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Key value for chemical safety assessment

Additional information

There are no genetic toxicity studies for Benzenesulfonic acid, mono-C20-24 (even)-sec-alkyl derivs., para-, sodium salts (generic name: C20 -24 sodium sulfonate) available. Therefore, data on structurally similar analogues is taken to cover information requirements for this endpoint.

Genetic toxicity data (in vitro) for read across substances:

In the key study, a reverse gene mutation assay in bacteria, the strains TA98, TA100, TA1535 and TA 1537 of S. typhimurium and E. coli WP2uvrA were exposed to a calcium sulfonate read across substance, (Analogue of CAS 70024-69-0), at concentrations of 100, 330, 1000, 3330 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (Sanitised, H., 1989; according to OECD 471). A Dose Range-finding Study was conducted using tester strains TA98 and TA100, and dose levels of test material ranging from 0.003 to 10 mg/plate were used.No cytotoxicity was observed in the dose range-finding study with tester strains TA100 and WP2uvrA with or without metabolic activation as evidenced by normal background lawn and no reduction in the number of revertants/plate. The S9 optimization study was performed using TA98 and TA100 with the highest non-cytotoxic dose of test article, (10,000 µg/plate) and concentrations of S9 mix of 25-400 µL. In the absence of any effect 25 µL S9 mix/plate was used in the mutagenicity study. In the main study there were two treatment sets for each tester strain, with (+S9) and without (-S9) metabolic activation. Each of the tester strains was dosed with five concentrations of test substance, vehicle controls, and a positive control. Three plates/dose group/strain/treatment set were evaluated. The results of the initial assay were confirmed in a second independent experiment. 100 µL of test material, positive control or vehicle control were added to each plate along with 100 µL of tester strain, S9 mix (if needed) and 2.0 mL of top agar. This was overlaid onto the surface of 25 mL minimal bottom agar in a petri dish. Plates were incubated for 48 hours at 37°C. The condition of the bacterial background lawn was evaluated for cytotoxicity and test article precipitate. The test material formed a stable emulsion with the vehicle and the dilutions were well dispersed in the top agar. However after incubation test material was visible at all dose levels in the top layer. The test material was not cytotoxic to any tester strain. In the repeat study statistically significant increases in revertant colonies were observed in TA1535 without metabolic activation and in WP2uvrA with metabolic activation. However since these findings were not found during the first experiment they were not considered biologically significant. The positive control for each respective test strain exhibited at least a 3-fold increase (with or without S9) over the mean value of the vehicle control for a given strain, confirming the expected positive control response. Dosing solution analysis confirmed that high dose concentration was acceptable.Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the pre-incubation modification of the Salmonella/microsome test.

In a supporting study with a calcium sulfonate read across substance, (CAS 68783-96-0), the test material was applied (doses of 250, 500, 1000, 2500 and 5000 µg/plate in the initial assay, and 1000, 2000, 3000, 4000 and 5000 in the repeat assay) in agar to the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (Sanitised, L., 1995; according to OECD 471). As metabolic activation system, S9 aroclor induced rat liver was used. Tetrahydrofuran was the vehicle for the test material, and DMSO was the vehicle for the positive controls (9-aminoacridine, 2-nitrofluorene and 2-aminoanthracene and N-methyl-N-Nitro-N-Nitrosoguanidine).Prior to study initiation the solubility of the test substance in the vehicle (tetrahydrofuran, 2 vehicle controls for each strain) was confirmed. In addition, one non-treated control and a positive control were tested for each strain. In the main study there were two treatment sets for each tester strain, with and without metabolic activation. Three plates/dose group/strain/treatment set were evaluated. The results of the initial assay were verified by repeating the assay at dose levels of 1000, 2000, 3000, 4000 and 5000 µg/plate. After 2 days of incubation all plates in the initial and repeat assays were evaluated for gross toxic effects and total revertant colony numbers.The test substance did not induce significant increases in revertant colonies (equal to or greater than three times the THF control) in any of the tester strains, at any dose level, with or without metabolic activation in the initial or repeat assays.

