Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study planned
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: 2,3-epoxypropyl isopropyl ether

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

- Available GLP studies
Genetic Toxicity in vitro: Chromosome aberration (micronucleus test): human lymphocytes ±S9: positive (OECD 487, GLP)
The test item demonstrated toxicity in the preliminary toxicity test and the main experiment where near optimum toxicity was achieved in each of the exposure groups. The test item induced statistically significant increases in the frequency of binucleate cells with micronuclei, in the three exposure groups of the Main Experiment. The test item was considered to be able to induce chromosome breaks and/or gain or loss to human lymphocytes in vitro.

- Available non-GLP studies
Genetic Toxicity in vitro:
Gene mutation (Bacterial reverse mutation test / Ames test): S. typhimurium TA 100, TA 1535, TA 98, TA 1537, ±S9 (hamster, rat): S. typhimurium TA 100, TA 1535, positive ±S9, S. typhimurium TA 98: negative ±S9 (rat), S. typhimurium TA 98: ambiguous +S9 (hamster), S. typhimurium TA 1537: negative ±S9 (equivalent to OECD 471)
Strains TA98, TA100, TA1535, and TA 1537 of S. typhimurium were exposed to Isopropyl glycidyl ether in water at concentrations of 0, 100, 333, 1000, 3333, 10000 µg/plate in the presence and absence of mammalian metabolic activation (hamster and rat liver S9 mix) in the preincubation method. Isopropyl glycidyl ether was tested up to limit concentration 10000 µg/plate. There was a dose-related increase of induced mutant colonies over background in strains TA100 and TA1535 with and without metabolic activation, whereas the test item was negative in strains TA98 and TA1537. The positive controls induced the appropriate responses in the corresponding strains.

SOS Chromotest: E. coli PQ37: positive
The substance yielded positive results in the SOS chromotest in E. coli PQ37.

SCE assay: V79 cells –S9: positive (equivalent to EU method B.19)
Isopropyl glycidyl ether induced sister chromatid exchanges in V79 Chinese hamster cells without an external metabolic activation system.

SLRL assay: postmeiotic and meiotic germ cells of male Drosophila melanogaster: positive (sex-linked recessive lethal assay)
Isopropyl glycidyl ether was shown to be mutagenic in tests for sex-linked recessive lethal mutations and reciprocal translocations in Drosophila melanogaster

- Historical human data: On the substance to be registered, there is no concrete human data available. During the complete manufacturing and handling history of the substance however there are no clinical cases or poison incidents known, no indication on a possible genotoxic insult in vivo in humans is given.
For the structurally related substance glycerol polyglycidyl ether, there is some human data regarding mortality and some cancer types available. This study has identified no clear mortality or carcinogenic effect of glycerol polyglycidyl ether so far.
In this study, mortality was studied among 8,878 employees who worked at any time from 1965 to 1988 at a synthetic fibers plant that used a finishing agent containing glycerol polyglycidyl ether (Lanes et al., 1994; Am J Ind Med. 1994 May; 25(5): 689-96.). Some glycidyl ethers are mutagenic and tumorigenic in laboratory animals. The main route of exposure to workers was inhalation of the spray mist, although there was also skin contact. The study was initiated by the plant to measure any possible effect of exposure to glycerol polyglycidyl ether on the occurrence of cancer among employees. Expected numbers of deaths were computed in the cohort using age-, race-, and gender-specific annual mortality rates for the United States (U.S.) population and the local county population. Since the national rates and local rates were similar, analyses using the more stable U.S. rates were used. Cohort members contributed person-time to the analysis from the date of hire until either the date of death or December 31, 1988. Then standardized mortality ratios (SMRs) were computed as the ratio of the number of observed deaths in the cohort to the number of deaths expected if employment were unrelated to mortality. Effect estimates were standardized for age, race, gender, and calendar time. The precision of the effect estimates was assessed by computing 95% confidence limits using a Poisson approximation.
There were identified 553 deaths in the cohort and the standardized mortality ratio (SMR) from all causes of death combined was 0.80. For most causes of death, mortality rates in the cohort were similar to mortality rates in the U.S. population. Among categories with at least five observed deaths, the largest effect estimate was for cancer of the central nervous system (SMR = 1.77), and the SMR for lung cancer was 0.94. The cancer categories of central nervous system (brain) and “other” lymphopoietic cancers (lymphoma and myeloma) showed weak associations with duration of employment. In case-control analyses in which work history data were utilized to compute effect estimates by duration of exposure, no increased risk of lung cancer or brain cancer among employees with more than 5 years of exposure was found. Effect estimates for lymphoma and myeloma tended to increase with duration of exposure, although there were only seven deaths in this category and the effect estimates were very imprecise. To date, this study has identified no clear carcinogenic effect of glycerol polyglycidyl ether, but plausible induction periods have not yet elapsed. The cohort should continue to be monitored to obtain more precise estimates after moderate or long induction times.
Summarizing, there is no clear evidence for tumours induced over background in humans, which could possibly indicate a relevance of the positive genetic toxicity tests in vivo.

