Bis[(3,4-epoxycyclohexyl)methyl] adipate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (Guzzie, 1988)

Under the conditions of this study the test material was considered to be mutagenic in the presence of metabolic activation with Salmonella typhimurium strain TA1535 and marginally mutagenic with Salmonella typhimurium TA100.

Read-across to structurally similar substance (CAS No. 2386 -87 -0)

Ames Test (Machigaki, 1991)

Under the conditions employed in this test, the test material, was considered to be a mutagen in the presence of metabolic activation when tested in the  Salmonella strains TA100 and TA1535 which indicated a base pair mutagenic response. In the Salmonella strains TA 98 and TA1527 and E.coli WP2 uvrA negative responses were observed in the presence of metabolic activation. Without metabolic activation there were no positive responses in any of the strains tested.

Mouse Lymphoma Assay (Beilstein, 1984)

A mouse lymphoma assay (Beilstein, 1984) reports a positive response, both in the absence and presence of metabolic activation.  A clastogenicity assay performed in vitro in CHO cells (Slesinskiet al., 1980) reports consistently negative results both in the absence and presence of metabolic activation. Slesinski et al. (1980) also report a positive and ‘highly significant’ result in a sister chromatid exchange (SCE) assay in CHO cells; this assay was performed in the absence of metabolic activation only. The same authors report an equivocal result in an unscheduled DNA synthesis (UDS) assay using cultured primary rat hepatocytes. A very weak increase in the level of DNA damage indicated at lower concentrations in this study was not replicated at higher concentrations.

UDS Assay (Slesinski, 1980)

Based on the results, the test material appeared to produce a very weak response in the present UDS test with cells treated over a 1000-fold range of test concentrations. The test material was considered questionable-to-weakly active in producing DNA damage in the tests with hepatocytes.

Clastogenicity Assay (Slesinski et al., 1980)

The test material was consistently inactive as a mutagenic agent for CHO cells when tested with or wlthout an S9 metabolic activation system over a 16-fold range of concentrations. Although small increases in the numerical frequency of mutants were obtained at some test concentrations of the test material, these results appeared to be a random effect without statistical or probable biological significance.

SCE Assay (Slesinski, 1980)

The test material produced highly significant increases in the frequency of SCE when tested over a 32-fold range of concentrations in tests without addition of an S9 metabolic activation system.  Evidence of a dose-related effect on the SCE frequency following exposure to the test material indicated that the test material should be considered significantly active in the present in vitro assay.

The results of the in vitro studies therefore indicate a genotoxic potential for the substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Read-across to structurally similar substance (CAS No. 2386 -87 -0)

-Mouse Micronucleus Assay (Wing & Machado, 1991)

Based on the results of this study, the test material did not induce micronuclei in bone marrow erythrocytes of mice under the conditions of this test.

-Rat Liver UDS Assay (San & Sly, 1999)

Based on these results, the test material did not induce a significant increase in the mean number of net nuclear grain counts in hepatocytes isolated from treated animals, and was concluded to be negative in the unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo.

-MutaMouse Assay (Masumori, 2016)

 The study report concludes that the test material induces gene mutations in the stomach of transgenic mice under the conditions of this study. This interpretation of a marginal response which is clearly within the background control range, is considered to be questionable.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test (Guzzie, 1988)

The potential of the test material to cause mutagenic activity was investigated in a study similar to OECD 471, under GLP conditions.

Test doses for the Ames test were chosen from data obtained in a preliminary study using Salmonella typhimurium strain TA100. Preliminary cytotoxicity results of tests performed without the presence of a rat-liver S9 metabolic activation mixture showed that a concentration of 10 mg/plate produced a complete absence of growth of the background lawn of bacteria while a lower dose of 5 mg/plate allowed sparse growth of the bacterial lawn. A higher dose of 30 mg/plate was not soluble in the overlay agar which resulted in confluent growth of the background lawn. In the preliminary test with S9, a dose of 10 mg/plate produced sparse growth of the background lawn. Higher doses were not soluble which permitted confluent growth of the background lawn except around the areas where the chemical formed precipitate. Based on these results, the test material was tested with five concentrations ranging from 0.1 to 5 mg/plate in the absence of S9, and from 0.3 to 10 mg/plate in the presence of S9, using triplicate cultures at each dose level.

For the main mutagenicity tests, a sterile tube containing 2 mL of top agar, a 100 µL aliquot of the appropriate bacterial culture was added followed by the addition of 100 µL of the appropriate solvent, control, or test material solution. Either 0.5 mL of S9 mix or 0.5 mL of phosphate-buffered saline (PBS) was added for tests with and without metabolic activation, respectively. The top agar mixture was then poured onto a VB-E plate. Each dose was tested in triplicate and with all five bacterial strains. Sterility checks were done on the PBS and/or S9 mix, all solvents, and the highest concentration of each test material. The plates were transferred to a darkened 37 °C incubator after hardening and incubated for 48 to 72 hours. Concurrent solvent and positive controls were tested in each experiment.

In tests performed without S9, no indication of mutagenicity was observed with any of the strains, either by evidence of a dose-response relationship or a doubling of the average number of colonies over the average solvent control value. All five strains showed signs of treatment-related inhibition of growth of the background lawn with at least the highest dose level of test material tested (5 mg/plate). In the presence of S9, highly positive (> 10-fold) and dose-related increases in relative numbers of revertant colonies were observed with TA1535. Positive and dose-related mutagenic activity was also observed in the preliminary test performed with TA100 in the presence of S9. However, only weak mutagenic activity was evident in the definitive test performed with S9 using strain TA100.

All five bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without S9 metabolic activation. Negative (solvent) controls were also tested with each strain, and in general, the average number of spontaneous revertants was within the historical ranges at the laboratory. All positive and negative controls were tested concurrently with the test material. Concurrent sterility testing showed that the S9 mix, PBS, the test material and the solvent control agent were sterile.

Under the conditions of this study the test material was considered to be mutagenic in the presence of metabolic activation with Salmonella typhimurium strain TA1535 and marginally mutagenic with Salmonella typhimurium TA100.

Read-across to structurally similar substance (CAS No. 2386 -87 -0)

Ames Test (Machigaki, 1995)

In a study conducted by Machigaki (1991), the test material was examined for its potential to be mutagenic when tested in a bacterial assay using 5 strains of bacteria, including Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and the Escherichia coli WP2 uvrA- strain. The study was performed in accordance with the standardised guideline OECD 471, under GLP conditions.

The study was performed using the following concentrations, 156, 313, 625, 1250, 2500 and 5000 µg per plate. Each concentration was tested in triplicate and each assay was conducted twice. Each strain was tested using the appropriate suitable positive control, relevant for each bacterial strain. Each assay was done both in the presence and absence of metabolic activation in the form of S9 mix.

Under the conditions employed in this test, the test material, was considered to be a mutagen in the presence of metabolic activation when tested in the Salmonella strains TA100 and TA1535 which indicated a base pair mutagenic response. In the Salmonella strains TA 98 and TA1527 and E.coli WP2 uvrA negative responses were observed in the presence of metabolic activation. Without metabolic activation there were no positive responses in any of the strains tested.

Mammalian Cell Mutation Assay (Beilstein, 1984)

In a study conducted by Beilstein (1984), the test material was tested for its potential to cause mutagenic effects on L5178Y/TK+/- mouse lymphoma cells in vitro, both in the presence and absence of microsomal metabolic activation and the appropriate positive controls were also used. A mutagenic effect of a substance was demonstrable on comparison of the number of colonies in the treated and control cultures.

The test material was examined at 7 concentrations, 12.5, 25, 50, 100, 150, 200 and 250 µg/mL. The results of this study indicate that the test material was considered to be mutagenic as the colony count of the test material was greater than 2.5 at the top three concentrations in the test in the presence of metabolic activation and the 4 highest concentrations in the test conducted in the absence of metabolic activation indicated mutagenic activity.

