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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data from the read across substance Docusate sodium did not induce mutations in the Ames test up to concentrations close to toxic range, both with/without liver metabolic activation (S9) system. A marginal increase in chromosome aberrations was observed in CHO cells in the presence of S9 only and at the highest dose levels near cytotoxicity, which was likely related to an indirect mechanism. Read across with a category group substances did not reveal chromosome aberrations up to cytotoxic concentrations, nor were there positive effects in the mouse lymphoma assay. Finally, an in vivo Micronucleus assay with Docusate sodium tested up to the maximum tolerated dose level of 2000 or 1000 mg/kg bw/day in males and female rats, respectively, that produced clinical signs of toxicity and cytotoxicity in bone marrow, following repeated oral administration showed no genotoxic properties in the rat bone marrow micronucleus test at a sampling time of 24 hours after the last dosing.

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

Genetic toxicity in vivo

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

Additional information

A key study for bacterial mutagenicity was performed in an Ames reverse mutation test with 5 Salmonella typhimurium strains (Cytec, Clare 1993); the study was conducted according to OECD 471 and GLP guidelines, and was considered to be reliable, adequate and relevant. After a range-finder experiment showing cytotoxicity at the highest concentration of 5000 µg/plate, maximum test concentrations of 1000 and 2500 µg/plate were chosen for the main experiment 1. In experiment 1, concentrations were close to the limit of toxicity, therefore for experiment 2, concentrations for all strains were maximally 2000 µg/plate without S9 and 2500 µg/plate with S9. In both experiments, Docusate sodium did not result in statistically significant increases in revertant number of colonies, both with and without S9. In addition, a literature study was available with Docusate sodium tested at concentrations of 1, 10 and 100 µg/plate, which did also not result in statistically significant increases in revertant number of colonies, both with and without S9 (Literature, Bonin & Baker 1980). It is not further taken into account due to the low concentrations.

 

A key study was available for the mutation assay at the thymidine kinase locus (TK+/-) in mouse lymphoma L5178Y cells with sodium dihexylsulfosuccinate (CAS 2373-38-8); the study was conducted according to OECD 476 and GLP guidelines, and was considered to be reliable, adequate and relevant (Cognis, Wollny 2006). The assay was performed in two independent experiments, using two parallel cultures each. Both main experiments were performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment (experiment IA) was required to verify the results obtained in experiment I and to cover highly toxic concentrations using an adjusted concentration range. The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with regard to the molecular weight of the test item. The dose range of the main experiments was limited by toxicity of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item. In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

 

Weight of evidence was available for chromosome aberration potential from read across substances in the same category:

- Docusate sodium (CAS No. 577-11-7) was tested in V79 CHO cells (Cytec, Marshall 1993); the study was conducted according to OECD 473 and GLP guidelines, and was considered to be reliable, adequate and relevant. The highest dose level used, 470 µg/mL was close to the solubility limit of Docusate sodium in culture medium. Treatment of cultures with Docusate sodium in the absence of S9 resulted in frequencies of cells with aberration which were similar to and not significantly different from those in concurrent negative controls. Numbers of aberrant cells in all treated cultures fell within (or very close to) the historical negative control range. Cultures treated with Docusate sodium in the presence of S9 in Experiment 1 had significantly increased frequencies of cells with aberrations at the highest dose level chosen for analysis (120 µg/mL) which fell outside the historical negative control range. In contrast, cultures treated and sampled under these conditions in Experiment 2 had normal frequencies of aberrant cells at all dose levels analysed, however the highest concentration failed to induce 50% mitotic inhibition. This part of the assay was therefore repeated on 2 further occasions using a smaller interval, however, it was not possible to achieve 50-75% mitotic inhibition. The highest scorable dose in the second trial was 130 µg/mL and slides from these cultures were analyzed. Due to applicant and author, small but statistically significant increases in cells with aberrations were seen at both 125 and 130 µg/mL, however the effect was considered to be of marginal biological significance because numbers of aberrant cells fell outside the normal range in only a single replicate at the highest dose. Author further mentioned that most likely aberration induction involved an indirect mechanism. Therefore induction of chromosome aberration is not concluded based on the opinion of the applicant. This is underlined by a read across chromosome aberration studies below.