- (Q)SAR: Due to the epoxy group of the molecule, the substance possible binds directly to proteins and DNA, which is indicated by SAR Toxtree estimation (Ideaconsult Ltd (2004-2013). Estimation of Toxic Hazard – A decision Tree approach, version 2.6.6, http://toxtree.sourceforge.net/). Other Modelling tools for human endpoints, such as the OECD QSAR toolbox, estimate possible effects by comparison of the substance in question with analogues. This approach is adequately covered in the section “Grouping and read-across”.

- In vitro methods: For details, see above (available (non-)GLP studies); in brief:
Genetic Toxicity in vitro: Chromosome aberration (micronucleus test): human lymphocytes ±S9: positive (OECD 487, GLP)
Gene mutation (Bacterial reverse mutation test / Ames test): S. typhimurium TA 100, TA 1535, TA 98, TA 1537, ±S9 (hamster, rat): S. typhimurium TA 100, TA 1535, positive ±S9, S. typhimurium TA 98: negative ±S9 (rat), S. typhimurium TA 98: ambiguous +S9 (hamster), S. typhimurium TA 1537: negative ±S9 (equivalent to OECD 471)
SOS Chromotest: E. coli PQ37: positive
SCE assay: V79 cells –S9: positive (equivalent to EU method B.19)
SLRL assay: postmeiotic and meiotic germ cells of male Drosophila melanogaster: positive (sex-linked recessive lethal assay)

- Weight of evidence: See above (Historical human data, available (non-)GLP studies) and below (Grouping and read-across) for details to the respective sections.
The available in vitro studies indicate that 2,3-epoxypropyl isopropyl ether must be considered as genotoxic in vitro in various test designs, both gene and chromosome mutations as well as various indicator tests. Similarly, information on structural analogues, also derived from various test systems, lead to the same conclusion, i.e. that glycidyl ethers in general need to be considered as genotoxic in vitro.
However, in order to assess the relevance of those results for genotoxic events in vivo, including germ cell mutagenicity and carcinogenicity in humans possibly arising from the substance to be registered, 2,3-epoxypropyl isopropyl ether, further information is needed, as in vivo systems have a large amount of possibly modifying systems, including Cytochrome P450 metabolism, various redox-systems or possibly elevated hydrolysis after oral application. Due to the lack of in vivo data for the substance itself, which is the reason for the present testing proposal, animal repeated dose / carcinogenicity data and epidemiological studies on structural analogues are taken into consideration. Here however, no consistent effects were found throughout the group of glycidyl ethers. Glycerol polyglycidyl ether could not be proven human carcinogen in the available epidemiological study, n-Butylglycidyl ether lead to no neoplastic lesions in a 90 day rat inhalation study, Diglycidyl ether of substituted glycerine gave no clear carcinogenic response in different species, Alkyl glycidyl ether showed no neoplastic lesions in all studies available. Glycidol however led to neoplastic and non-neoplastic lesions in a 2 year study in rats and mice.
The only in vivo micronucleus test which is known to the registrant on a substance of the group of glycidyl ethers, i.e. n-Butylglycidyl ether, gave a positive result. The transferability of this result to the substance to be registered, 2,3-epoxypropyl isopropyl ether, cannot be confirmed due to the non-consistent outcomes of various animal repeated dose / carcinogenicity data and epidemiological studies on structural analogues. Further, no study is known to the registrant evaluating the genotoxic events in vivo in direct target / first impact organs, in this case the stomach, via Comet assay.
So following the weight of evidence approach, it is considered that all available information on the substance itself and structural analogues allow no clear conclusion on the possible outcome of the in vivo micronucleus / Comet assay, and actual testing is required, and so the general adaption possibilities of REACH Annex XI are not adequate to generate the necessary information.

