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in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study planned
Justification for type of information:

- Name of the substance on which testing is proposed to be carried out


- Available GLP studies
No in vivo studies with the registered substance are available
- Available non-GLP studies
No in vivo studies with the registered substance are available
- Historical human data
No data available
- (Q)SAR
The mutagenic potential of the test substance was screened using the OECD QSAR Toolbox v4.5 (assessed December 2022). The following profilers were checked, including all metabolites predicted by the simulators ‘in vivo Rat metabolism’ and ‘Rat liver S9 metabolism’ (no empirical metabolism data is available):
• DNA binding by OASIS: negative
• DNA binding by OECD: negative
• Carcinogenicity (genotox and nongenotox) alerts by ISS: negative
• DNA alerts for AMES, CA and MNT by OASIS: negative
• in vitro mutagenicity (Ames test) alerts by ISS: negative
• in vivo mutagenicity (Micronucleus) alerts by ISS: positive for the structural alert ‘H acceptor-path3-H acceptor’. It should be noted that this alert is associated with the possibility that the structure allows non-covalent binding to DNA via any two hydrogen bond-accepting atoms attached to two adjacent atoms. This represents a very unspecific structural alert for mutagenicity.
- In vitro methods
In a recent Ames assay (Guideline and GLP conform) with the registered substance the Salmonella strains TA 100 and TA 98 tested positive for in vitro mutagenicity in the pre-incubation experiments. These strains, as well as all remaining strains were negative in the plate incorporation experiment.
In an older Ames assay, the substance tested negative. This assay was performed to the standards of its time, however it does not meet current standards because one of the required strains is missing. Among the strains tested in the older Ames assay were the two positive strains from the recent assessment (TA 100 and TA98), however only the plate incorporation assay was performed in the older study.
A recently performed MLA (Guideline and GLP conform) showed no mutagenic activity in mammalian cells.
An in vitro chromosome aberration test was performed in 2007 and gave a negative result. This is a high quality GLP study which meets current standards.

- Grouping and read-across
Hexamethylenediamine, a structurally similar amine, was tested for chromosomal aberration in vivo (similar to OECD 475, under GLP). No chromosomal aberrations were observed. The chloride salt of hexamethylenediamine was tested in a micronucleus assay (comparable to OECD 474, under GLP). No increase of micronucleus formation was observed.
Albeit the negative outcome, these assays do not address the same mechanism of genotoxicity which is indicated by mutation in Salmonella strains TA100 and TA98 (base substitutions and frameshift mutations). Additionally, available in vitro data for the mutagenicity of hexamethylenediamine is negative, which questions the validity as a read across source for the registered substance.
No in vivo data on other structurally sufficiently similar substances is available.
The available in vivo mutagenicity studies are therefore not sufficient to close the data gap by read-across.

- Weight of evidence
The positive result in the Ames test in the strains TA98 and TA100 occurred only in the pre-incubation assay and not in the plate incorporation. The mutation frequency was only hardly above the thresholds set in the criteria for a positive response. All other available data from Ames tests with similar substances (two amine groups on a C6 backbone with no other functional groups) uniformly gave a negative result.
Altogether, the recent Ames test on cyclohex-1,2-ylenediamine does not give rise to a particularly high level of concern and a negative outcome of the proposed in vivo testing is expected. Yet, no sufficiently convincing case can be made to disregard the positive in vitro result. According to Regulation (EC) No 1907/2006 (REACH) Annex VII and VIII, section 8.4, a positive in vitro genotoxicity test mandates the proposal of appropriate in vivo testing to ECHA.


Waiving according to column 2 is not applicable to the registered substance.


Based on the recommendations given in the Endpoint specific guidance, Chapter R.7a (Version 6.0, July 2017) for substances that appear preferentially to induce gene mutations, the transgenic rodent (TGR) assay, the rat liver UDS assay or the in vivo comet assay may be used to address this data gap.

