Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

GENETIC TOXICITY IN VITRO

 

A summary of data on genotoxicity in vitro is presented in Section 4.1.2.7.1 of the EU-RAR (2001, page 61ff).

The key studies for this endpoint are nearly identical to the ones selected in EU RAR (see robust study summary in IUCLID Section 7.6.2). However, the study is presented in IUCLID data set under name CMA (Chemical manufactures Association), while EU RAR cited the name of the study director.

Only for gene mutation in bacteria and in vivo micronucleus test recent studies performed by NTP have been selected.

 

Bacterial test systems detecting gene mutations

The key study for this endpoint was the Ames-Test recently performed by NTP (2009, see robust study summary in IUCLID Section 7.6.2). In this guideline study according to OECD 471 using Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 at concentrations 0, 1, 3, 10, 33, 100, 166 and 333 µg/plate cumene was found to be negative, both with and without metabolic activation system (S9 mix).

 

Studies in EU RAR

Lawlor and Wagner (1987, cited in EU RAR, Note: Study is presented in IUCLID data set under name CMA) tested cumene in an Ames test using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at concentrations 33, 67, 100, 333, 667, 1000 and 2000 µg/plate. The assay was carried out with and without metabolic activation system (S9 mix). At 2000 μg/plate was a decrease in revertant colony. Under the conditions of the study, cumene did not cause a positive response, with or without metabolic activation, in any of the tested strains.

In addition, Florin et al. (1980) in a screening of tobacco smoke constituents for mutagenicity using the Ames' test, cumene was assayed qualitatively using strains TA98, TA100, TA1535 and TA1537 with and without with and without metabolic activation system (S9 mix), at 3μmol/plate and the results were negative. Cumene was tested quantitatively at concentrations used of 0.03, 0.3, 3 and 30μmol/plate using strains TA98 and TA100 with and without metabolic activation system (S9 mix). Cumene not found to be mutagenic and was toxic to the bacteria at concentrations 3μmol/plate.

 

Chromosome mutations in mammalian cells

A Chromosome Aberrations study in Chinese Hamster Ovary (CHO) cells was carried out by Putman (1987a, cited in EU RAR, Note: Study is presented in IUCLID data set under name CMA). This assay was conducted both with and without metabolic activation system (S9 mix) at 6 cumene dose levels from 20 to 225 µg/mL. Toxicity was observed at the high dose tested in each treatment condition (with and without S9 mix).

Under the conditions of the assay, cumene did not induce structural or numerical chromosome aberrations in CHO cells when tested in the absence of an exogenous metabolic activation system. A statistically significant increase in number of aberrations per cell was observed at 156 µg/mL in the presence of S9 mix when compared to the vehicle control; however, a statistically significant increase was not observed when compared to the historical control range. This increase was considered to be due to the low F127 control values and not an increase in the cumene-treated cultures. Cumene was concluded to be negative in the CHO chromosome aberrations assay.

 

Gene mutations in mammalian cells

A gene-mutation (HGPRT) study in Chinese Hamster Ovary (CHO) cells was carried out by Yang (1987, cited in EU RAR, Note: Study is presented in IUCLID data set under name CMA). Six dose levels from 100 to 225 µg/mL were selected for mutagenicity assays in the absence and presence of S9 mix. Treatment period of 18 hours was decided for the non-activated portion of the mutation study.

The assay at dose levels of 100-125 µg/ml yielded relative cloning efficiencies of 29 - 110%. Dose levels of 225, 200, 175 and 150 µg/ml yielded relative cloning efficiencies of <10%.

None of the mutant frequencies of cumene treated groups were increased significantly above the controls. The activity of cumene in the mutation assay after a 5 hours treatment in the presence of S9 is not increased significantly above the controls.

The positive and negative controls fulfilled the requirements for a valid test.

 

Aneuploidy in mammalian cells

No information regarding this endpoint is available

 

Assays indicating DNA damage

Cumene was tested in the Unscheduled DNA Synthesis Test using rat primary hepatocytes (Curren 1987, cited in EU RAR, Note: Study is presented in IUCLID data set under name CMA).

Based on the results of the initial UDS test and cytotoxicity test, cumene was tested at thirteen dose levels ranging from 1 to 128 µg/ml and was fully evaluated at six dose levels of 1.0, 2.0, 4.0, 8.0, 16.0 and 24.0 µg/mL. Doses of test article >24 µg/mL proved too toxic for evaluation for UDS.

After eighteen to twenty hours of exposure, cells were evaluated for UDS. The results of the UDS assay indicate that under the test conditions, the cumene did not cause a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the control), at any dose level. Therefore, the cumene is considered negative in this study. The positive control, 2-acetylaminofluorene (2-AFF), induced significant increases in the mean number of rat nuclear grain counts over that in the solvent control.

