Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

not mutagenic

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The following data were obtained for Similar Substance 01. It is expected that the Target substance will present similar mutagenic potential to this read across substance. Moreover, a read-across approach using data from Similar Substance 02 and Similar Substance 03 was used to complete the in vitro mammalian cell mutagenicity endpoint for which Similar Substance 01 did not have experimental data. Justification for the use of a Read Across approach is provided in Section 13 of IUCLID.

IN VITRO BACTERIAL REVERSE MUTATION TEST

Similar Substance 01 was examined for its ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The test was performed according to the OECD Guideline 471 (1997) and the EU method B.13/14 of EC 440/2008. The five tester strains TA1535, TA1537, TA98, TA100 (S. tuphimurium) and WP2 uvrA (E. coli) were used. Experiments were performed both in the absence and presence of metabolic activation, using rat liver S9 fraction pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in sterile water for injection. The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals. On the basis of toxicity test results, the test item was assayed at five dose levels from 313 to 5000 µg/plate in the Main Assay using the plate incorporation method. Neither precipitation of the test item, nor toxicity was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.

Similar Substance 01 induced dose-related and large increases in the number of revertant colonies in TA1537, TA98 and TA100 tester strains, both in the absence and presence of S9 metabolism. Positive increases in the number of revertant colonies (2.9-fold) were also observed with TA1535 tester strain in the presence of S9 metabolism. All the validity criteria were met. Since a clear, positive response was observed, no further experiment was undertaken. It can be concluded that Similar Substance 01 induces reverse mutation in bacteria both in the absence and presence of S9 metabolism, under the reported experimental conditions.

Similar Substance 02 and Similar Substance 03 were also found to be positive for in vitro bacterial cell mutagenicity. Similar Substance 02 was tested using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli strain WP2 uvr A, both with and without metabolic activation, according to the EU Method B.13/14 (2008). S. typhimurium strains TA 1537 and TA 100 as well as E. coli strain WP2 uvr A were found to be positive to genotoxicity both in the presence and absence of metabolic activation. Similarly, Similar Substance 03 was tested for bacterial cell mutagenicity using five S. typhimurium strains (TA 1535, TA 1537, TA 1538, TA 98 and TA 100), according to the OECD Guideline 473 (1983) and the EU Method B.14 (1984), and tested positive for mutagenicity in S. typhimurium strains TA 1537, TA 1538 and TA 100 in both the presence and absence of metabolic activation, and TA 98 with metabolic activation only. The presence of consistently positive bacterial mutagenicity results in all three substances across the board supports the use of the read-across approach for estimating the bacterial mutagenic potential of the Target Substance and the necessity of further mutagenic testing.

IN VITRO MICRONUCLEUS TEST IN HUMAN LYMPHOCYTES

Similar Substance 01 was assayed for the ability to induce micronuclei in human lymphocytes following in vitro treatment, according to the OECD Guideline 487 (2016). Three treatment series were included in the study. A short term treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. A harvest time of approximately 32 hours, corresponding to approximately two cell cycle lenghts, was used. A long term (continuous) treatment was also performed in the absence of S9 metabolism only, until harvest at approximately 31 hours. Solutions of Similar Substance were prepared in complete culture medium. Based on the type of substance, the top concentration selected for treatment was 5000 µg/ml. Eight lower dose levels spaced by a factor of 1.5 were used for the three hour treatment in the absence and presence of S9 metabolism. An additional dose level (130 µg/ml) was included for the continuous treatment in the absence of S9 metabolism. The experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. CytoB was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

In order to evaluate Similar Substance 01 concentrations at adequate levels of cytotoxicity, covering a range from the maximum (55 ± 5 %) to slight or no toxicity, the following dose levels were selected for the scoring of micronuclei: for short term treatment 2220, 988, 439 µg/ml (with S9) and 3330, 1480, 658 µg/mL (without S9); for long term treatment 439, 293 and 195 µg/ml (without S9).

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with Similar Substance 01, no statistically significant increase in the incidence of micronucleated cells was observed at any dose level nor concentration related increase of cells bearing micronuclei was observed in any treatment series. All the validity criteria were met. It can therefore be concluded that Similar Substance 01 does not induce micronuclei in human lymphocytes following in vitro treatment, under the reported experimental conditions.

A micronucleus assay was also performed on Similar Substance 02 according to the same guideline, which demonstrated that Similar Substance 02 also did not induce micronuclei in human lymphocytes. Two similar experiments were performed on Similar Substance 03. Both were chromosome aberration assays on Chinese hamster V79 cells according to the OECD Guideline 473 (1983), but used different concentration ranges of 0.8 to 30.0 µg/ml in one study, and 0.3 to 2.5 mg/ml in the other. Both studies demonstrated that Similar Substance 03 did not produce any relevant increase in structural or numerical aberrations after treatment either without or with metabolic activation. The homogeneity of chromosome damage results across all three read-across substances supports the use of the read-across approach for determining the potential of the Target Substance to induce chromosome damage.