 

In another supporting study with a calcium sulfonate read across substance, (CAS 61789-86-4), the test material was applied (All strains - except TA1537 - with and without S9 (-S9) at 5000 1500, 500, 150, 50 and 15 µg/plate, and at 2500, 750, 250, 75, 25 and 7.5 µg/plate for TA1537 only without S9) in agar to the S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 (Loveday, 1988a; according to OECD 471). As metabolic activation system, S9 aroclor induced rat liver was used. Tetrahydrofuran was the vehicle for the test material, and DMSO or sterile distilled water was the vehicle for the positive controls (9-aminoacridine, 2-aminoanthracene, benzo(a)pyrene, 4 -nitroquinoline-N-oxide, 4 Nitrofluorene and N-ethyl-N-nitro-N-nitrosoguanidine). Negative control and positive controls were tested for each bacterial strain.The test sample caused no visible reduction in the growth of the bacterial background lawn at any dose level and was tested up to the maximum recommended dose level of 5000 µg/plate. No test sample precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. The test sample was considered to be non-mutagenic under the conditions of this test.

 

Mammalian cell gene mutation data (in vitro) for read across substances

In a key mammalian cell gene mutation assay, mouse lymphoma L5178Y cells were exposed to a calcium sulfonate read across substance, (CAS 68783-96-0), at concentrations of 0, 500, 1000, 1500, 2000, 4000 and 5000 µg/mL with and without S9 metabolic activation (Sanitised, D., 1984; according to OECD 476). As vehicle DMSO was used, as positive controls 7,12 -dimethylbenzanthracene and ethylmethanesulphonate were used.

Prior to study initiation the solubility of the test substance and of the positive control materials in the vehicle (DMSO) was confirmed. A pre-test dose range finding study was conducted at concentrations up to 10,000 ug/mL with and without metabolic activation. In the main study there were two treatment sets for each concentration of test substance, with (+S9) and without (-S9) metabolic activation. DMBA (positive control) was tested with activation and EMS (positive control) was tested without activation. The test material was added to cells with and without activation and incubated for four hours. Cells were then washed and placed in suspension cultures for two days with a cell population adjustment at 24 hours. The cells were then plated in a restrictive media containing trifluorothymidine (TFT) which allows TK-/-cells to grow. Cells were also plated in a non-restrictive media that indicated cell viability. Plates were incubated at 37°C in a humidified 5% CO2 atmosphere for 10-12 days. Following incubation all plates were scored for total number of colonies/plate. The frequency of mutation by dose was determined by comparing the average number of colonies in the mutagenicity plates to the average number of colonies in the corresponding viability plates. None of the cultures treated with test material with or without activation exhibited mutant frequencies significantly different from the average mutant frequency of the negative (solvent) controls at a per cent total growth of 10% or greater. Positive and vehicle control group responses were appropriate and met the criteria outlined above. So the test substance was not mutagenic in this assay with or without metabolic activation.

