control group. It can thus be concluded that the test substance does not induce chromosomal damage in this in vivo micronucleus test.


Overal conclusion in vitro and in vivo:

Based on all tests results from in vitro and in vivo studies it could be concluded that the substance is not genotoxic by any of the mechanisms described.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification