Sodium dibutyldithiocarbamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sodium dibutyldithiocarbamate (SDBC) in its manufactured form (as 47.5% aqueous solution) was not mutagenic in Ames test with and without metabolic activation up to dose levels of 5000 µg per plate (2375 µg per plate for pure substance). No data on its ability to induce gene mutations and chromosome aberrations in mammalian cells were available; however, based on the read-across with its structural analogues sodium dimethyldithiocarbamate (SDMC), and sodium diethyldithiocarbamate (SDEC) which are not genotoxic (based on in vivo studies), SDBC is concluded to have no genotoxic potential.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

DNA base pairs

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

derived from rat liver (S-9 mix)

Test concentrations with justification for top dose:

50, 158, 500, 1580 and 5000 µg per plate (for 47.5% aqueous solution of the substance), corresponding to 23.75, 75.05, 237.5, 750.5 and 2375 µg per plate for pure (anhydrous) substance

Vehicle / solvent:

- Solvent used: distilled water

Negative solvent / vehicle controls:

yes

Remarks:

with and without S-9

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Positive controls:

yes

Positive control substance:

9-aminoacridine

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 2 days

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: examination for the presence of a background lawn of non-revertant colonies

Evaluation criteria:

No data

Statistics:

No data

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY:
The lowest level of SDBC causing visible thinning of the background lawn of non-revertant cells was 5000 µg/plate. This was therefore selected as the top exposure level for use in the main assays.

Summary of the revertant colony means:

Plate N°		Addition (µg)	S-9mix: + present; - absent	Revertant colony means
				TA98 (Test 1)	TA98 (Test 2)	TA100 (Test 1)	TA100 (Test 2)	TA1535 (Test 1)	TA1535 (Test 2)	TA1537 (Test 1)	TA1537 (Test 2)
1	None; sterility check		+	0	0	0	0	0	0	0	0
2	SDBC sterility check	5000	-	0	0	0	0	0	0	0	0
3	SDBC	5000	+	14	24	29	28	6	7	8	6
4	SDBC	1580	+	28	33	70	55	14	12	7	7
5	SDBC	500	+	34	32	108	109	15	13	7	7
6	SDBC	158	+	38	32	111	108	15	13	7	6
7	SDBC	50	+	36	33	113	111	14	14	7	7
8	Distilled water		+	37	32	110	110	13	13	8	7
9	SDBC	5000	-	18	29	109	109	13	12	5	8
10	SDBC	1580	-	29	34	109	111	14	14	8	7
11	SDBC	500	-	33	35	112	111	15	15	8	6
12	SDBC	158	-	34	31	112	111	14	15	7	8
13	SDBC	50	-	33	34	112	111	14	16	7	7
14	Distilled water		-	36	31	110	111	14	16	8	8
15	Benzo(a)pyrene		-	31	26	104	102	12	16	6	6
16	Benzo(a)pyrene		+	260	393	590	470	846	494	218	171
17	2-Nitrofluorene		-	440	909	531	673	644	416	167	146
18	None; 10E-6 dilution of bacterial culture only		-	115	113	115	113	114	116	114	113

Conclusions:

Negative in the Ames test.

Executive summary:

The ability of sodium dibutyldithiocarbamate (SDBC) in its manufactured form (as 47.5% aqueous solution) to induce gene mutations in bacteria was studied in Ames test with Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at test concentrations 50, 158, 500, 1580 and 5000 µg per plate (corresponding to 23.75, 75.05, 237.5, 750.5 and 2375 µg per plate for pure (anhydrous) substance), in the presence and absence of metabolic activation (Life Science Research Ltd, 1990). The study was performed in accordance with OECD Guideline 471 and EPA OTS 798.5265 and under GLP. The lowest level of SDBC causing visible thinning of the background lawn of non-revertant cells was 5000 µg/plate. The substance did not induce a statistically significant increase in the number of revertants in any strain, both with and without metabolic activation, indicating that sodium dibutyldithiocarbamate (SDBC) is not mutagenic in Ames test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd, Margate, Kent, UK
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 161-198 g

Route of administration:

oral: gavage

Vehicle:

purified water

Duration of treatment / exposure:

one application of 10 mL/kg bw

Frequency of treatment:

n.a.