Chromosome aberration data (in vitro) for read across substances

In a key mammalian cell cytogenetics assay [chromosome aberration], Chinese hamster ovary (CHO, clone tested WBL) cell cultures were exposed to a calcium sulfonate read across substance, (CAS 68783-96-0), at concentrations of 0, 10, 20, 40, 80, 120 and 160 µg/mL with and without S9 metabolic activation (Sanitised, M., 1995; according to OECD 473). As vehicle Tetrahydrofuran (THF, for the test material) and acetone (for the positive controls) was used.The solubility of the test substance in the culture medium was established at concentrations of 10, 20, 39, 78, 156, 313, 625, 1250, and 2500 µg/mL. Visual and microscopic examinations were made for precipitation at 0, 30 and 180 minutes post preparation. Concentrations showing signs of insolubility at any of these time points were considered unsuitable for dosing.A Dose range finding study was conducted with the test substance and vehicle controls tested in duplicate cultures each with and without activation.Test substance tested at concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL. Cytotoxicity and mitotic indices were evaluated.In the main study there were two treatment sets for each concentration of test substance, with (+S9) and without (-S9) metabolic activation. 7,12-Dimethylbenz(a) anthracene - DMBA (positive control) was tested with activation and N-Methyl-N-Nitro-N-Nitrosoguanidine - MNNG (positive control) was tested without activation. Prepared cultures were treated with test substance or control material and were incubated for 16 hours. A repeat assay was performed using 16 and 40 hour harvest time points. Vehicle, MNNG and DMBA cultures were incubated for 16 hours only. Two to three hours prior to the 16 and 40-hour harvest the spindle inhibitor, Colcemid, was added to each culture to obtain a final concentration of 0.2 µg/mL. Harvested cells were evaluated microscopically for per cent confluency, morphology and estimated number of mitotic cells prior to harvest. The initial assay and the repeat assay (each after 16-hr incubations) with and without S9 were conducted with 10, 20 and 40 µL/mL. The repeat assay with and without S9 (after 16 and 40-hr incubations) was conducted with 10, 20 and 40 µg/mL. Slides were prepared for these groups using Giemsa stain. Two slides/treatment groups were evaluated. 200 metaphase cells (100 per culture) each containing 19-23 chromosomes per treatment group were scored. Chromosomes were counted for each cell. Chromosome aberrations, either chromosome or chromatid type were recorded. The following observations were recorded and excluded from the total aberration frequency: gaps, polyploid and endoreduplicated cells, pulverized chromosomes, Robertsonian translocations, translocations and abnormal monocentric chromosomes. The percentage of aberrant cells and the frequency of aberration (%) per treatment group were determined. In order for a test substance to be considered to have induced a positive response compared to vehicle control a statistically significant dose related increase in the percentage of aberrant cells along with a mean percentage of aberrant cells in excess of 5% in at least one treatment group were required. Or, a reproducible and statistically significant response in at least one treatment group with a mean % of aberrant cells exceeding 5% was observed. Test substance concentration verification was performed on the highest stock concentration in both the initial and repeated assays.Results were within 6% of nominal. Samples were homogeneous and stable for the intended period of use. Precipitate and/or cloudiness were present with and without metabolic activation at concentrations of 39 µg/mL and greater. In the culture medium solubility test precipitate and/or cloudiness were present with and without metabolic activation at concentrations of 39 µg/mL and greater. In the pre-test toxicity assay there was an 81% reduction (compared to vehicle control) in cell survival at 160 µg/mL without metabolic activation. The doses selected for the initial assay were 10, 20, 40, 80, 120 and 160 µg/mL. A greater than 50% reduction in cell survival and/or mitotic index was not observed in either the initial or repeat assays. Precipitation was observed at concentrations greater than 40 µg/mL in the chromosomal aberration assay. Therefore, 40 µg/mL was considered to be the limit of solubility for the test substance and was selected as the highest test concentration to be evaluated. There were no statistically significant differences in the number of chromosomal aberrations between the treated and vehicle control groups in either the initial or repeat assay at any dose level evaluated (10, 20 and 40 µg/mL with and without metabolic activation). In the initial 16-hour harvest, there were statistically significant increases with dose in the per cent of aberrant cells for both the activated and non-activated evaluations. These trends were not reproducible in the repeat 16-hour harvest and therefore were not considered biologically significant. Positive and vehicle control group responses were as expected. The positive control groups have frequencies of aberrations outside the normal range of the vehicle control and at least twice the vehicle control value. The test material was not clastogenic or genotoxic under the conditions of this study.