Under the experimental conditions of this study, the test material was considered to be mutagenic, both in the presence and absence of metabolic activation.

UDS Assay (Slesinski et al., 1980)

In a study conducted by Slesinski et al (1980), the test material was investigated for its potential to induce Unscheduled DNA Synthesis (UDS) in vitro using primary hepatocytes from rat liver cells. The hepatocytes were exposed to the test material at 6 concentrations, ranging from 1000 x 10E-4% to 1.0 x 10E-4% by volume.

The results of this study indicate that the lowest 3 concentrations produced highly numerically elevated levels of UDS activity. Because these values were not consistently significant in statistical comparisons to the concurrent solvent control, the results could not be definitively labelled as either positive or negative. The data were considered to be suggestive of a low level of activity and the test material appeared to be questionably-to-weakly active in the present test with the hepatocyte test system.

Clastogenicity Assay (Slesinski et al., 1980)

In a study conducted by Slesinski et al (1980), the test material was evaluated for its potential to induce mutagenic activity in an in vitro gene mutation study using Chinese Hamster Ovary (CHO) cells. The test was conducted in the presence and absence of metabolic activation in the form of S9 mix. The experiment was conducted twice, with concentrations in the 1st experiment ranging from 100 x 10E-4% to 6.25 x 10E-4% and in the 2nd experiment from 200 x 10E-4% to 12.5 x 10E-4%. The tests were conducted alongside appropriate positive, negative and solvent controls. Based on the results of this study the teat material was consistently inactive as a mutagenic agent for CHO cells when tested with or wlthout an S9 metabolic activation system over a 16-fold range of concentrations. Although small increases in the numerical frequency of mutants were obtained at some test concentrations of the test material, these results appeared to be a random effect without statistical or probable biological significance. As a result of this, the test material does not require classification according to Regulation EC No. 1272/2008.

SCE Assay (Slesinski et al., 1980)

In a study conducted by Slesinski et al in 1980, the test material, Epoxy Resin ERL-4221, was investigated for its potential mutagenic activity in the in vitro test, the Sister Chromatid Exchange test. The test was conducted in the absence of metabolic activation in the form of S-9 mix and the appropriate positive, negative and solvent controls. The test material was tested over a range of 6 concentrations, from 100 x 10E-4% to 3.125 x 10E-4%.

The test material produced highly significant increases in the frequency of SCE when tested aver a 32-fold range of concentrations in tests without addition of an S9 metabolic activation system. Evidence of a dose-related effect on the SCE frequency following exposure to Epoxy Resin ERL-4221 indicated that the test material should be considered significantly active in the present in vitro assay.

Mouse Micronucleus Assay (Wing & Machado, 1991)

In a study conducted by Wing and Machado (1991), the test material was examined for its ability to induce micronuclei in mouse bone marrow erythrocytes. The test material was diluted in peanut oil and administered intraperitoneally to 18 Swiss Albino mice of each sex. The dose levels used were 0 .50, 1.00 and 2.25 g/kg. Bone marrow smears of 5 animals per sex pre treatment group were made at approximately 24, 48 and 72 hours after treatment.

Clinical signs of toxicity, including decreased motor activity, collapse, weakness, ataxia and laboured breathing were observed in both sexes at the 2.25g/kg dose level. Cytotoxicity was noted in females treated with 0.50 and 2.25g/kg and sampled at 48 hours.

A statistically significant increase in micronucleated polychromatic erythrocytes was noted in males treated with 1.00 g/kg and sampled at 48 hours; however, this increase was not dose-responsive and was not considered to be biologically significant.

Based on the results of this study, the test material did not induce micronuclei in bone marrow erythrocytes of mice under the conditions of this test.