 

Other in vitro investigations

The key study for this endpoint is identical to the one selected in EU RAR (see robust study summary in IUCLID Section 7.6.2). However, the study is presented in IUCLID data set under name CMA (Chemical manufactures Association), while EU RAR cited the name of the study director.

 

Although not mutagenicity assays, a cell transformation assay, using BALB/3T3 Mouse Embryo cells in the absence of exogenous metabolic activation was conducted by Putman (1987b, cited in EU RAR, Note: Study is presented in IUCLID data set under name CMA).

The toxicity test was performed for the purpose of selecting dose levels for the transformation assay and was based upon colony-forming efficiency.

Cells were exposed to nine doses from 50 to 500 µg/mL as well as medium, solvent and positive controls for 3 days at 36+/-1ºC. After 7 - 10 days from initiation of treatment, the concurrent cytotoxicity dishes were fixed, stained and scored for colony formation. After 4 - 6 weeks incubation from initiation of treatment, the transformation dishes were scored for morphologically transformed Type II and Type III foci.

Treatment with cumene at concentrations of 250 - 500 µg/mL was completely toxic to BALB/3T3 cells. The four lowest concentrations tested (50, 100, 150 and 200 µg/mL) yielded survival levels of 102%, 87%, 19% and 4% respectively. Although survival at 200 µg/mL was low (4%), the cell monolayer did reach confluence and was scored for morphologically transformed foci.

No increase in Type III foci were observed in the cumene treated cells compared to the vehicle (F127)-treated cells. Cumene was concluded to be negative in the BALB/3T3 cell transformation.

 

GENETIC TOXICITY IN VIVO

The key studies for this endpoint were the Micronucleus-Tests recently performed by NTP (2009, see robust study summaries in IUCLID Section 7.6.). In these guideline studies according to OECD cumene was found to be negative. Although the authors judged the two independent tests after ip injection positive, this vote is not supported by the applicant after detailed review of the test results as well as one external reviewer of the NTP report. The background level in the control was relatively low, there was no dose-response relationship, and in one test also the trend was insignificant. In addition, the increase in MN/PCE was small, but significant in only one or two doses. The investigation after inhalation exposure in mice (in course of the 90 day inhalation study) was negative.

As supporting study, the study cited in EU RAR was used. Cumene was tested in micronucleus test in CDR-1 (ICR) BR Swiss mice (Gulf Oil Corporation 1985b, cited in EU RAR). Cumene dissolved in 5 g - % in paraffin oil was administered by gavage for an exposure period of 2 days. Half of the animals were sacrificed on day 3 and the remaining on day 4; 1000 polychromatical erythrocytes and all mature erythrocytes were examined for each animal; 10 mice/sex/group.

In micronucleus assay, done according to GLP standards in bone marrow, cumene did not affect the ratio of polychromatic to normochromatic erythrocytes nor increase the frequency of micronucleated erythrocytes at doses of 250, 500 and 1000 mg/kg bw d; therefore, indicating no potential for clastogenicity in vivo.

 

The overall conclusion taking into account all findings as a weight of evidence is that cumene is not considered to be positive for in vivo mutagenicity, based on the results of the Micronucleus-Tests presented.

Short description of key information:
In vitro test systems
Cumene was found to be not mutagenic in bacterial gene mutation tests. Cumene did not induce structural or numerical chromosome aberrations as well as Chromosome mutations in mammalian cells. A test for induction of aneuploidy is not available. Assays indicating DNA damage revealed that cumene was negative when testing in the Unscheduled DNA Synthesis Test using rat primary hepatocytes.
Although not mutagenicity assays, a cell transformation assay, using BALB/3T3 Mouse Embryo cells, performed with cumene was negative.


In vivo test systems
There was a weak positive result in micronucleus tests performed by NTP. However, this vote was not supported by the applicant after detailed review of the test results as well as one external reviewer of the NTP report. The background level in the control was relatively low, there was no dose-response relationship, and in one test also the trend was insignificant. In addition, the increase in MN/PCE was small, but significant in only one or two doses. The investigation after inhalation exposure in mice (in course of the 90 day inhalation study) was negative.

In addition, cumene was found negative in another micronucleus test, which has been added as supporting study. In this study, cumene did not affect the ratio of polychromatic to normochromatic erythrocytes nor increase the frequency of micronucleated erythrocytes; therefore, indicating no potential for clastogenicity in vivo.
The overall conclusion taking into account all findings as a weight of evidence is that cumene is not considered to be positive for in vivo mutagenicity, based on the results of the micronucleus tests presented.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data indicated that classification for genotoxicity is not warranted.