Data on DNA damage and/or repair - micronucleus assay for read across substances

In the key in vivo study, a mouse bone marrow micronucleus assay, Swiss Albino Crl:CD-I (ICR) BR mice were treated once via the peritoneum with a calcium sulfonate read across substance (Analogue of CAS 70024-69-0), at doses of 0, 100, 200, 400 and 500 mg/kg bw (Sanitised, I., 1989, OECD 474). Bone marrow cells were harvested at 24, 48 and 72 hours post-treatment.  A range-finding study was conducted at 200, 400 and 600 mg/kg. Mortality and physical observations were evaluated. During the dose range-finding study mortality (9 of 10 animals) was observed at 600 mg/kg but not at lower dose levels. Signs of toxicity observed at all dose levels included reduced faeces, reduced food consumption, hyperactivity and phonation. Decreased motor activity was observed at 400 and 600 mg/kg. Based on these results dose levels of 100, 200, 400 and 500 mg/kg were selected for the main study. In the main study the vehicle was peanut oil and the dose volume 5 mL/kg. As positive control substance triethylenemelamine was used. All animals were observed frequently for physiological or behavioural abnormalities on the day of dosing and at least twice daily thereafter. Body weights were taken on the first day of the study prior to treatment and at sacrifice. Macroscopic pathology performed on all animals at sacrifice. Five/sex from each treatment group and vehicle control group were sacrificed for bone marrow sampling 24, 48 and 72 hours post treatment. Positive controls sampled at 24 hours only. NCE/PCE ratio and %PCE of total erythrocytes were calculated by counting a total of > 1000 erythrocytes/animal. A total of 1000 PCE /animal were evaluated for the presence of micronuclei. The number of micronuclei in NCEs was also determined. During the main study, toxicity was observed at 400 and 500 mg/kg. At 500 mg/kg 5 males and 4 females of 15/sex died prior to the scheduled sampling time. At 400 mg/kg 1 of 18 treated females died on day 3. Other clinical signs of toxicity included palpebral closure, decreased motor activity and weakness. Cytotoxicity was observed in both sexes. A statistically significant increase in NCE/PCE ratio was observed in males at 500 mg/kg at 24 hours. Elevated ratios were also observed in individual animals of both sexes in other groups. Altered proportions of erythrocytes to nucleated cells were noted for both sexes in the treated groups. No biological or statistical significant increase in the number of micronucleated-PCE was observed in any treated group compared to the vehicle control. All values for individual animals were within the expected range of micronucleated-PCE/1000 PCE expected for control animals. The variability in response observed in the treated animals was similar to that observed in the vehicle controls. The positive control exhibited a statistically significant increase in micronuclei as expected. Chemical analysis confirmed that the dosing solution preparation procedure utilized for this study resulted in homogeneous solutions of appropriate concentration. As there was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time, under the conditions of this study the test material did not induce micronuclei in bone marrow erythrocytes of mice.

In a supporting in vivo study, a mouse bone marrow micronucleus assay, Swiss Albino mice were treated trice (three treatments approximately 24 h apart followed by sacrifice 24 h after final dose) via oral gavage with a calcium sulfonate read across substance (Analogue of CAS 68783-96-0), at doses of 0, 500, 1000 and 2000 mg/kg bw in peanut oil (Sanitised, N., 1995, OECD 474). Bone marrow cells were harvested at 24 hours after the last treatment. A range-finding study was conducted at 500, 1000 and 2000 mg/kg. Mortality and physical observations were evaluated. All dose range-finding animals survived and were free of clinical signs. Bone marrow toxicity was not observed at any dose levels tested. Therefore 2000 mg/kg was selected as the high dose for the micronucleus assay. The mid and low doses were selected to be 1/2 and 1/4 of the high dose. In the main study the vehicle was peanut oil and the dose volume 5 mL/kg. As positive control substance cyclophosphamide was used. All animals were observed frequently for physiological or behavioural abnormalities on the days of dosing and at least twice daily thereafter. Five/sex from each treatment group and vehicle control group were sacrificed for bone marrow sampling 24after the last treatment. NCE/PCE ratio and %PCE of total erythrocytes were calculated by counting a total of > 1000 erythrocytes/animal. A total of 1000 PCE /animal were evaluated for the presence of micronuclei. The number of micronuclei in NCEs was also determined. In the main study, all vehicle, positive control and treated animals were normal after dosing and remained healthy until sacrifice. There were no dose related increases or statistical differences in micronuclei formation observed at any dose level. Cytotoxicity was not observed since there was no statistically significant decreases in the percentage of polychromatic erythrocytes compared with the vehicle control. The positive control induced a statistically significant increase in mean micronucleated PCEs in both sexes compared with the vehicle controls which indicated the positive control was clastogenic and responded appropriately. The positive control also induced cytotoxicity. Chemical analysis confirmed the uniformity and stability of the test material in peanut oil for at least 9 days at all 3 concentrations. Concentration verification analysis confirmed that each dose level was within 3% of nominal concentration. The test substance was not genotoxic under the conditions of the study.