- Grouping and read-across
The structural analogue for 2,3-epoxypropyl isopropyl ether, i.e. 1,2,3-Propanetriol, glycidyl ethers, is a mixture of different components with average 2-3 epoxy functional groups per molecule, to be more accurate, with 2,3-epoxypropyl side chains. Those are identical to the side chain of 2,3-epoxypropyl isopropyl ether, which is considered to be the toxicologically more relevant functional group. Also, the isopropyl chain is related to the partially substituted propane moiety of 1,2,3-Propanetriol, glycidyl ethers. Both substances are rather small molecules, indicating a similar distribution pattern, which is supported by the liquid state, high water solubility, similar vapour pressure and low logPow, especially in ecotoxicity tests. They are both not readily biodegradable and sufficiently hydrolytically stable over the usual short-term exposure period in ecotoxicity testing. In consequence, 1,2,3-Propanetriol, glycidyl ethers may serve as a read-across substance for 2,3-epoxypropyl isopropyl ether. Similar considerations apply to glycidyl ethers in general, which may so be used for grouping, too. Those epoxides become systemically available via all routes of exposure due to their optimal molecular weight, solubility in water and in organic solvents and logPow. The absorption through the skin may be hindered for the epoxides which logPow is negative. Their bioavailability is evident by their toxicity pattern of acute and repeated toxicity animal studies. As an example, the available OECD 422 on the substance to be registered itself may be cited.

Below there is some data cited, gained from various structurally related substances:
n-Butylglycidyl ether (CAS 2426-08-6):
Positive (in a number of in vitro and in vivo Micronucleus Test, Screening Assessment, Canada, 2010)
No neoplastic lesions (90-day inhalation study; HPV, 2001 (High Production Volume (HPV) Challenge Program. Test Plan and Robust Study Summaries for n-Butyl Glycidyl Ether submitted to the US Environmental Protection Agency by Epoxy Resin Task Group (ERSTG), original submission, 2001; AR201-13352A));
Suspected carcinogen (Screening Assessment Canada, 2010)

Glycidol (CAS 556-52-5):
Positive (Ames Test, Mouse Lymphoma Assay with L5178Y/TK cells (without metabolic activation, SCE and Chr. Aberrations in CHO cells) (NTP TR 374, 1990 (Toxicology and Carcinogenesis Studies of Glycidol (CAS No. 556-52-5) in F344N rats and B6C3F1 (Gavage studies). NIH Publication No. 90-2829))
Neoplastic and non-neoplastic lesions (2-y, rats, mice; NTP TR 374, 1990 (Toxicology and Carcinogenesis Studies of Glycidol (CAS No. 556-52-5) in F344N rats and B6C3F1 (Gavage studies). NIH Publication No. 90-2829))

Diglycidyl ether of substituted glycerine (CAS 63089-76-9):
Positive (TA 100, Wade et al., 1979); Positive (TA 100, TA 98), Negative (TA 1531, TA 1533)
(Pattys, 1981 (Epoxy Compounds. Hine, Rowe, White, Darmer and Youngblood. Polyglycidyl Ether of Substituted Glycerine (GE-100) pp.2237-2240))
No clear carcinogenic response in different species (Pattys, 1981 (Epoxy Compounds. Hine, Rowe, White, Darmer and Youngblood. Polyglycidyl Ether of Substituted Glycerine (GE-100) pp.2237-2240))

Alkyl glycidyl ether (CAS 68609-97-2):
Positive (TA 1535); Negative (bacterial strains, at HGPRT locus in CHO cells; Chromosome aberration (in vitro and in vivo) HPV, 2002 (High Production Volume (HPV) Challenge Program. Test Plan and Robust Study Summaries for Alkyl (C12-C14) Glycidyl Ether submitted to the US Environmental Protection Agency by Epoxy Resin Task Group (ERSTG), revised submission))
No neoplastic lesions in all studies available (HPV, 2002 (High Production Volume (HPV) Challenge Program. Test Plan and Robust Study Summaries for Alkyl (C12-C14) Glycidyl Ether submitted to the US Environmental Protection Agency by Epoxy Resin Task Group (ERSTG), revised submission))