-Sodium dihexylsulfosuccinate (CAS No. 2373-38-8) was tested according to OECD 473 and GLP guidelines covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (Cognis, Schulz 2003). A pre-test for cytotoxicity testing was performed up to 5000 µg/mL, resulting in reduced cell numbers after 4h treatment with 625 µg/mL and above in the absence and the presence of S-9 mix. Considering the toxicity data of the pre-test, 800 µg/mL (without S9) and 1000 µg/mL (with S-9) were chosen as top concentrations in the main experiment I. Dose selection of experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24h exposure with 312,5 µg/mL and above. Therefore 600 µg/mL was chosen as top treatment concentration for continuous exposure in the absence of S-9 and 800 µg/mL in the presence of S-9. In both experiments, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed after treatment with the test item. No increase in frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate positive controls induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, the test material was considered to be non clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.

-Sodium bis (C11 -14 -isoalkyl, C12-rich) sulfosuccinate (CAS No. 848588-96-5) was tested in a Micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals (Flügge, 2013). The test was carried out employing 2 exposure times without S9 mix: (4 hours and 20 hours) and one exposure time with S9 mix (repeated). The harvesting time was 24 hours after the end of exposure. Each treatment was conducted in duplicate. The test item was completely dissolved in aqua ad iniectabilia, which also served as the vehicle control. Based on a preliminary experiment, cytotoxicity was noted starting at a concentration of 250 µg test item/mL in the experiment without and with metabolic activation. Hence, 250 µg/mL were employed as the top concentration for the main test without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure). There was no increase in micronuclei up to the cytotoxic concentration when compared to control both with and without metabolic activation. Under the present test conditions, the test item tested up to cytotoxic concentrations, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of any chromosomal damage in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant damage.

 

Read across with category members/structural analogues was performed for genotoxicity (chromosome aberration) but also for other endpoints (e.g. sensitisation, repeated dose toxicity), demonstrating a similar toxicological profile for Docusate sodium and Sodium dihexylsulfosuccinate and other analogues.

 

Summarising the results, no positive genotoxicity was observed with Docusate sodium and category member Sodium dihexylsulfosuccinate (CAS 2373-38-8 and CAS 845888-96 -5). As indicated in section 7.7, there is also information from a 24 -month carcinogenicity test in rats with Docusate sodium, which was negative for increased tumor incidence.

 

An in vivo cytogenetics assay was requested based on ECHA Communication number CCH-D-2114321066-61-01/D. Docusate sodium was used as read across substance for the registration of Potassium 1,2-bis(2-ethylhexyloxycarbonyl)ethanesulphonate (CAS 7491-09-0). Based on the results of the in vitro chromosomal aberration (OECD 473 -) study with Docusate sodium, which showed increases in the proportion of cells with structural aberrations, ECHA requested an in vivo cytogenetics assay (mammalian erythrocyte micronucleus test, mammalian bone marrow chromosomal aberration test or mammalian alkaline comet assay). The new study with Docusate sodium was conducted for both the registrations of both Potassium 1,2-bis(2-ethylhexyloxycarbonyl)ethanesulphonate and Docusate sodium, and is described below.

 

A key in vivo bone marrow micronucleus test was performed with Docusate sodium in the rat for the detection of damage to the chromosomes or the mitotic apparatus following repeated oral administration of the test item on two days at a 24-hour interval. Blood samples and bone marrow samples were taken for possible bioanalysis.

In a preliminary dose-range-finding study at dose levels of 500, 1000 and 2000 mg/kg bw, the maximum tolerated dose (MTD) leading to a reduction in the proportion of immature erythrocytes among total erythrocytes was determined to be 2000 mg/kg bw/day. No signs of systemic toxicity were noted up to the highest reasonable dose level of 2000 mg per kg bw/day. No animal died prematurely.