Post exposure period:

2-4 h, 12-14 h

Remarks:

Doses / Concentrations:
400, 1000 mg/kg bw
Basis:
other: concentration in vehicle: 40, 100 mg /mL

No. of animals per sex per dose:

4

Control animals:

yes

Positive control(s):

2-4 h preparation interval: 2-Acetylaminofluorene (2-AAF), 75 mg/kg bw, in corn oil,
12-14 h preparation interval: Dimethylnitrosamine (DMN), 10 mg/kg bw, in water

Tissues and cell types examined:

- Tissue: Liver
- Type of cells: Hepatocytes

Details of tissue and slide preparation:

- Number of animals: 4 per dose (hepatocytes were prepared from 3)
- Number of cells: 4.5×105 cells per slide

Evaluation criteria:

- Parameters: 3H-incorporation (silver grain formation)

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

One animal (2-4h, 400 mg/kg SDDC) had a cell viability of 44%. Because of slide analysis that was considered to have no effect on the test system.

Table 7.6.2-B1: Net nuclear grain values and percentage of cells in repair Treatment Period Dose [µg/mL] NNG [mean ± SD] % of cells in repair (NNG ≥ 5) [mean ± SD] vehicle 2-4 h – -0.9 ± 0.2 1.0 ± 1.0 SDDC 2-4 h 400 -1.0 ± 0.6 1.0 ± 1.0 SDDC 2-4 h 1000 -1.1 ± 0.6 1.0 ± 1.0 DMN 2-4 h 10 10.9 ± 1.6 69.0 ± 2.6 vehicle 12-14 h – -1.0 ± 0.2 1.0 ± 0.0 SDDC 12-14 h 400 -0.6 ± 0.3 1.0 ± 1.0 SDDC 12-14 h 1000 -0.7 ± 0.5 4.0 ± 2.0 2-AAF 12-14 h 75 16.0 ± 1.0 97.7 ± 0.6 NNG = net nuclear grains

Conclusions:

It was concluded that SDMC does not induce UDS detectable to the liver of rats under the experimental conditions employed in the present test.

Executive summary:

In the present in vivo genotoxicity study,  SDMC (41.4% w/w aqueous solution) was tested for its ability to induce unscheduled DNA synthesis (UDS) in the livers of orally dosed male rats. The test was performed according to OECD 476 and under GLP.  To determine if there were any substantial inter-sex differences in toxicity both male and female animals were tested in an initial toxicity range-finder experiment. Groups of four male rats (Han Wistar (Crl:WI (GlxIBRL/Han) BR) were treated once with the vehicle (purified water), SDDC (at 400 mg/kg or 1000 mg/kg) or the required positive control, by oral gavage, at a dose volume of 10 mL/kg. The positive controls used were 75 mg/kg 2-acetamidofluorene (2-AAF) suspended in com oil (Experiment 2) and 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (Experiment 1). No clinical signs of toxicity were observed in Experiment 1 (2-4 hours) or Experiment 2 (12-14 hours). Approximately 2-4 hours (Experiment 1) or 12-14 hours (Experiment 2) after dosing, animals were sacrificed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from three animals in each dose group and were treated with [3H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and of these, three were dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically after development of the emulsion and staining, and the net grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of two of the three slides, each animal and dose group. Negative (vehicle) control animals gave a group mean NNG value of less than zero with only 1% cells in repair. Group mean NNG values were increased by 2-AAF and DMN treatment to more than 10.9 and more than 50% cells found to be in repair. In this study the vehicle control NNG value was consistent with both published and historical control data, and the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid. Treatment with 400 or 1000 mg/kg SDMC did not produce a group mean NNG value greater than -0.6 nor were any more than 4% cells found in repair at either dose. It was concluded that SDMC did not induce UDS detectable under the experimental conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The ability of sodium dibutyldithiocarbamate (SDBC) in its manufactured form (as 47.5% aqueous solution) to induce gene mutations in bacteria was studied in Ames test with Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at test concentrations 50, 158, 500, 1580 and 5000 µg per plate (corresponding to 23.75, 75.05, 237.5, 750.5 and 2375 µg per plate for pure (anhydrous) substance), in the presence and absence of metabolic activation (Life Science Research Ltd, 1990). The study was performed in accordance with OECD Guideline 471 and EPA OTS 798.5265 and under GLP. The lowest level of SDBC causing visible thinning of the background lawn of non-revertant cells was 5000 µg/plate. The substance did not induce a statistically significant increase in the number of revertants in any strain, both with and without metabolic activation, indicating that sodium dibutyldithiocarbamate (SDBC) is not mutagenic in Ames test.