1,2-Epoxybutane (106-88-7):
1,2-Epoxybutane induced morphological transformation, sister chromatid exchanges, chromosomal aberrations and mutation in cultured animal cells; however, in a single study, it did not induce unscheduled DNA synthesis in rat primary hepatocytes. It induced sexlinked recessive lethal mutations and translocations in Drosophila melanogaster, mitotic recombination in yeast, and mutations in yeast and fungi. 1,2-Epoxybutane induced DNA damage and mutations in bacteria.
1,2-Epoxybutane was tested for carcinogenicity by inhalation exposure in one study in mice and in one study in rats, producing nasal papillary adenomas in rats of both sexes and pulmonary alveolar/bronchiolar tumours in male rats. It did not induce skin tumours when tested by skin application in one study in mice.
(1,2-EPOXYBUTANE, IARC Monographs, 71-27, 1989)

Further similar data can be found in the following literature:
Waechter et al., Epoxy Compounds—Olefin Oxides, Aliphatic Glycidyl Ethers and Aromatic Monoglycidyl Ethers, in Patty's Toxicology, 6th Ed, 2012:
Although all of the compounds described in this chapter were mutagenic to bacteria (excluding epoxidized glycerides) as well as positive in other in vitro genotoxicity assays, not all have produced genotoxicity in in vivo studies. Ethylene oxide was positive in the mouse micronucleus assay and mouse dominant lethal assay. In contrast, propylene oxide, although positive in all the in vitro assays in which it was tested, was negative in all of the in vivo mammalian assays where propylene oxide was administered via the relevant inhalation route. These negative mammalian studies include a mouse micronucleus assay (although positive by IP injection of high doses), mouse sperm cell analysis, and a rat dominant lethal assay. In addition, propylene oxide failed to cause chromosomal changes (SCE and chromosomal aberrations) in monkey lymphocytes following chronic exposures to 300 ppm. Other compounds showing positive or equivocal effects in vitro but negative effects in vivo are styrene oxide, and many of the glycidyloxy compounds used in epoxy resin formulations.
There has been no evidence of teratogenicity for glycidyl ethers or olefin oxides, except ethylene oxide (EO), when tested by oral or inhalation exposure in conventional developmental toxicity studies.
A number of these epoxide compounds have been found to be carcinogenic in rodents, although there has been no clear epidemiologic evidence for cancer in the workplace. In rats and/or mice, many epoxy compounds produce a carcinogenic response in the tissues of first contact. These compounds include ethylene oxide, butylene oxide, propylene oxide, styrene oxide allyl glycidyl ether, phenyl glycidyl ether, and neopentyl glycol diglycidyl ether. A few of them, such as ethylene oxide, butadiene dioxide, and vinylcyclohexene dioxide, have produced tumors at sites other than the “portal of entry.”

Waechter et al., Epoxy Compounds—Aromatic Diglycidyl Ethers, Polyglycidyl Ethers, Glycidyl Esters, and Miscellaneous Epoxy Compounds, in Patty's Toxicology, 6th Ed, 2012
Generally, in vitro genetic toxicity testing of the epoxide compounds has resulted in positive (genotoxic) responses; the majority of the studies of genotoxic potential have been carried out using bacteria. These results are not surprising because many of these compounds have been tested in strains TA1535 and TA100 of S. typhimurium or in other gene mutation assays that are specifically sensitive to base-pair substitution. Metabolic activation was not required for most of the epoxides, which showed mutagenic effects in these tests. Many other in vitro assays examining both gene mutation and chromosomal effects have been employed to test the epoxy compounds, including assays in E. coli, yeast, Chinese hamster ovary cells (CHO/HPGRT), mouse lymphoma cells, and cultured human lymphocytes; the results have usually been mixed or positive. Fewer epoxy compounds have been tested using in vivo assays for genotoxic effects, although some have been extensively studied.
Glycidol was positive in the Drosophila sex-linked recessive lethal assay and mouse micronucleus assay and produced chromosomal aberrations in the bone marrow of mice dosed orally or intraperitoneally. Glycidaldehyde was positive in the Drosophila sex-linked recessive lethal assay. In contrast, epichlorohydrin, also a low-molecular-weight epoxy compound, was negative in both the mouse micronucleus test following intraperitoneal administration and the mouse dominant lethal assay following oral or intraperitoneal administration, although it was positive in many of the in vitro assays. Another compound showing positive or equivocal effects in vitro but negative effects in vivo is the diglycidyl ether of bisphenol A.

