Sodium diethyldithiocarbamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sodium diethyldithiocarbamate (SDEC) was negative in Ames test with Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, chromosome aberration assay with CHO cells and sister chromatid exchange assay with CHO cells. The results of an in vitro mouse lymphoma assay were positive, although irregular dose-responses were obtained. No in vivo genotoxicity studies were available for assessment. However, based on the negative results of carcinogenicity study with rats and mice, the substance is considered to be non-genotoxic. It shall be mentioned that the form of the test material was not specified, but tests were performed up to cytotoxic concentrations.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Principles of method if other than guideline:

Method; Other: Salmonella preincubation assay (modification of the standard plate incorporation assay)
Method; Other: Ames BN, McCann J, Yamasaki E (1975): Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat Res 31:347-364.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9, Aroclor 1254-induced or hamster liver S-9, Aroclor 1254-induced

Test concentrations with justification for top dose:

0.0, 33.0, 100.0, 333.0, 1000.0, 3333.0 and 10000.0 µg/plate

Vehicle / solvent:

- Solvent used: DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for TA 1535 and TA 100

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

for TA98

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for TA97 and TA 1537

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for all strains with hamster and rat liver metabolic activation systems

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C followed by 48 hours at 37°C

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

The criteria used for data evaluation were as follows: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) nonmutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range

Dose	TA 100						TA 1535
	NA		10% HLI		10%RLI		NA		10% HLI		10%RLI
µg/plate	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM
0.0 (DMSO)	96	1.5	104	3.0	97	4.2	8	1.2	8	1.3	13	0.3
33.0	75	2.6	94	4.7	82	5.9
100.0	80	1.5	83	1.9	85	3.2	11	1.2	15	1.9	12	3.8
333.0	83	5.6	85	12.5	81	1.2	8	1.5	9	1.8	15	1.7
1000.0	65	12.1	102	5.8	97	7.0	7	1.7	10	2.7	10	1.5
3333.0	0	0.0	2	1.5	1	1.0	5	1.5	7	0.9	7	2.4
10000.0							0	0.0	1	0.9	0	0.5
POS	384	20.4	2285	33.2	2285	35.2	117	8.4	131	29.6	37	6.6

 

Dose	TA 1537						TA 98
	NA		10% HLI		10%RLI		NA		10% HLI		10%RLI
µg/plate	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM
0.0 (DMSO)	10	1.3	3	0.3	7	1.2	11	1.2	12	2.6	16	3.1
33.0
100.0	1	0.3	3	0.6	6	0.3	12	1.9	12	2.0	15	2.6
333.0	1	0.6	3	0.3	3	2.8	7	1.9	17	2.3	18	1.5
1000.0	1	0.6	2	0.9	2	0.7	8	1.2	15	5.2	14	4.2
3333.0	0	0.3	1	0.9	2	0.3	7	1.8	11	2.5	10	1.8
10000.0	0	0.0	1	0.3	1	0.3	0	0.0	10	1.5	8	1.5
POS	28	3.8	89	8.5	130	15.5	92	10.2	883	25.4	127	13.3

Abbreviations:

POS: positive control; NA, not activated; RLI, rat liver S-9, Aroclor 1254-induced; HLI, hamster liver S-9, Aroclor 1254-induced

Conclusions:

No increased number of revertants was found, indicating that the substance SDEC is not mutagenic in Ames test.

Executive summary:

In a Salmonella preincubation assay, according to Ames et al. (1975), strains TA1535, TA 1537, TA 98 and TA 100 of S. typhimurium were exposed to SDEC at of 0.0, 33.0, 100.0, 333.0, 1000.0, 3333.0 and 10000.0 µg/plate, with and without metabolic activation. The test substance was tested up to the required limit concentrations (up to 5000 µg/plate). The compound did not induce any significant increase in the observed numbers of revertant colonies in any of the tester strains used, neither in the presence or absence of metabolism (S9). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. In conclusion, the test substance SDEC is not-mutagenic in the Ames test.  

