N,N-dicyclohexylbenzothiazole-2-sulphenamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The data from the bacterial mutation assays indicated no genotoxic potential of DCBS (Monsanto Co.1984 & JETOC 1994). This finding is confirmed by the results from a mammalian cell mutation assay (HGPRT assay, Monsanto Co. 1985) and an UDS assay using primary rat hepatocytes (Monsanto Co. 1985). An in vitro chromosome aberration assay with CHL cells revealed no clastogenic effects of DCBS; but an increase of polyploidy was noted in the long-term treatment without metabolic activation (JETOC 1998). According to recommendation given in the current OECD guideline 473 (in vitro chromosomal aberration assay) this test system is not designed to measure numerical aberrations and should not routinely be used for that purpose. Beside this, no dose-response relationship was noted and thus the increase in polyploidy was considered as ambiguous. The increase in micronuclei and polyploidy was however confirmed in a follow-up in vitro micronucleus assay (JETOC 1998).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A testing proposal is included in this dossier to generate compliant in vivo genotoxicity study. An in vivo mammalian erythrocyte micronucleus test (OECD TG 474) combined with an in vivo mammalian alkaline comet assay (OECD TG 489) in rats with oral administration is proposed.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

Table 7.6.1/1 : Summary of key genotoxicity results:

 

Test n°	Test Guideline / Reliability	Focus	Strains  / cells tested	Metabolic activation	Test concentration	Statement
1 (Monsanto, 1984)	Ames Test (OECD 471, GLP) WoE, rel.2	Gene mutation	S. thyphimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538	-S9 +S9	Tested up to 5000 µg/plate in DMSO	-S9: not mutagenic + S9: not mutagenic
2 (JETOC, 1994)	Ames Test (OECD 471, GLP) WoE, rel.2	Gene mutation	S. thyphimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvrA	-S9 +S9	Tested up to 300 µg/plate in acetone (precipitation)	- S9: not mutagenic + S9: not mutagenic
3 (JETOC, 1988)	CAT (OECD 473, GLP) K, rel.2	Chromosomal aberration	Chinese Hamster lung CHL/IU	-S9 +S9	Tested up to 0.82 mg/mL (cytotoxicity limit / 24/48 hrs  -S9) or up to 3.5 mg/mL (= 10 mM / 6 hrs  +/- S9) in 0.5% CMC	- S9 (24/48 hrs): ambiguous - S9 (6 hrs): not clastogenic + S9 (6 hrs): not clastogenic
4 (JETOC, 1988)	MNT (no guideline, GLP) K, rel.2	Chromosomal aberration	Chinese Hamster lung CHL/IU	-S9	Tested up to 0.82 mg/mL (cytotoxicity limit / 48 hrs  -S9) in 0.5% CMC	- S9: clastogenic / aneugenic
5 (Monsanto, 1985)	hprt (eq. OECD 476, GLP) K, rel.1	Gene mutation	Chinese Hamster Ovary CHO	-S9 +S9	Tested up to 500 µg/mL in acetone (precipitation)	-S9: not mutagenic + S9: not mutagenic

 

In vitro data:

Gene mutation assays:

The mutagenic potential in bacteria of the test substance DCBS was evaluated in a GLP study. Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were used. Treatment by the plate-incorporation method was done for doses up to 300 µg/ plate, because of precipitation at 300 µg/plate and higher. In this study no biologically relevant and dose dependent increases in revertants was evaluated in any of the tester strains evaluated with and without metabolic activation (Monsanto Co. 1984 / Test No. 1).

In another GLP and guideline study the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E coli WP2uvrA were used. No toxicity was detected up to 5000 µg/plate with and without metabolic activation, whereas precipitation was observed at 312.5 µg/ plate and higher. No increase in mutagenicity was noted in any of the tester strains evaluated in presence or absence of metabolic activation (JETOC 1994 / Test No. 2).