No data on the ability of SDBC to induce chromosome aberrations or gene mutations in mammalian cells were available for assessment. However, Article 13 of the REACH legislation states that, in case no appropriate animal studies are available for assessment, information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. In vitro and in vivo studies on genotoxicity were available for the structural analogues of SDBC, SDMC and SDEC.

In vitro mutagenicity studies in mammalian cells

SDEC

In an in vitro mouse lymphoma assay, performed according to a procedure similar to OECD guideline 476, the ability of SDEC to cause genotoxicity was tested in mouse lymphoma L5178Y cells (McGregor, Brown et al. 1991). The exact form of SDEC (pure or in aqueous solution) was not specified. In total 4 experiments were performed without metabolic activation, using the following test concentrations: 1st experiment 0, 0.3125, 0.625, 1.25, 2.5, 5 and 10 μg/mL; 2nd experiment 0, 0.5, 1, 2, 3 and 4 μg/mL; 3rd experiment 0, 0.063, 0.125, 0.25, 0.5 and 1 μg/mL; 4th experiment 0, 0.031, 0.063, 0.0125, 0.25, 0.5 and 1 μg/mL. All of the trials were judged to be positive and manifested irregular dose-related responses. In the 1st and 2nd trials survival was greater and the induced mutant fraction lowest at 1-2 μg/mL; survival was lower and mutant fractions were higher at both lower and higher concentrations. In the two subsequent trials, 1 μg/mL was the highest dose tested, but lower doses were both more toxic and more mutagenic, at least from 0.063 μg/ml upwards. The mutagenic responses were essentially reproducible and mirrored the survival trend.

SDMC

The mutagenic potential of the test substance 40% SDMC (aqueous solution) was assessed in an in vitro mammalian cell mutation assay in CHO cells conducted according to OECD TG 476 (Morry et al. 1986).  The study consisted of a preliminary toxicity test.The cells were exposed to the test substance in the absence or presence of metabolic activation (S9 mix) in concentrations to 0.1 to 1 or 0.1 to 10 ug/mL, respectively, for 5 hours. Survival at the highest dose without S9 (1.0 µg/mL) indicated significant cytotoxicity. The relative cell survival is in the three highest concentrations less than 10%. With S9 the relative cell survival is in the two highest concentrations less than 10%. Exposure of the CHO cells to the test item did not result in increase of mutagenicity under the conditions of this test.

 

In vitro cytogenicity studies in mammalian cells

SDMC

The potential of SDMC to induce structural chromosomal aberrations in cultured mammalian cells was tested in vitro in a GLP study, performed according to OECD 473 (Jenkinson et al. 2002). Duplicate cultures of human lymphocytes, treated with the test material (SDMC 41.4% w/w aqueous solution), were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 1% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 2% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels of test material used during the course of the study were selected based on the results from a preliminary toxicity test. The test material did not induce any significant dose-related increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition. The test material was shown to be non-clastogenic to human lymphocytes in vitro.

