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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In two reverse gene mutation assays in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, delta-3 -carene was found as non mutagenic in the presence or absence of metabolic activation.

In a study conducted according to OECD Guideline 476 in compliance with GLP (in vitro HPRT cell mutation assay) delta-3 -carene did not demonstrate any mutagenic potential.

In a study performed according to OECD Guideline 487 in compliance with GLP delta-3 -carene did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a reverse gene mutation assay in bacteria, performed according to Guideline OECD 471 and in compliance with GLP, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant ofEscherichia coli, strain WP2uvrA (pKM101), were exposed to the test item diluted in DMSO at the concentrations below.

First Test (Plate incorporation method):

5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA (pKM101), with and without S9-mix

Second Test (Pre-incubation method):

5, 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA (pKM101), with and without S9-mix

Additional Second Test (Pre-incubation method):

0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98, TA 100 and TA 1535 strains, without S9-mix

0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 1537 and WP2 uvrA (pKM101) strains, without S9-mix

0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100 and TA 1535 strains, with S9-mix 

Metabolic activation system used in this test is S9 mix (10% v/v S9 fraction): S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

In the first test, toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or with a reduction in revertant colony numbers) was seen in strains TA98, TA100, TA1535 and WP2uvrA (pKM101) following exposure to the test item at 5000 μg/plate and in strain TA1537 at 1500 μg/plate and above in the absence of S9 mix. In the presence of S9 mix, toxicity (observed as thinning of the background lawn of non revertant colonies, and/or with a reduction in revertant colony numbers) was seen in strains TA98, TA100 and TA1535 at 5000 μg/plate and in strains TA1537 and WP2uvrA (pKM101) at 1500 μg/plate and above.

In the second test in the absence of S9 mix, toxicity (observed as a reduction in revertant colony numbers and/or thinning of the background lawn of non-revertant colonies) was seen in strains TA98, TA100 and TA1535 at 50 μg/plate and above, and in strains TA1537 and WP2uvrA (pKM101) at 150 μg/plate and above. In the presence of S9 mix, toxicity (observed as a reduction in revertant colony numbers and/or thinning of the background lawn of non-revertant colonies) was seen in strain TA1535 at 15 μg/plate and at 500 μg/plate and above, in strains TA98 and TA1537 at 500 μg/plate and above, in strain TA100 at 150 μg/plate and above, and in strain WP2uvrA (pKM101) at 1500 μg/plate and above. As the required four non-toxic concentrations were not achieved in any of the strains in the absence of S9 mix, or in strains TA100 and TA1535 in presence of S9 mix, an additional test was performed using modified dose concentrations.

In the additional test in the absence of S9 mix, toxicity (observed as a reduction in revertant colony numbers and/or thinning of the background lawn of non-revertant colonies) was seen in strains TA98, TA100 and TA1535 at 50 μg/plate and above, in strain TA1537 at 150 μg/plate and above, and in strain WP2uvrA (pKM101) at 500 μg/plate. In the presence of S9 mix, toxicity (observed as a reduction in revertant colony numbers and/or thinning of the background lawn of non-revertant colonies) was observed in strain TA100 at 150 μg/plate and above, and in strain TA1535 at 500 μg/plate.

No evidence of mutagenic activity was seen at any concentration of the test item in any mutation test. The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed tothe test itemin the presence and absence of a metabolic activation system. Metabolic activation system used in this test was 10% (v/v) S9 fraction; S9 fraction was obtained from the liver homogenates of male Sprague-Dawley derived rats induced with phenobarbital and 5,6-benzoflavone.

 Preliminary Toxicity Test: 1.33, 2.66, 5.32, 10.64, 21.29, 42.57, 85.14, 170.29, 340.58 and 681.15 μg/mL; 3 h exposure with and without S9-mix; 20 h continuous exposure without S9-mix

Main Test

3 h exposure to the test item formulations without S9-mix: 2, 20, 40, 50, 60, 65, 70, 75, 80, 90 and 100 μg/mL

3 h exposure to the test item formulations with S9-mix: 60, 120, 180, 220, 260, 300, 340 and 680 μg/mL

20 h continuous exposure to the test item without S9-mix: 2, 20, 35, 40, 45, 50, 55 and 60 μg/mL

Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.