In the main study, dose levels of 500, 1000 and 2000 mg/bw/day and the vehicle (tap water) were administered two times at 0 and 24 hours. Females were compared to males for the vehicle control and high dose group due to differences in toxicity observed between male and female rats in the 2-week dietary toxicity study, where males showed increased liver and kidney weights whereas this was not marked in females. Hence, in this study each group consisted of 5 male rats and, in addition, 5 females each were employed for the vehicle control and high dose groups (1000 and 2000 mg/kg bw/day). The administration volume was 20 mL/kg bw.

The male animals treated with 500 or 1000 mg/kg bw/day revealed soft faeces on Test days 2 and 3, respectively. 1000 mg/kg bw/day caused slightly reduced motility, slight ataxia and slight dyspnoea in the males 6 hours after the first administration until Test day 3. The high dosed male and female animals with 2000 mg Docusate Sodium per kg bw/day revealed soft, mucous and evil-smelling faeces, slightly to moderately reduced motility, slight to moderate ataxia and slight to moderate dyspnoea 6 hours after administration until Test day 3 in all 5 of 5 male and 5 of 5 female animals. Four of 5 female animals died prematurely within three days and were not suitable for the micronucleus assay. Hence, 1000 mg/kg bw/day were administered to 5 female rats as the maximum tolerated dose level for females. 1000 mg/kg bw/day caused pilo-erection and soft faeces on Test days 2 and 3 in all 5 female rats. All high dosed animals (males: 2000 mg per kg bw/day and females: 1000 mg/kg) and, in addition, 3 of 5 male animals dosed with 1000 mg per kg bw/day lost body weight within 3 days.

Bone marrow was sampled on Test day 3 (24 hours after the last dose for the vehicle control and test item-treated animals and 48 hours after single administration for the positive reference item). The PCE/NCE ratios of the high dosed male (2000 mg/kg bw/day) and female animals (1000 mg/kg bw/day) were statistically significantly decreased (at p < 0.01) and of the males treated with 1000 mg/kg bw/day significantly decreased at p < 0.05 compared to the control animals which demonstrate cytotoxicity and, hence, bone marrow exposure to the test item. Therefore no further bioanalysis was necessary.

Docusate sodium did not increase the incidence of micronucleated polychromatic erythrocytes (PCE) at any of the three tested dose levels of 500, 1000 or 2000 mg test item/kg bw/day. The incidence of micronucleated PCE was 0.9 or 1.0 per 1000 PCEs for the high dosed samples for males (2000 mg/kg bw/day) and females (1000 mg/kg bw/day). The corresponding micronucleus frequency of the vehicle control (negative reference) was 0.9 or 1.0 per 1000 PCEs (males and females, respectively). Administration of cyclophosphamide (positive reference item) significantly increased the number of micronucleated PCE to 29.8 per 1000 PCEs.

In conclusion, under the present test conditions, Docusate sodium tested up to the maximum tolerated dose level of 2000 or 1000 mg/kg bw/day in males and females, respectively, following repeated oral administration showed no genotoxic properties in the rat bone marrow micronucleus test at a sampling time of 24 hours after the last dosing.

In the same system, cyclophosphamide (positive reference item) induced significant damage.

 

Based on the combination of in vitro and in vivo studies, Docusate sodium is not considered to have genetic toxicity potential. This lack of genetic toxicity potential will also apply to to Potassium 1,2-bis(2-ethylhexyloxycarbonyl)ethanesulphonate CAS No 7491-09-0 as the only differ in the sodium ion being replaced by a potassium ion. In aqueous condition both will be dissociated so the different metal ion will have no influence on potential for genetic toxicology.

Justification for classification or non-classification

As the mutagenicity experiments did not indicate genotoxicity potential for the docusate sodium the source substance for read across to Potassium 1,2-bis(2-ethylhexyloxycarbonyl)ethanesulphonate CAS No 7491-09-0, classification for genotoxicity is not warranted.