Rat Liver UDS Assay (San & Sly, 1999)

In a study conducted by San and Sly (1999), the test material was investigated for its ability to induce mutations in an unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo in male Sprague-Dawley rats. The assay was performed in two phases. The first phase, the dose range-finding assay, was used to determine the maximum tolerated dose. The second phase, the UDS assay, was used to evaluate the potential of the test material to induce unscheduled DNA synthesis in hepatocytes of exposed male rats. In the dose range finding assay, male rats were exposed to 1000, 2000, 4000 and 5000 mg test material/kg bw and in the definitive UDS test, the highest dose administered was the maximum tolerated dose of 2000 mg/kg bw, in addition to 500 and 1000 mg test material/kg bw.

No mortality was observed in any test material treated or control treated rats. All animals appeared normal following dose administration.

However, diarrhoea was observed in one animal treated with 2000 mg/kg bw (12-16 hour exposure) and one DMN treated (2-4 hour exposure) animal prior to sacrifice. The test material did not induce a significant increase in the mean number of net nuclear grain counts in hepatocytes isolated either 2 to 4 hours or 12 to 16 hours after dose administration.

Based on these results, the test material did not induce a significant increase in the mean number of net nuclear grain counts in hepatocytes isolated from treated animals, and was concluded to be negative in the unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo.

MutaMouse Assay (Masumori, 2016)

A somatic and germ cell mutation assay was performed with the test material using male transgenic mice (MutaMouse). The study was performed in accordance with the standardised guideline OECD 488, under GLP conditions. The study was conducted to assess the potential of the test material to induce gene mutations in the liver, stomach, nasal cavity and germ cells, using the lacZ gene as a mutation reporter gene. The dose levels of the test material used in the main study were based on the results of a dose range-finding study in which CD2F1/Slc mice were gavaged for 7 days with 0, 125, 250, 500, or 1000 mg/kg bw/d CEL2021P in corn oil. No signs of toxicity were observed in any of the dose groups. A dose level of 1000 mg/kg bw/d was therefore selected as the high dose level for the main study.

The test material was administered by gavage (in corn oil) to groups of six male transgenic mice orally for 28 consecutive days. A control group was treated with the vehicle alone and a positive control group was administered ENU by intraperitoneal injection at 100 mg/kg bw/d for two days. Three days following administration of the final dose, the liver, stomach, nasal cavity, testes and vas deferens/cauda epididymis were removed. Mutation frequencies in the liver, stomach, nasal tissue and germ cells were determined for five mice per group.

The mutation frequencies in the nasal tissue and germ cells of all groups treated with the test material did not show any statistically significant increase compared to the vehicle control group. A slight (but statistically significant) increase in the mean mutation frequency was seen in the liver of mice administered 1000 mg/kg bw/d; however the value was within the background control range and was also within the laboratory’s acceptable range (defined as the mean of the background control range ±3 sd). A slight (but statistically significant) increase in the mean mutation frequency was seen in the stomach of mice administered 1000 mg/kg bw/d. This value was within the background control range, but marginally exceeded the laboratory’s acceptable range and was therefore considered by the laboratory to represent a positive response. The mutation frequencies in the liver, stomach, nasal tissue and germ cells of the positive control group were statistically significantly increased compared with the negative control group, demonstrating the sensitivity of the assay.

 The study report concludes that the test material induces gene mutations in the stomach of transgenic mice under the conditions of this study. This interpretation of a marginal response which is clearly within the background control range, is considered to be questionable.

Justification for classification or non-classification

Classification under the CLP Regulation is required for substances causing germ cell mutagenicity. This hazard class is primarily concerned with substances having potential to cause mutations in the germ cells of humans that may be transmitted to progeny. The results from tests in vitro and in mammalian somatic and germ cells in vivo are considered when classifying substances in this hazard class. Substances may be classified in Category 1A (appropriate substances known to induce heritable mutations in the germ cells of humans); in Category 1B (substances regarded as if they induce heritable mutations in the germ cells of humans); or in Category 2 (substances which cause concerns owing to the possibility that they may induce heritable mutations in the germ cells of humans).