In another supporting in vivo study, a mouse bone marrow micronucleus assay, B6C3F1 mice were treated once via oral gavage with a calcium sulfonate read across substance (Analogue of CAS 61789-86-4), at the doses of 5 g/kg undiluted (Loveday, 1988b). A total of 80 mice, 40 males and 40 females, were used for the study. Ten animals (5 males and 5 females) were used per test group. Bone marrow cells were harvested at 18, 24 and 48 hours after the last treatment.  In the main study no vehicle was used and the dose volume did not exceed 10 mL/kg. Extra heavy U. S. P. mineral oil was used as the negative control. As positive control substance cyclophosphamide was used. Cyclophosphamide is tested at 50 and 75 mg/kg in female mice and at 25 and 50 mg/kg in male mice to ensure a positive response in both sexes. Only one positive control group is analysed for each sex. One male mouse, 63, dosed with cyclophosphamide had some food in its cage at the time of dosing; but this did not affect the study since a positive result was observed in this animal. Following each exposure period (18, 24 and 48 hours), mice were killed by cervical dislocation and the femurs were removed and the ends cut. Bone marrow cells were collected by flushing the cavity with a phosphate buffered saline solution (pH 7.4). The cells are concentrated by centrifugation and the cell pellet resuspended in a small volume of phosphate buffer. The cells are smeared on clean slides and air-dried. The slides are stained with acridine orange (0.125 mg/mL). Slides were made by placing one drop of suspension on a clean slide and spreading the cells evenly with a second slide. At least two slides were made from each animal. Slides were stained with acridine orange a few minutes prior to analysis using a fluorescent microscope. Slides are scored using a fluorescent microscope (FITC filter combination, excitation wavelength 495 nm, emission wavelength 520 nm). One thousand polychromatic erythrocytes (PCE) from each animal are scored for micro-nuclei. The ratio of PCE to normochromatic erythrocytes (NCE) are determined for each animal by counting a minimum of 1,000 erythrocytes . The NCE are not scored for micronuclei. The mean micronucleated PGEs for the negative control male mice were 2.6, 3.0 and 2.4 per 1000 PGEs for the three time periods. The average number of micronucleated PCEs for the positive control male mice was 14.5. The mean micronuc1eated PCEs for the test chemical were 1.7, 4.3 and 3.6 per 1000 PCEs for the three time points, respectively. These means are not significantly higher than the negative control values. The mean number of micronucleated PCEs for the negative control female mice were 1.9, 2.1 and 2.9 per 1000 PCEs for the three time periods, while the positive control female mice had an average of 20.5. The frequencies of micronucleated PCEs in female mice treated with the test chemical were 2.9, 3.2 and 2.1 per 1000 PCEs for the three time points, respectively. These means are not significantly higher than the negative control values. Based on these data, the calcium sulfonate read across substance, which was tested at the maximum dose of 5 g/kg, did not induce a significant increase in micronuclei in bone marrow cells from either male or female B6G3Fl mice. Thus, the calcium sulfonate read across substance (61789-86-4) was negative in this in vivo assay.


Justification for selection of genetic toxicity endpoint
No study is selected since all results are negative.

Short description of key information:
Genetic toxicity in vitro: OECD 471, Analogue of CAS 70024-69-0, negative.
Mammalian cell gene mutation: OECD 476, CAS 68783-96-0, negative.
Chromosome aberration: OECD 473, CAS 68783-96-0, negative.
Micronucleus test: OECD 474, Analogue of CAS 70024-69-0, negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the all negative results of in vitro and in vivo studies available for the read-across substances, Benzenesulfonic acid, mono-C20-24 (even)-sec-alkyl derivs., para-, sodium salts (generic name: C20-24 sodium sulfonate) is considered to be neither mutagenic nor genotoxic. According to the European regulation (EC) No. 1272/2008 C20-24 sodium sulfonate does not meet the criteria for classification and will not require labelling as a mutagen.