SDEC

The ability of sodium diethyldithiocarbamate (SDEC) to induce chromosome aberrations and sister chromatid exchange in Chinese hamster ovary (CHO) cells was examined (Loveday, Lugo et al. 1989). In the first study, the following test concentrations have been used: without metabolic activation, 0.0; 0.1; 0.3 and 1.0 µg/mL; with metabolic activation (rat liver S9 fraction), 0.0; 15.2; 50.7 and 152.0 µg/mL. It is not clear which form of the substance was tested, although the purity is stated to exceed 99%. The substance was tested up to cytotoxic concentrations. Under the conditions of this test, SDEC does not induce chromosomal aberrations.  In the sister chromatid exchange assay, two experiments without and one with metabolic activation (rat liver S9 fraction) were performed, using the following concentrations: without metabolic activation, 1st experiment: 0.0; 0.05; 0.075 and 0.1 µg/mL; second experiment 0.0, 0.075, 0.1 and 0.15 µg/mL; with metabolic activation: 0.0; 0.318; 0.9540 and 3.1800 µg/mL. The initial experiment without S9 yielded a positive response at the highest dose tested, when an extended harvest time (30 hr instead of 26 hr) was used. However, this response was not reproducible, and the chemical was judged negative.

  

In vivo genotoxicity testing

SDMC

The potential genotoxicity of SDMC was also investigated in vivo. In a mouse bone marrow micronucleus test, performed according to OECD 474 (GLP), the test material (41.4% w/w aqueous solution SDMC) was administered orally by gavage at 350, 700 or 1400 mg/kg/day to groups of six male mice killed 24 hours after the second administration. Clinical observations of eye closure and lethargy were noted in all dosed mice at levels of 700 and 1400 mg/kg/day. Additionally, abnormal breathing and two mortalities were observed in animals tested at 1400 mg/kg/day. The negative (vehicle) control was purified water and cyclophosphamide (CPA), served as the positive control. Mice treated with SDDC at all dose levels exhibited group mean ratios of PCE to NCE and frequencies of micronucleated PCE that were similar to the values for the vehicle control group and which also fell within normal historical control ranges. There were no statistically significant increases in micronucleated PCE in the test article treated groups compared to the concurrent vehicle control. Following treatment at 1400 mg/kg/day there was a notable decrease in PCE to NCE ratio compared to the vehicle control group. This is considered indicative of test article induced toxicity in the bone marrow and thus confined exposure of the target tissue to the test article. It is concluded that SDMC did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 1400mg/kg/day(the maximum tolerated dose for this study).

In another in vivo genotoxicity study,  SDMC (41.4% w/w aqueous solution) was tested for its ability to induce unscheduled DNA synthesis (UDS) in the livers of orally dosed male rats. The test was performed according to OECD 476 and under GLP.  To determine if there were any substantial inter-sex differences in toxicity both male and female animals were tested in an initial toxicity range-finder experiment. Groups of four male rats (Han Wistar (Crl:WI (GlxIBRL/Han) BR) were treated once with the vehicle (purified water), SDDC (at 400 mg/kg or 1000 mg/kg) or the required positive control, by oral gavage, at a dose volume of 10 mL/kg. The positive controls used were 75 mg/kg 2-acetamidofluorene (2-AAF) suspended in com oil (Experiment 2) and 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (Experiment 1). No clinical signs of toxicity were observed in Experiment 1 (2-4 hours) or Experiment 2 (12-14 hours). Approximately 2-4 hours (Experiment 1) or 12-14 hours (Experiment 2) after dosing, animals were sacrificed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from three animals in each dose group and were treated with [3H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and of these, three were dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically after development of the emulsion and staining, and the net grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of two of the three slides, each animal and dose group. Negative (vehicle) control animals gave a group mean NNG value of less than zero with only 1% cells in repair. Group mean NNG values were increased by 2-AAF and DMN treatment to more than 10.9 and more than 50% cells found to be in repair. In this study the vehicle control NNG value was consistent with both published and historical control data, and the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid. Treatment with 400 or 1000 mg/kg SDMC did not produce a group mean NNG value greater than -0.6 nor were any more than 4% cells found in repair at either dose. It was concluded that SDMC did not induce UDS detectable under the experimental conditions employed.

Based on these results, sodium dibutyldithiocarbamate (SDBC) is also concluded to be non-genotoxic in vivo.

Justification for classification or non-classification

Based on the negative results in the Ames test with sodium dibutyldithiocarbamate (SDBC) and negative in vivo studies with a structural analogue, SDMC, the substance should not be classified as genotoxic, according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.