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Additional information

Valid experimental data were available to assess the genetic toxicity of the test substance in-vitro and in-vivo.



- Gene mutation in bacteria:

The test article was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA1538 at concentrations ranging from 10 to 5000 µg per plate (CIBA, 1990). The tests were conducted, using the plate incorporation method, on agar plates in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity. Positive control compounds demonstrated the sensitivity of the assay and the metabolising, potential of the S9 mix. No mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition. Precipitation was not observed. Toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in strain TA 1538 at 1000.0 and 5000.0 (µg/plate with metabolic activation and in strain TA 98 at 5000.0 µg/plate with and without metabolic activation.

- Chromosome aberration in mammalian cells:

The test substance was submitted for testing and evaluation of clastogenic potential. The test substance was assessed for its potential to produce structural chromosome aberrations in V79 cells of the Chinese hamster at concentrations of 0 (solvent "saline G"), 5, 50 and 100 µg/ml in the presence or absence of rat liver S9 mix (CIBA, 1990). In two parallel cultures 100 metaphases were scored at 7, 18,and 28 hours. In the absence of S9 mix no increase in the frequency of cells with aberrations was noted at any interval. In the presence of S9 mix, however, at fixation intervals 7 h and 28 h aberration rates which were statistically significantly different from the matched solvent controls were obtained at 50 µg/ml; at 18 h there was a slight incidence of cells with exchanges (1.5%) as compared to the matched control (0%). Although the test substance appeared to induce structural aberrations in the V79 test system in presence

of S9 mix, it is doubtful whether the results warrant the consideration that the test article is mutagenic in this chromosomal aberration assay, for the following reasons. The response observed is not clearly concentration-dependent. Treatment with 50 and 100 µg/ml was cytotoxic and reduced the plating efficiency of the V79 cells. Also the mitotic index was reduced after treatment at fixation intervals of 7 h and 18 h in the presence of S9 mix. Aberrant cells (excluding gaps) occurred in the solvent control at 2.5% and exchanges up to 1.0% in solvent controls without S9.

That the results obtained in vitro with V79 cells have no significance in vivo, where metabolic activation is expected to occur, is demonstrated by the negative outcome of the bone marrow chromosome aberration assays in rodents. In particular, the micronucleus assay in bone marrow cells of NMRI mice appears to be important in this respect. In this assay (MNT, CIBA 1990) the mice received a maximum tolerated oral dose of 5000 mg/kg the test substance which caused an increase in polychromatic erythrocytes in the bone marrow, without any increase in micronuclei of interphase cells. This result is significant in that no evidence of clastogenicity was obtained under conditions which produced a clear cytotoxic effect on the bone marrow cells. It is concluded therefore that the test substance is devoid of genotoxic potential in vivo.

in vivo:

- Chromosome aberration:

MNT: This study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (CIBA 1990). The test article was dissolved in NaCl-solution (0,9%). This solvent was used as negative control. The volume administered orally was 10 ml/kg b.w.. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w.. After treatment with the test article the number of NCEs per 1000 PCEs was enhanced as compared to the corresponding negative controls thus indicating that the test substance induced cytotoxic effects. In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at preparation intervals 24 hours and 72 hours after application of the test article. Biometric analysis demonstrated a statistically significant difference between control and test article data at preparation interval 48 hours. However, this biometric result is considered to be of no relevance, because the negative control value at this preparation interval was very low as compared to the actual negative control rates and in comparison to the historical laboratory control value. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. This data is supported by a second nucleus anomaly test done by CIBA in 1974 with Chinese hamsters.

Dominant Lethal Study: The compound was administered orally in single doses to male albino mice (NMRI-derived) which were then mated to untreated females over a period of six weeks (CIBA, 1976). At the end of each week the females were replaced by new ones. Doses of 500 and 1500 mg/kg were given. At day 14 of gestation females were examined. The females mated to males which had been treated with the compound did not differ from the females mated to controls, neither in mating ratio nor in the number of implantations and embryonic deaths (resorptions). No evidence of dominant lethal effect was observed in the progeny of male mice treated with the test substance.

Chromosome aberration test: The test substance was administered by gavage to male and female chinese hamsters (CIBA, 1974). Treatment consisted of one daily application on 2 consecutive days. 2 hours after the second application the animals were injected i.p. with colcemid (10 mg/kg) and sacrificed 4 h later. From the bone marrow chromosome preparations were made. The chromosome displays from the animals of the dosage groups showed no chromatid-type or chromosome-type aberrations. In the

control group one chromatid-type aberration per 400 metaphases (0.25 %) was found. This incidence is within the frequency observed in animals of the breed used and considered spontaneous in origin. By contrast, a "positive control" experiment with cyclophosphamide (64 mg/kg) yielded 23.5 % cells with chromatid-type aberrations and 0.5 % cells with chromosome-type aberrations. 8.8 % of the cells showed pulverizations. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamster treated with the test substance.

- DNA-damage/repair:

The test article was assessed in the in vivo/ in vitro UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats (RCC, 1991). The test article was formulated in Aqua bidest. After a treatment period of 4 and 16 hours, respectively, the animals were narcotized and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to ³HTdR which is incorporated if UDS occurs. The test article was tested at the following dose levels: 4 hour treatment period: 1000 mg/kg b.w. 16 hour treatment period: 100 and 1000 mg/kg b.w. For each dose level, including the controls, hepatocytes from three treated animals were assessed for the occurrence of UDS. No toxic reactions of the animals occured at any of the treatment periods or dose groups. In addition, neither the viability nor the in vitro attachment of the hepatocytes was dramatically affected due to the in vivo pre-treatment with the test article. No dose level of the test article revealed UDS induction in the hepatocytes of the treated animals as compared to the current negative controls. Neither the nuclear grains nor the resulting net grains were enhanced due to the in vivo treatment of the animals with the test article for 4 hours or 16 hours, respectively. An appropriate reference mutagen (2-AAF, 100 mg/kg b.w.) was used as positive control. In vivo treatment with 2-AAF revealed distinct increases in the number of nuclear and net grain counts. In conclusion, it can be stated that during the described study and under the experimental conditions reported, the test article did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats.

Short description of key information:
in vitro: Ames: negative, CIBA 1989, OECD TG 471
chromosome aberration: equivocal-probably secondary effect related to high cytotoxicity, CIBA 1990, OECD TG 473
in vivo: MNT: negative, CIBA 1990, OECD TG 474
UDS: negative, CIBA 1991
Dominant Lethal Study: negative, CIBA 1974
Chromosome aberration: negative, CIBA 1974

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genetic toxicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.


Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008.