 

The negative response seen in the bacterial test systems was confirmed in a mammalian cell mutation assay. No increase in mutants was noted in a HGPRT assay with CHO-K1 cells with and without metabolic activation (Monsanto Co. 1985 / Test No. 5). In addition, an UDS using primary rat hepatocytes revealed a non-genotoxic potential of DCBS in this assay (Monsanto Co.1985).

 

Chromosomal aberration assay:

No increase in aberrant cells was noted in a chromosome aberration assay (OECD TG 473) with CHL cells. The CHL cells were treated with 0.9, 1.8 and 3.5 mg/ml in the short-term treatment (in presence and absence of S9-mix) and with 0.21, 0.41 and 0.82 mg/ml in the long-term treatment (without S9-mix). Cytotoxicity was noted without metabolic activation at 0.41 mg/ml and higher. A significant increase of polyploidy was noted in the long-term treatment (polyploidy: 0.13 % control, 3.38 % 0.21 mg/ml, 6.00 % 0.41 mg/ml and 1.64 % 0.82 mg/ml. However, no dose response-relationship was demonstrated (JETOC 1998 / Test No. 3).

In a limited documented in vitro micronuclei assay (only secondary literature available) an increase in micronuclei and polyploidy was noted in CHL cells. The cells were treated with the test substance DCBS in a long-term treatment (48 hours) without metabolic activation. An increase in both micronuclei and cells with multi nuclei was noted at 0.21 mg/ml and higher (JETOC 1998 / Test No. 4).

 

In vivo data:

The genotoxic potential of DCBS was evaluated in an in vivo bone marrow chromosome aberration assay. Male and female Sprague-Dawley rats were administered with 1000 mg/kg bw test substance once via gavage. Groups of 5 males and 5 females (treatment group and vehicle control each) were sacrificed at 6, 24 and 48 hours following test substance administration. The body weight and clinical observations were recorded during the study. The bone marrow cells were prepared and at least 50 mitotic cells per animal were analysed for cytogenetic aberrations. Clinical signs were observed for all 1000 mg/kg bw treated animals at 24 and 48 hours after application. The clinical signs observed were depression, red stains on nose/eyes, soft faeces, slight depression, urine stains, and rough coat. No animal died during the study. A significant change in body weight was seen for the 48 hours treatment group. No statistically significant differences between the mean mitotic indices of the test groups and the vehicle control were seen. A statistically significant increase in percent aberrant cells and the mean number of aberrations per cell was seen in the positive control group (cyclophosphamide). Results from the 6- and 24 hours sacrifice data show that no statistically significant increase in the frequency of chromosome aberrations compared to control values was seen in the groups treated with DCBS. No statistically significant increase in aberrant cells was noted for the 48 hour treatment group (1 % aberrant cells) compared to historical control data (0.27% aberrant cells), whereas comparison to the current negative control (0% aberrant cells) revealed a statistically relevant difference using the non-parametric analysis. Further statistical analysis was performed on the 48-hours results and comparisons were made with historical control data and data from the current solvent control. The author concluded that the statistical significant increase in aberrations seen at 48 hours is based upon comparison with control value of zero. Based on these findings, the authors concluded that the test substance DCBS is not clastogenic. Moreover, the average number of chromosomes in the examined metaphases was determined for each animal and all treatment groups were statistically compared to the control group. Rats have a normal diploid chromosome number of 42. No statistically significant differences between the mean chromosome numbers of the test group and the vehicle control were noted (Monsanto Co 1985).

This study does not match current requirements for in vivo mammalian bone marrow chromosome aberration test (50 cells analysed instead of 200; highest dose level limited to 1000 mg/kg bw instead of 2000 mg/kg bw).

A testing proposal is therefore included in this dossier to generate compliant in vivo genotoxicity study. An in vivo mammalian erythrocyte micronucleus test (OECD TG 474) combined with an in vivo mammalian alkaline comet assay (OECD TG 489) in rats with oral administration is proposed.

Justification for classification or non-classification

Further in vivo testing is proposed to conclude on classification or non-classification of DCBS.