- Substance-tailored exposure driven testing
Route of exposure and target organs are specified below. The testing proposal allows assessing the in vivo genotoxicity of 2,3-epoxypropyl isopropyl ether in both the organ of first impact (stomach via Comet assay) and systemic exposure (Mammalian Erythrocyte Micronucleus Test). To assess the latter, the oral route of exposure is considered most suitable. In addition, results derived from the organ of first impact may be transferred to a certain extent also to other possibly exposed organs, e.g. respiratory tract and skin, although exposure of workers is minimized by appropriate safety precautions. So the proposed test design is most suitable to address all possible events in question.

- Approaches in addition to above
All relevant approaches were already considered above.

- Other reasons
No other reasons.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
First of all it should be noted that the proposed testing as such is not explicitly demanded under REACH. However, according to REACH Annex VIII column 2, appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII. The substance was tested positive in a Chromosome aberration (micronucleus test) in human lymphocytes ±S9 according to OECD 487 under GLP, and was tested positive in two out of four strains both detecting transitions and transversions (base substitutions at the G:C basepair).
There are indications that IPGE acts as direct mutagen and clastogen by direct DNA-addition via the epoxy group, but taking into account its potential to also bind directly to Proteins or being modified in vivo via additional systems not being present in in vitro genotoxicity tests, a definitive conclusion on its mutagenicity in vivo cannot be drawn. Provided that IPGE reaches non-modified, i.e. with intact epoxy group, the DNA, similar adducts may occur. However, as in vivo systems have a large amount of possibly modifying systems, including Cytochrome P450 metabolism, various redox-systems or possibly elevated hydrolysis after oral application, no conclusion may be drawn on a possible in vivo mutagenicity.
Further, various data on structural analogues indicate that no clear conclusion can be drawn from positive results in vitro for relevance in vivo. Most analogues were tested positive in various in vitro genotoxicity tests, but only in some cases positive results were obtained in carcinogenity studies. A combined in vivo micronucleus / Comet assay may not be considered adequate to a carcinogenity study, but gives sufficient information on the relevance of positive in vitro data for both local and systemic genotoxicity in vivo. So, before determining the classification, an in vivo genotoxicity test must be performed ahead, and all the available, well-evaluated information does not suffice to draw clear conclusion for the substance to be registered, 2,3-epoxypropyl isopropyl ether.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
The study is aimed to be performed according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test), in order to clarify the relevance of the positive results in the in vitro micronucleus test. Further, to give indications on the relevance of the positive results in the available Ames test, additional information should be gained with the proposed OECD 474 study with an integrated Comet Assay in vivo related to the OECD Guideline for Testing of Chemicals, Section 4, No. 489, adopted September 26, 2014, „In Vivo Mammalian Alkaline Comet Assay“, on the target / first impact organ(s). Data gained from the available OECD 422 study in rats, and literature data on glycidyl esters in general, indicate that the stomach is most relevant here. The Comet assay detects single DNA strand breaks in various tissues and organs of exposed animals. The substance is presumed to exhibit its mode of action via direct DNA-addition via the epoxy group, leading i.a. to single-strand breaks of the affected DNA. Hence, the Comet assay is considered to be a suitable method the verify the in vivo relevance of both effects noted in vitro, i.e. clastogenic activity and mutagenicity.

Data source

Materials and methods

Test guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-epoxypropyl isopropyl ether
EC Number:
223-672-9
EC Name:
2,3-epoxypropyl isopropyl ether
Cas Number:
4016-14-2
Molecular formula:
C6H12O2
IUPAC Name:
2-[(propan-2-yloxy)methyl]oxirane
Test material form:
liquid

Results and discussion

Applicant's summary and conclusion