Sodium 5-N-butylbenzotriazole

Endpoint summary

Additional information

In vitro Bacterial Mutation Assay

In this in vitro assessment of the mutagenic potential of Butyl benzotriazole-sodium salt, Slamonella typhimurium (strains TA 1535, TA1537, TA1538, TA 98 and TA 100) were exposed to the test substance, diluted in water whixh was also used as negatice control.

Tests were performed, in presence and abscence of metabolic activation system (Aroclor 1254 induced rat liver homogenate S-9). In the preliminary toxicity test, toxicity was observed at dose levels upper than 370 µg/plate of Butyl benzotriazole-sodium salt (1% of Butyl benzotriazole-sodium salt 37% solution). A top dose level of 370 µg/plate of Butyl benzotriazole-sodium salt was chosen for the mutation study. Other dose levels used in the mutation assays were 111, 37, 11.1 and 3.7 µg/plate of Butyl benzotriazole-sodium salt (respectively 0.3, 0.1, 0.03 and 0.01% of Butyl benzotriazole-sodium salt 37% solution)

No evidence of mutagenic activity was seen at any dose level of Butyl benzotriazole-sodium salt ineither mutation test.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the the metabolising activity of the liver preparation.

It is'concluded that no evidence of mutagenic potential of Butyl benzotriazole-sodium salt was obtained in this bacterial test system at the dose

In vitro Metaphase Chromosome Analysis

Butyl benzotriazole-sodium salt was tested in vitro to determine whether it would cause chromosomal aberrations in a mammalian cell Iine derived from Chinese hamster ovary tissue. The celIs were routinely grow and subcultured in tissue culture medium at 37°C in a humid atmosphere containing 5% carbon dioxide. They were incubated with a range of concentrations of the test compound together with solvent control and posltive control compounds both with and without supplementary metabolic activation (rat S-9 mix).

The mitotic indices of the cultures treated with the test compound and solvent control were calculated. On the basis of these data the dose levels chosen for the metaphase analysis were 30.8, 15.4, 7.7 and 3.8 %g/ml in the absence of metabolic activation and 125, 100, 50 and 20 µg/ml in its presence.

In the absence of metabolic activation butyl benzotriazole-sodium salt caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level wben compared with the solvent control. However, in the presence of metabolic activation statistically signiflcant increases were observed at the highest two dose levels, 100 and 125 µg/ml (P < 0.01 and P < 0.001, respectívely). The values of 10.01 and 10.5% excluding and including gap damage, respectively, at 100 µg/ml and 11.5% and 12.0% excluding and including gap damage, respectively, at 125 µg/ml, lie outside these historical control range observed in this laboratory i.e. 0 to 5.5% excluding gap damage and 0 to 6.5% including gap damage.

Both positive control compounds caused Iarge statistically hiqhly significant increases in chromosomal damage, demonstrating the sensitivity of the test system and the efficacy of the S-9 mix.

It is concluded that butyl benzotriazole-sodium salt has shown evidence of clastogenic activity in the presence of metabolic activation but not in its absence.

In vivo mammalian cell gene mutation test :

In this assessment of the effect of butyl benzotriazole-sodium salt on the incídence of micronucleated polychromatic erythrocytes in mice, a dosage of 600 mg/kg bodyweight was administered as a single oral dose by intragastric gavage. (A preliminary toxicity test had indicated that the maximum tolerable dosage of butyl benzotriazole-sodium salt was approxÍmately 600 mg/kg).

Negative and positive control groups were dosed in an identical manner, orally by intragastric gavage. The negative control group received the vehicle, distilled water. The positive control group was treated with mitomycin C, at 12 mg/kg bodyweight.

Bone marrow smears were obtained from the negative control and test compound groups at 3 sampling times; these being 24,48 or 72 hours after dosing. Bone marrow smears were obtained from the positive control group 24 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

At all sampling times, mice treated with butyl benzotriazole-sodium salt did not show any significant increase in the frequency of micronucleated polychromatic erythrocytes.

There was no significant decrease in the ratio of polychromatic to normochromatic erythrocytes after treatment of the animals with

butyl benzotriazole-sodium salt.

The positive control compound, mitomycin C, produced large, highly signifícant increases in the frequency of micronucleated polychromatic erythrocytes together with decreases in the ratio of polychromatic to normochromatic erythrocytes.

It is concluded from the results obtained that butyl benzotriazole-sodium salt shows no evidence of mutagenic potential or bone marrow cell toxicity when administered as a single oral dose in this in vivo test procedure.

Justification for selection of genetic toxicity endpoint
One study can not be selected as there are 2 in vitro and one in vivo key studies available.

Short description of key information:
Bacteria reverse mutation assay: perforemed according to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100. Negative Ames (with and without metabolic activation)
In vitro mammalian chromosome aberration test: performed according to OECD Guidance 473 in Chinese Hamster Ovary cells . Positive in vitro clastogenic activity in the presence of metabolic activation, but not in its absence.

In vivo mammalian cell gene mutation test : performed according EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test) and according to OECD Guideline for Testing of chemicals No. 474 ,Genetic Toxicology : Micronucleus Test" (1982).
Negative in vivo bone marrow micronucleus

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The positive result in the in vitro cytogenetics assay gives some cause for concern with respect to mutagenicity. In accordance with the testing strategy, a bone marrow micronucleus test has been conducted. A negative result was obtained.

According to EC criteria, the notified substance need not be classified as germ cell mutagenis.