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Additional information

In vitro

In a reverse gene mutation assay (95-0252-DGM), Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to 4 -Hydroxy TEMPO at concentrations of 0, 50, 160, 500, 1600, or 5000 µg/plate (vehicle: water) in the presence and absence of mammalian metabolic activation (S9 mix) in the standard plate test (plate incorporation method). In TA 100 and TA 1537 a weak mutagenic activity of the test compound was observed with metabolic activation. In the first experiment a weak mutagenic response was also seen in TA 100 without metabolic activation, but in the second experiment the mutagenicity without metabolic acitvation did not reach significance in this strain. A weak positve result in TA 98 with S9 in experiment 1 was not reproducible in the second experiment. The substance was therefore weakly mutagenic in the Ames test inducing base pair and frame shift mutations in the presence of rat liver S9 mix. The positive controls induced the appropriate responses in the corresponding strains. The study satisfies the requirements of Test Guideline OECD 471 for the evaluation of in vitro mutagenicity (bacterial reverse gene mutation).

Three published results of Ames tests (Gallez etal_1992, Sies and Mehlhorn_1986, Sosnovsky_1992) are listed in support for the weak mutagenicity of the test substance in the Ames assay (mutagenicity was not statistically significant in most instances).

In a forward gene mutation assay (95-0248-DGM), Chinese hamster ovary (CHO) cells were exposed to 4 -Hydroxy-TEMPO at concentrations of 0, 150, 450, 750, 1100 or 1500 µg/mL (test 1), or 0, 400, 700, 1000, 1300 or 1600 µg/mL (test 2) in the presence and absence of mammalian metabolic activation (S9 mix). Cell toxicity was determined in a pre-test at concentrations of 1800 µg/mL or above. The negative controls were consistent with the historical control data. The positive controls 3-methylcholanthrene (with S 9 mix) and ethyl methane sulfonate (without S9 mix) revealed the expected highly significant increases in mutation rates. Treatment of CHO cells with 4-OH-TEMPO in two independent experiments, both with and without S9 mix did not result in any reproducible biologically significant increase in the mutant frequency of the HPRT locus. There was no indication of a mutagenic effect of the test substance in this cell system. The study satisfies the requirements of Test Guideline OECD 476 for the evaluation of in vitro mutagenicity.

In vivo

In an in vivo mouse micronucleus assay (95-0250-DGM), male and female NMRI mice were orally (gavage) exposed to a single dose of 4 -Hydroxy TEMPO at a concentration of 1200 mg/kg bw (vehicle: saline solution). The maximum tolerated dose (MTD) was determined prior in a toxicity test. The post exposure period was 24 or 48 hrs. The following main signs of toxicity were observed: hunched posture, sedation, in males: staggering gait, convulsions. One male of the 48 h sampling group and one male of the satellite group died. No statistically or biologically significant increase in micronucleated polychromatic erythrocytes at the sampling times of 24 and 48 h was observed in male or female animals. No alterations of the ratio between polychromatic and normochromatic erythrocytes (PCE/NCE ratio) were detected. There was no indication of a clastogenic effect. The positive control induced the appropriate responses. The study satisfies the requirements of Test Guideline OECD 474 for the evaluation of in vivo mutagenicity (resp. clastogenicity: induction of micronuclei).

In an in vivo unscheduled DNA synthesis (UDS) assay (2003-6674-DGM), male rats were orally (gavage) exposed to a single dose of 4 -Hydroxy TEMPO at a concentration of 800 or 2000 mg/kg bw (vehicle: water). The maximum tolerated dose was determined prior in a toxicity test. The post exposure period was 24 or 48 hrs (experiment 1) or 2 to 4 hrs (experiment 2). No clinical signs of toxicity or loss of body weight were observed in the main study. The study was valid according to the criteria defined. The data indicate that treatment of male rats dosed once via oral gavage with 800 or 2000 mg/kg test substance did not result in increased UDS in hepatocytes isolated approximately 12-14 or 2-4 hours after dosing. The study satisfies the requirements of Test Guideline OECD 486 for the evaluation of in vivo mutagenicity (unscheduled DNA synthesis).


Justification for selection of genetic toxicity endpoint
Endpoints selection for genotoxicity from GLP compliant guideline studies: 95-0252-DGM and 95-0248-DGM (in-vitro) as well as 95-0250-DGM and 2003-6674-DGM (in-vivo).

Short description of key information:
The genetic toxicity in vitro of 4-Hydroxy TEMPO was tested in a GLP compliant OECD 471 guideline study (Ames test, classification: weakly mutagenic with metabolic activation), and in a GLP compliant OECD 476 guideline study (In vitro Mammalian Cell Gene Mutation Test, classification: not mutagenic). Thus, in conclusion the test substance is classified not mutagenic.
The genetic toxicity in vivo of 4-Hydroxy-TEMPO was tested in a GLP compliant OECD 474 guideline study (mouse micronucleus test, classification: not mutagenic) and in a GLP compliant OECD 486 guideline study (unscheduled DNA synthesis, classification: not mutagenic).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Although Ames tests results indicate a weak genotoxicity in the presence of a rat S9-mix in vitro, 4-Hydroxy TEMPO should not be subject to classification for mutagenicity according to EU Directive 67/548/EEC and according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008 due to the unanimously negative results in the HPRT assay as well as the negative results of the in vivo assays (mouse micronucleus test and unscheduled DNA synthesis).