Preliminary toxicity test.In all exposure conditions the highest concentration tested was 681.15 μg/mL and precipitate was observed by eye at the end of treatment at 681.15 μg/mL. After 3 h treatment in the absence of S9-mix, a reduction in CBPI compared with vehicle control values, equivalent to 64.5% cytotoxicity, was obtained with the test item at 85.14 μg/mL. At higher tested concentrations overt toxicity was observed. After 3 h treatment in the presence of S9-mix, a reduction in CBPI compared with vehicle control values, equivalent to 58.8% cytotoxicity, was obtained with the test item at 681.15 μg/mL. After 20 h treatment in the absence of S9-mix, a reduction in CBPI compared with vehicle control values, equivalent to 41.7% cytotoxicity, was obtained with the test item at 42.57 μg/mL. At higher tested concentrations overt toxicity was observed. These results were used to select concentrations for the main test.

Main test. Following 3 h treatment in the absence of S9 mix, a reduction in CBPI equivalent to 51.5% cytotoxicity was obtained with the test item at 75 μg/mL. Concentrations of the test item selected for micronucleus analysis were 2, 60 and 75 μg/mL. Following 3 h treatment in the presence of S9 mix, a reduction in CBPI equivalent to 58.1% cytotoxicity was obtained with the test item at 180 μg/mL. Concentrations of the test item selected for micronucleus analysis were 60, 120 and 180 μg/mL. In the absence of S9 mix following 20 h treatment, a reduction in CBPI equivalent to 52.6% cytotoxicity was obtained with the test item at 40 μg/mL. Concentrations of the test item selected for micronucleus analysis were 2, 20 and 40 μg/mL.

In both the absence and presence of S9 mix, following 3 h treatment, and in the absence of S9 mix, following 20 h treatment, test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls.

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed todelta-3-carenefor 3 h, with and without metabolic activation (10% v/v S9); S9 fraction prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone.

Preliminary toxicity test: 10.64, 21.29, 42.57, 85.14, 170.29, 340.58, 681.15 and 1362.3 μg/mL, 3 h exposure without and with metabolic activation 

Mutation tests:

3 h exposure without metabolic activation: 25, 50, 55, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5 and 80 μg/mL

3 h exposure with metabolic activation: 21, 42, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118, 124 and 130 μg/mL 

Additional Mutation tests:

3 h exposure without metabolic activation: 6.5, 13, 26, 28, 30, 32, 34, 36, 38 and 40 μg/mL

In apreliminary toxicity test, precipitate was observed by eye at the end of treatment at concentrations of 170.29 μg/mL and above and this was, therefore, the highest concentration plated for determination of relative survival (RS) in both the absence and presence of S9 mix. Exposure to the test item for 3 h at concentrations from 10.64 to 170.29 μg/mL in both the absence and presence of S9 mix resulted in RS values from 123 to 0% and 100 to 1% respectively. Concentrations for the main test were based upon these data.

In the additional main mutation test in the absence of S9 mix, cells were exposed to the test item at concentrations from 6.5 to 40 μg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 92 to 19% when compared with the vehicle control and the test item did not induce a statistically significant increase in mutant frequency.

In the main mutation test in the presence of S9 mix, cells were exposed to the test item at concentrations from 21 to 130 μg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 92 to 0% when compared with the vehicle control and the test item did not induce a statistically significant increase in mutant frequency.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.

Justification for classification or non-classification

In two reverse mutation assays in bacteria (Ames test) performed according to OECD Guideline 471 and in compliance with GLP, delta-3 -carene was not mutagenic in S. typhimurium strains TA1535, TA 1537, TA 98 and TA100 and in Escherichia coli strain WP2 uvrA (pKM101) in presence and absence of metabolic activation, up to limit or cytotoxic concentrations.

The resultst were also negative in a chromosome aberation test performed in human lymphocytes according to OECD Guideline 487 and in compliance with GLP, in presence and absence of metabolic activation.

 

In addition, negative results were obtained in a gene mutation test (HPRT) in CHO cells, performed according to OECD Guideline 476 and in complinace with GLP, in presence and absence of metabolic activation.

 

As the results obtained with delta-3 -carene were negative in two Ames tests, in a gene mutation test (HPRT) in CHO cells and in one chromosome aberration tests (in Human lymphocytes), the test item is not classified according to CLP Regulation (EC) n° 1272/2008.