However, in the meantime the OECD testing guideline (TG) 470 for the Mammalian Erythrocyte Pig-a Gene Mutation Assay (Pig-a Assay) was released on 30 June 2022. This assay detects gene mutations, including base pair substitutions and frame shifts. Therefore, from a mechanistical point of view, the Pig-a Assay or the TGR would be the most appropriate in vivo assays to follow up the observed gene mutations in Salmonella strains TA100 and TA98 (base substitutions and frameshift mutations) of the in vitro bacterial reverse mutation assay (AMES test/OECD TG 471; Robison et al., 2021). An analysis included in the Detailed Review Paper on the Pig-A assay from the Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology (OECD, Series on Testing and Assessment No. 315: The in vivo erythrocyte Pig-a gene mutation assay – Part 1; July 2020) compared the Pig-a Assay performance and existing in vivo genetic toxicity assays. A high degree of concordance was displayed, e.g. 96% when Pig-a Assay results were compared to the bone marrow TGR assay results (total of 28 chemicals). The Pig-a Assay protocol is especially suitable in the case of relatively weak genotoxic agents, due to its characteristics of accumulation and persistence of mutation (Chen et al., 2019 and Zhu et al., 2022). Therefore, the Pig-a-Assay seems to be the test of choice in this case considering, that the available in vitro genotoxic test results for cyclohex-1,2-ylenediamine, as well as the QSAR analyses, indicate at most a weak mutagenic potential.

The ECHA guidance states further, that ‘the choice of any of these three assays can be justified only if it can be demonstrated that the tissue(s) studied in the assay is (are) sufficiently exposed to the test substance…. This information can be derived from toxicokinetic data or, in case no toxicokinetic data are available, from the observation of treatment-related effects in the organ of interest.’ Even though the Pig-a Assay is not mentioned in the ECHA guidance this also applies here.
No toxicokinetic data is available which allows deducing target organs for possible mutagenicity. In a combined repeated dose and reproduction / developmental screening study by oral gavage of the registered substance, macro- and microscopic changes were noted in the liver (pale discoloration, hepatocellular vacuolization). Other target organs with minor findings were the lungs (alveolar inflammation), adrenal glands (vacuolization of the zona fasciculata), the kidneys (weight change in conjunction with basophilia) and the thymus (weight change in some animals in conjunction with atrophy). In the 90-day toxicity study there was an adverse reduction of body weight gain and adverse histopathological findings in the male reproductive organs, which were associated with reduced sperm count, motility and increased numbers of abnormal sperm in males receiving 500 mg/kg bw x day and epididymal vacuolation, associated with reduced epididymal sperm count in males receiving 150 mg/kg bw x day. Adverse findings were reported in the hemolymphoreticular system (spleen, lymph nodes and Peyer’s patches) of both sexes receiving 500 mg/kg bw x day. The hematological examination showed alterations in some parameters (e.g., low lymphocyte, eosinophil, basophil and large unstained cell counts in both sexes, in females decrease of hematocrit and hemoglobin concentration which associated with slightly low mean cell hemoglobin and mean cell hemoglobin concentration and high reticulocyte count and red cell distribution). Further non-adverse findings are not detailed here. Based on these findings, a systemic availability of the test substance can be assumed with sufficient certainty and ‘can be considered as line of evidence for bone marrow exposure’ according to the cross sectional guidance document from the scientific committee ‘Clarification of some aspects related to genotoxicity assessment’ (EFSA, 2017), section However, further analytical testing might be required to prove bone marrow exposure separately, in case of a negative Pig-a Assay result.

Taken together, an in vivo Mammalian Erythrocyte Pig-a Gene Mutation Assay (OECD TG 470) is the proposed follow up in vivo test regarding the observed in vitro AMES test results.