The criteria used for classification are described below:

Classification in Category 1A is on the basis of human data and is therefore not relevant for this substance.

Classification in Category 1B may be based on a positive result from an in vivo mammalian germ cell mutagenicity test, or on a positive result from an in vivo mammalian somatic cell mutagenicity test together with further toxicokinetic data showing systemic availability and germ cell exposure. Classification in Category 1B is not appropriate for the substance given the clearly negative result in germ cells in the recently conducted transgenic rodent assay.

Classification in Category 2 may be based on positive results in an in vivo mammalian somatic cell mutagenicity test, indicating mutagenic effects in somatic cells; or may be based on positive results in a mammalian somatic cell genotoxicity test in vivo supported by positive in vitro mutagenicity data. The CLP Guidance notes that ‘…where there is evidence of only somatic cell genotoxicity, substances are classified as suspected germ cell mutagens’; i.e. they should be classified in Category 2. It is further stated that ‘…if positive results in vitro are supported by at least one positive local in vivo somatic cell test, such an effect should be considered as enough evidence to lead to classification in Category 2’.

In this case, therefore, consideration focuses on the need for classification of the substance for germ cell mutagenicity in Category 2. In the case of this substance, clearly negative responses are reported in somatic cells in vivo in a mouse bone marrow micronucleus assay, a rat liver UDS assay and in the transgenic mouse assay in the liver and nasal tissue. The transgenic mouse assay, however, reports a positive result for the forestomach, based on the laboratory’s statistical evaluation criterion. Any conclusion on whether the substance is classified in Category 2 therefore hinges on interpretation of the response seen in the forestomach in this study, which may be taken to indicate that the substance has site of contact mutagenic activity.

The result in the forestomach is considered by the laboratory to be positive according to their statistical interpretation criterion, but even this is marginal (the mutation frequency of 78.5e-6 is compared to an ‘acceptable range’ maximum of 78.0e-6. Furthermore, the mutation frequency in the forestomach is well within the historical control range for this tissue (reported by the laboratory to be 31.1-84.7e-6). The interpretation of this result as positive based purely on a statistical analysis is questionable. It is notable that the test guideline (OECD 488) states that there are ‘several criteria for determining a positive result’, further stating that ‘…biological relevance of the results should be the primary consideration’and that‘appropriate statistical methods may be used as an aid in evaluating the test results’. The marginal response in the forestomach, seen only at the highest dose level of 1000 mg/kg bw/d and in a tissue with a highly acidic pH, is not considered to be clear biological significance and is also of questionable relevance to other potential (and more relevant) site of contact tissues such as the skin or respiratory tract. In this respect it is notable that a mouse skin-painting carcinogenicity study for the substance demonstrates an absence of site-of-contact carcinogenicity following long-term exposure to high dose levels of the substance.

Guidance on application of the CLP criteria states that the classification of substances ‘shall be based on the total weight of evidence available’ and additionally states that the decision should use expert judgement. For substances classified on the basis of a single study, it is stated that this study ‘shall provide clear and unambiguously positive results’. Furthermore it is stated that ‘the relevance of the route of exposure used in the study….compared to the most likely route of human exposure shall also be taken into account’. In the case of this substance, three in vivo studies of somatic cell mutagenicity are available. Two of these studies provide clearly negative results and a third study (the transgenic mouse study) gives negative results in all of the tissues examined, with the single exception of a marginal response in the forestomach where a positive response is concluded based purely on a statistical criterion. For the reasons discussed, the response in the forestomach is not considered to provide ‘clear and unambiguously positive results’. The relevance of the finding in the forestomach is also questionable compared to the likely routes of occupational exposure (dermal and inhalation), particularly when considered in light of the negative mouse carcinogenicity (skin painting study).

In conclusion, based on a detailed assessment of the genotoxicity data and comparison with the classification criteria, it is concluded that the available data are not sufficient to classify the substance for germ cell mutagenicity according to CLP.