Bis(1,2,2,6,6-pentamethyl-4-piperidyl) [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]butylmalonate

Endpoint summary

Additional information

The substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium (Ciba-Geigy 1979). The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 22 5, 675 and 2025 μg

/0.1 ml. Acetone was used as vehicle. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes from arochlor 1254 treated animals and co-factors). In the experiments on strains TA 98, TA 1535 and TA 15 37 without microsomal activation, comparison of the numbers of back-mutant colonies in the controls and the cultures treated with the various concentrations revealed a reduction in the colony count due to a growth-inhibiting effect of the compound at the concentrations of 225

μ

g/0.1 ml and above. This effect was also observed in the experiments with microsomal activation on Strains TA 98 and TA 1537 at the concentrations of 675 and 2025

μ

g/0.1 ml. The test is valid with restrictions because only four tester strains were used and because no independent repeat experiment was performed. Suitable positive and negative control incubations were included. The test substance was found to be non mutagenic in bacteria.

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro in a GLP compliant study following OECD testing guideline 476 (BASF 2012a). The study is therefore valid without restriction. Three independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated in this study: 1st Experiment

without S9 mix (4-hour exposure period)

0; 0.016; 0.031; 0.063; 0.125; 0.25; 0.5; 1.0; 2.0 μg/mL

with S9 mix (4-hour exposure period)

0; 0.313; 0.625; 1.25; 2.5; 5.0; 10.0; 20.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period)

0; 0.008; 0.016; 0.031; 0.063; 0.125; 0.25; 0.5; 1.0 μg/mL

with S9 mix (4-hour exposure period)

0; 0.938; 1.875; 3.75; 7.5; 15.0; 20.0 μg/mL

3rd Experiment (24-hour exposure period)

0; 0.063; 0.125; 0.25; 0.5; 1.0; 2.0; 4.0; 8.0 μg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the

frequencies of forward mutations. In all experimental parts in the absence and presence of metabolic activation evaluated for

gene mutations the highest concentrations were clearly cytotoxic. Due to lacking cytotoxicity the 2nd Experiment in the absence of metabolic activation was discontinued.On the basis from the results of the present study, the test substance did not cause any

relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in three experiments performed independently of each other.

The substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the absence and the presence of a metabolizing system. The study was performed according to OECD guideline 473 and under GLP. It is therefore valid without restrictions.

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiment, the following doses were tested and the test groups in bold type were evaluated:

1st Experiment

4-hour exposure, 18-hour sampling time, without S9 mix

0; 0.391; 0.781; 1.563; 3.125; 6.25; 12.5; 25; 50 μg/mL

4-hour exposure, 18-hour sampling time, with S9 mix

0; 0.391; 0.781; 1.563; 3.125; 6.25; 12.5; 25; 50 μg/mL

2nd Experiment

4-hour exposure, 18-hour sampling time, without S9 mix

0; 0.049; 0.098; 0.195; 0.391; 0.781; 1.563; 3.125 μg/mL

3rd Experiment

18-hour exposure, 18-hour sampling time, without S9 mix

0; 0.049; 0.098; 0.195; 0.391; 0.781; 1.563; 3.125 μg/mL

18-hour exposure, 28-hour sampling time, without S9 mix

0; 0.049; 0.098; 0.195; 0.391; 0.781; 1.563; 3.125 μg/mL

4-hour exposure, 28-hour sampling time, with S9 mix

0; 0.391; 0.781; 1.563; 3.125; 6.25; 12.5; 25 μg/mL

A sample of 100 metaphases for each culture was analyzed for chromosomal aberrations, except for the positive control cultures and the highest dose groups scored at 28 hours sampling time with and without S9 mix (3.125 and 0.781 μg/mL, respectively) where only

50 metaphases were scored due to clearly increased aberration rates. The vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance caused a statistically significant and biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps at 28 hours sampling time in the absence and presence of a metabolizing system. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either. Thus, under the experimental conditions described, the substance is considered to have a chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation at prolonged 28 hours sampling time.

As a follow-up study to the postive in-vitro clastogenicity test, a micronucles study in vivo in mice was performed. The study followed OECD guideline 474 and was performed under GLP. The test item was suspended in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w. The volume of the positive control administered was 10 mL/kg.

24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Seven males per test group were evaluated for the occurrence of micronuclei except for the negative and positive control groups with five animals each. Per animal 4000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg b.w..
48 h preparation interval: 2000 mg/kg b.w..

The highest dose (2000 mg/kg b.w.; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

Clinical symptoms in the main experiment included ruffled fur, reduced spontaneous activity and eyelid closure in the animals treated with the high and mid dose of test item. Additional diarrhoea was observed for particular animals. The animals treated with the low dose showed ruffled fur only. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that Tinuvin 144 did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

The observed systemic toxicity at the tested doses is indicative for a systemic distribution of the test item. Thus, bioavailability of the test item under the tested conditions is assumed. In addition, plasma analysis was performed at the laboratories of the sponsor.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. The substance is considered to be non clastoenic in vivo.

Justification for selection of genetic toxicity endpoint
This the highest TIER study.

Short description of key information:
Ames (non-GLP, similar to OECD 471) and hprt (OECD 476, GLP): non mutagenic (Ciba-Geigy 1979 and BASF 2012a). Chromosome aberration in vitro (OECD 473, GLP): clastogenic (BASF 2012b); Micronucleus test in vivo (OECD 474, GLP) negative (BASF 2015)

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. The substance is not mutagenic in bacteria and in cultivated mammalian cells. It was positive in an in-vitro clastogenicity assay, but the in-vivo clastogenicity assay was negative. As a result the substance is not considered to be classified for mutagenicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

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.

The substance is not mutagenic in bacteria and in cultivated mammalian cells. It was positive in an in-vitro clastogenicity assay, but the in-vivo clastogenicity assay was negative.

As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive EC944/2013.