Pursuant the 3R principles (Directive 2010/63/EU), combined testing with an already commissioned Extended-One-Generation-Toxicity-study (OCED TG 443) is considered scientifically valid (as laid out in the OECD TG 470, it is specifically designed to be included in repeated dose toxicity studies) and feasible. Thus, the Pig-a Assay is planned to be integrated in the respective dose range finding study by collecting blood samples of six male animals from control or each of the three treatment groups after 25 days of exposure during the pre-mating phase (only 6 animals from the 8 animals/group will be sampled as this is sufficient according to testing guideline). The animals will be dosed with 0, 300, 500, or 800 mg/kg bw x day via gavage. The high dose group was determined based on the available substance data and is in line with the current strategy for dose range finding studies for reproductive toxicity studies. Even though it is slightly below the limit dose of the OCED TG 470 (i.e. 1000 mg/kg bw x day) it is applicable as male animals are the more sensitive sex (cf. findings from the 90-day toxicity study and the fact that in the accompanying dose range finding study, in which the substance was administered for two weeks up to 1000 mg/kg bw x d revealed findings at this dose in males which suggested that this dose would be too high for longer-term administration). It is planned, that 200 µL blood per animal will be sampled on two occasions a) pre-dose sampling and b) on treatment day 25 to give the animals at least 48 hours rest before pairing, and thus not to interfere with the purpose of the DRF study itself. Although, a 28-day repeat-dose protocol is recommended within the OECD TG 470, alternate dosing schedules can be utilised, especially in instances when they facilitate integration with other toxicology studies. With 25-day exposure no reasonable change in Pig-a sensitivity is expected, as previously proven by Dertinger et al. (2019) and also shorter exposure durations are included in the OECD testing guideline. Pre-dose sampling is planned to identify possible outliers, due to rule out naturally occurring high mutant or ‘jackpot’ animals. Since only limited laboratory historical control data are currently available at the CRO, a positive control of six animals, treated with N-ethyl-N-nitrosourea (ENU) is foreseen as well. The general study design is in accordance with OECD TG 470 and blood samples will be collected, stored, and later on analysed as described in the respective testing guideline.

Overall, although the Pig-a Assay according to OECD TG 470 is not yet listed in the Endpoint specific guidance, Chapter R.7a it is fully in line with
• the required test strategy for in vivo follow up genotoxicity testing in this case and
• with 3R principles in general.

Chen G, Wen H, Mao Z, et al. Assessment of the Pig-a, micronucleus, and comet assay endpoints in rats treated by acute or repeated dosing protocols with procarbazine hydrochloride and ethyl carbamate. Environmental and Molecular Mutagenesis. 2019-1 2019;60(1):56-71. doi:doi:10.1002/em.22227

Dertinger SD, Avlasevich SL, Torous DK, et al. 3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig-a gene mutation assessments: Proof-of-concept with 13 reference chemicals. Environmental and molecular mutagenesis. 2019-10 2019;60(8):704-739. doi:doi:10.1002/em.22312

EFSA, European Food Safety Authority. Opinion of the Scientific Committee on a request from EFSA on the "Clarification of some aspects related to genotoxicity assessment". The EFSA Journal. 2017;15(12):e05113. doi:

OECD, Organisation for Economic Co-Operation and Development. The in vivo erythrocyte Pig-a gene mutation assay. Part 1: Detailed Review Paper and Retrospective Performance Assessment Prepared by Heflich et. al for the Organisation for Economic Cooperation and Development Working Group of the National Coordinators of the Test Guidelines Programme. OECD Publishing, Paris.; 2020.
Robison TW, Heflich RH, Manjanatha MG, et al. Appropriate in vivo follow-up assays to an in vitro bacterial reverse mutation (Ames) test positive investigational drug candidate (active pharmaceutical ingredient), drug-related metabolite, or drug-related impurity. Mutation Research - Genetic Toxicology and Environmental Mutagenesis. 2021-8 2021;868:503386. doi:doi:10.1016/j.mrgentox.2021.503386

Zhu X, Huo J, Zeng Z, et al. Determination of potential thresholds for N-ethyl-N-nitrosourea and ethyl methanesulfonate based on a multi-endpoint genotoxicity assessment platform in rats. Environmental Science and Pollution Research International. 2022-12 2022;29(56):85128-85142. doi:doi:10.1007/s11356-022-21605-z

Data source

Materials and methods

Test guideline
other: OECD guideline 470 (Mammalian Erythrocyte Pig-a Gene Mutation Assay)

Test material

Constituent 1
Chemical structure
Reference substance name:
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Results and discussion

Applicant's summary and conclusion