As data on the test item regarding the in vitro gene muation potential in mammalian cells was also required, and no relevant study was available, the gene mutation potential can be assumed using a read-across approach with two other similar substances. Specifically, a mammalian gene mutation assay was performed on hprt and xprt genes of Chinese hamster V79 lung fibroblast cells using Similar Substance 02 and Similar Substance 03 according toto the OECD Guideline 476 (2016 and 1984, respectively), and was used in a read-across approach as detailed in IUCLID 13. Mammalian gene mutation assay results were as follows:

IN VITRO GENE MUTATION ASSAYS

In the first study, a solubility/sterility preliminary test was first performed on Similar Substance 02 to determine whether 2.0 and 5.0 mg/ml test item was soluble in DMEM medium; no sterility test was necessary. A preliminary test for mycoplasma was performed at concentrations of 0.25, 0.5, 1.0, 2.0 and 5.0 mg/ml. A preliminary test for cytotoxicity was also performed using concentrations of 0.01, 0.1, 0.2, 0.5, 1.0 and 2.0 mg/ml without metabolic activation only. pH of the cultivation medium with various concentrations was checked. In the mutation assay, Chinese hamster lung fibroblasts (V79) were exposed to concentrations of 0.25, 0.5, 1.0 and 2.0 mg/ml (based on results from the cytotoxicity pre-test) for 3 hours both with and without S9 rat liver metabolic activation. A further concentration of 5.0 mg/ml alongside controls was tested in a subsequent additional experiment. Negative (solvent: DMEM) and positive (DMBA, EMS50 and EMS100) controls were tested alongside Similar Substance 02 concentrations.

Similar Substance 02 was found to be soluble at both 2.0 and 5.0 mg/ml in DMEM. No cytotoxicity was observed in the cytotoxicity pre-test. All media after cultivation of cells were considered free of mycoplasma. In the mutagenicity assay, the highest cytotoxicity was 31.6 %, observed at 0.25 mg/ml with metabolic activation, which was considered an error due to lower toxicity at higher concentrataions. Cytotoxicity in the highest concentration was approximately 25 % with metabolic activation (2.0 mg/ml) and 24 % without metabolic activation (2.0 mg/ml). Medium cytotoxicity of approximately 45 % and 36 % was observed in the additional experiment (5.0 mg/ml), with and without metabolic activation, respectively. The plating efficiency was acceptible: more than 2 million cells per replicate were influenced; in 2 cases, less than 2 million cells were seeded for further cultivation, however, the sum of cells in both replicates fulfilled the condition of 2 million transferred cells, so no impact is expected. Up to the highest investigated dose (5.0 mg/ml), no relevant increase in mutant colony numbers was observed in experiments either with or without metabolic activation. The positive controls showed a distinct increase in induced mutant colonies. Similar Substance 02 did not induce point mutations under the experimental conditions at the HPRT locus in Chinese hamster V79 cells. Therefore, Similar Substance 02 is considered to be non-mutagenic in this HPRT assay.

In the second study, using Similar Substance 03, a preliminary dose-finding experiment was performed using concentrations of 0.01, 0.1, 1, 3, 6, 10, 25 and 50 µg/ml. The mutagenicity test was performed in two independent experiments, using identical procedures, both with and without S9 liver microsomal activation. Chinese hamster V79 cells were exposed to the following concentrations: 5.0, 10.0, 25.0 and 50.0 µg/ml. Negative, solvent (DMSO) and positive (EMS: 1.0 mg/ml and DMBA: 15.4 µg/ml) controls were tested in parallel.

Plating efficiency was reduced in experiment I (both with and without metabolic activation) and experiment II (only with metabolic activation) at the highest concentration. Mutation rates were not increased in treated groups when compared with negative and solvent controls. No reproducible, dose-dependent increase in mutant colony numbers was observed and were within the range of the negative controls. Mutant frequencies were increased by approximately a factor of 3 in experiment II (without metabolic activation at the highest dose of 50 µg/ml and with metabolic activation at 25 µg/ml), however, these increases were not considered biologically relevant. Positive controls demonstrated a distinct increase in induced mutant colonies. Therefore, Similar Substance 03 is considered to be non-mutagenic in this HPRT assay.

