Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: Negative (OECD TG 471, GLP)

Micronucleus assay: Negative (OECD TG 487, GLP)

Mouse Lymphoma Assay: Negative (OECD TG 490, GLP)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames test

The test substance was evaluated in a bacterial reverse mutation assay performed according to OECD 471 and following GLP using Salmonella typhimurium strains TA98, TA100, TA 1535 and TA1537 and Escherichia coli strain WP2 uvrA both in the presence and absence of an exogenous metabolic activation system (S9-mix). The test substance was evaluated using an initial plate incorporation assay and a confirmatory preincubation procedure in triplicate with and without metabolic activation. In the first experiment, test substance concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate were assessed. In the second experiment, test substance concentrations of 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate (All Salmonella strains), and 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate (WP2uvrA) were assessed. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence and presence of metabolic activation, initially from 500 μg/plate. In the second experiment, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of metabolic activation, initially from 50 μg/plate. In the presence of metabolic activation, the lowest test item concentration at which toxicity was initially observed was strain dependant and started from 150 μg/plate. No test item precipitate was observed on any of the plates. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. One statistically significant value was noted (TA98 at 50 μg/plate in the presence of metabolic activation (S9-mix)). This value was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. Therefore, the test material was considered to be non-mutagenic under the conditions of this test.

Micronucleus assay

The test substance was evaluated in a micronucleus assay performed according to OECD 487 and following GLP using human lymphocytes both in the presence and absence of an exogenous metabolic activation system (S9-mix). Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle (quadruplicate cultures) and positive controls (duplicate cultures). Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the main experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the main experiment were 0, 16, 32, 64, 72, 80, 96, 128 µg/mL (4- and 24- hour exposure without S9) and 0, 32, 64, 96, 128, 160, 192, 256 µg/mL. All vehicle (dimethyl sulphoxide (DMSO)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item was toxic to human lymphocytes but did not induce any toxicologically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced approximately 50% reduction in CBPI. The test item, Terpinyl acetate (multi)was considered to be non-clastogenic and non‑aneugenic to human lymphocytes in vitro.

Mouse lymphoma assay

The test substance was evaluated in a mouse lymphoma assay performed according to OECD 490 and following GLP using the L5178Y mouse lymphoma cell lineboth in the presence and absence of an exogenous metabolic activation system (S9-mix). One main Mutagenicity Test was performed. In this main test, cells were treated with the test item at ten dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels selected for the main experiment were 0, 7.5, 15, 30, 35, 40, 45, 50, 55, 60, and 65 µg/mL (4- and 24-hour exposure without S9) and 0, 7.5, 15, 30, 60, 70, 80, 90, 100, 110, and 120 µg/mL (4-hour exposure with S9). The maximum dose levels in the main test were by test item-induced toxicity in all three of the exposure groups, as recommended by the guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the cell line. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that achieved optimum levels of toxicity in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, and approached or exceeded optimum levels of toxicity in the 4-hour exposure group in the presence of metabolic activation. The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor of 126E-6, consequently it is considered to be non-mutagenic in this assay.

Justification for classification or non-classification

Based on the negative results of the Ames test, the micronucleus assay and the mouse lymphoma assay, Terpinyl Acetate multi does not need to be classified for genotoxicity according to EU CLP (EC No. 1272/2008 and its amendments).