It is worth noting that bacterial mutagenicity study and chromosome damage study results permormed using Similar Substance 02 and Similar Substance 03 are comparable to those of Similar Substance 01. Specifically, the Ames test results were positive for bacterial mutagenicity (all three substances) and the chromosome aberration studies (Similar Substance 03) and micronucleus studies (Similar Substances 01 and 02) were negative for genotoxicity. Therefore, the use of the read-across approach for determining the mutagenic potential of the Target Substance is appropriate. Additionally, three in vivo mutagenicity studies were available for Similar Substance 03, and all three were found to be negative for mutagenicity. Specifically, two in vivo micronucleus assays (OECD 474, 1983) and an in vivo DNA repair synthesis assay were performed using the same batch of the in vitro tests.

IN VIVO MUTAGENICITY TESTS

In the first in vivo micronucleus assay (CCR, 1992), 6 male and 6 female Wistar rats were administered 1000 mg/kg bw of Similar Substance 03 in aqua deionised in a 10 ml/kg bw solution once per day for 28 days. Bone marrow cells were collected from each animal and 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. Animals in a negative control group were administered 10 ml/kg bw of aqua deionised alone in parallel; animals in a positive control group were administered 10 mg/kg bw cyclophosphamide in parallel. 5 animals of each sex per group were evaluated for cytotoxicity as number of normochromatic erythrocytes (NCE) per 1000 PCE (also referred to as the PCE:NCE ratio).

2 female and 1 male treatment animals died before the end of the study period. The PCE:NCE ratio of cells from treatment animals was increased compared to the negative controls indicating a substance-dependent effect on the target cells. No statistically significant enhancement in the frequency of the detected micronuclei was observed following administration of the test item. The number of micronuclei observed in treatment males was not substantially enhanced compared to those of control males. The number of micronuclei observed in treatment females was slightly enhanced, however, it was not considered to be of biological relevance, which was supported by biometric analysis of females. The cells positive control animals showed a distinct increase in induced micronuleus frequency. Therefore, under the experimental conditions reported, Similar Substance 03 did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat.

In the second in vivo micronucleus assay (CCR, 1988), a preliminary test for toxicity determined the highest possible dose of Similar Substance 03 of 4000 mg/kg bw was appropriate for the main study. Groups of 5 male and 5 female mice were administered Similar Substance 03 at a concentration of 4000 mg/kg bw in 4 % CMC as a 20 ml/kg bw solution by oral gavage. 24 hours (group 1), 48 hours (group 2) and 72 hours (group 3) after administration, bone marrow cells were collected from each animal and 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. Cytotoxicity was evaluated as number of normochromatic erythrocytes (NCE) per 1000 PCE. Animals in a negative control group were administered 20 ml/kg bw of 4 % CMC alone in parallel; animals in a positive control group were administered 30 mg/kg bw cyclophosphamide in parallel (24 hour sampling time only).

Similar Substance 03 did not induce cytotoxic effects as the PCE:NCE ratio was not affected compared to the corresponding negative controls. No enhancement in the frequency of the detected micronuclei was observed at any preparation interval following administration of Similar Substance 03. The positive control showed a distinct increase in induced micronuleus frequency. Therefore, under the experimental conditions reported, Similar Substance 03 did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

The potential of Similiar Substance 03 to induce DNA repair was evaluated in an in vivo/in vitro Unscheduled DNA Synthesis test (UDS assay, CCR, 1992) performed using rat hepatocytes. Male Wistar rats were administered the test item by oral gavage in concentrations of either 100 mg/kg bw (4 animals) or 1000 mg/kg bw (4 animals) as a 10 ml/kg bw solution in aqua bidist., then three animals from each group were sacrificed by liver perfusion 16 hours after treatment. Liver hepatocytes were isolated, cultured for four hours and radiolabelled, then nuclear and cytoplasm grain counts were performed visually. Additional groups of 4 animals each were administered either aqua bidest. alone (negative control); 100 mg/kg bw 2-acetylaminofluorine (positive control) or 1000 mg/kg of Similar Substance 03 (sacrificed after four hours).

No dose level of Similar Substance 03 revealed UDS induction in the hepatocytes of the treated animals compared to negative controls. Neither the nuclear grains nor the resulting net grains were enhanced due to the in vivo treatment of the animals with the test item for either 4 hours or 16 hours. PCEs of the positive control animals demonstrated a significant and dose-depended increase in nuclear grains compared to negative controls. Based on the absence of DNA repair, Similar Substance 03 does not induce DNA damage in this UDS assay and, therefore, is considered non-mutagenic under test conditions.

Justification for classification or non-classification

According to the CLP Regulation (EC) no. 1272/2008 a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known (including specific base pair changes and chromosomal translocations). The term ‘mutagenic’ and ‘mutagen’ will be used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms. For the purpose of the classification for germ cell mutagenicity, substances can be allocated in one of two categories:

- Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans, or

- Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Based on the experimental data available, the test item does not meet the classification criteria for genetic toxicity according to the CLP Regulation (EC) no. 1272/2008.