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The experimental procedure used was based upon those described by McGregor et al. (Environ. Mol. Mutagen. 1988, 15, 85-154; Environ. Mol. Mutagen. 1988,11, 91-118; Environ. Mol. Mutagen. 1987, 9, 143-160).

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase locus (tk)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Metabolic activation system:

rat S9

Test concentrations with justification for top dose:

Without S9: 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; 0, 0.031, 0.063, 0.0125, 0.25, 0.5 and 1 μg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: -S9: ethyl methanesulfonate (250 μg/mL), methyl methanesulphonate (15-20 μg/mL); + S9: 3-methylcholanthrene (2.5 μg/mL).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION

- Exposure duration: 4 hr
- Expression time (cells in growth medium): 2 days


NUMBER OF REPLICATIONS: vehicle control, four cultures; positive control, two cultures; at least five test compound concentrations, two cultures per concentration.


NUMBER OF CELLS EVALUATED: 3 x 10e6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency. The highest concentration used in preliminary toxicity test was 5 mg/ml, unless indicated otherwise by poor substance solubility.Four trial

OTHER
- Test compound concentrations were primarily two-fold dilutions from the highest testable concentration, as estimated from the toxicity test.

Evaluation criteria:

A response was considered positive when the dose-related trend and the response at one of the tree highest acceptable doses were statistically significant. A test was considered positive when, out of three trials, a positive trial was reproducible.

Statistics:

Dose trend test (Barlow et al., 1972) and a variance analysis of pair-wise comparisons of each dose against the vehicle control.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Four trials were performed in the absence of S9 mix, all of which were judged to be positive and manifested irregular dose-related responses. In the 1st and 2nd trials survival was greated 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.

Results of 4 trials with sodium diethyldithiocarbamate without S9 mix.

 

 

Concentration μg/ml	CE	RTG	MC	MF	AVE MF
0 (vehicle control)	74 87 75 100	106 95 83 117	97 130 127 108	43 50 56 36	46
0.3125	67 74	53 57	240 238	119 107	113
0.625	77 61	71 61	257 196	111 107	109
1.25	76 75	67 82	175 134	76 60	68
2.5	74 59	54 48	84 141	38 79	58
5	37 37	4 4	282 347	252 315	284
10	Lethal Lethal
EMC 250 μg/mL	60 64	67 64	558 464	311 243	277
MMS 20 μg/mL	31 37	19 27	342 352	370 314	342

 

Concentration μg/ml	CE	RTG	MC	MF	AVE MF
0 (vehicle control)	83 84 85 88	82 99 115 105	117 90 86 92	47 36 34 35	38
0. 5	66 67	25 29	339 281	172 140	156
1	51 58	39 60	153 201	99 98	99
2	58 49	36 32	291 208	168 143	155
3	53 45	10 8	521 539	328 401	364
4	Lethal Lethal
MMS 15 μg/mL	7r 13	3 5	88 101	409 266

 

r = Rejected when 50% > CE > 120% or RTG < 1%

 

Concentration μg/ml	CE	RTG	MC	MF	AVE MF
0 (vehicle control)	80 53 70 82	113 88 106 92	91 83 107 129	38 53 51 52	48
0.063	34 40	14 19	104 196	103 163	133
0.125	44 37	22 21	230 172	173 154	163
0.25	46 40	35 29	210 203	152 169	161
0.5	50 53	41 40	214 300	142 188	165
1	64 49	51 78	184 109	96 74	85
MMS 20 μg/mL	24 20	18 14	413 242	582 414	498

 

Concentration μg/ml	CE	RTG	MC	MF	AVE MF
0 (vehicle control)	62 67 69 58	94 93 107 106	71 64 84 91	38 32 40 52	41
0.011	46 54	25 51	124 163	90 100	95
0.063	40 48	11 13	194 326	163 228	195
0.125	39 47	11 12	213 265	182 188	183
0.25	73 56	18 14	299 213	137 126	126
0.5	65 54	28 22	239 211	122 131	126
1	62 58	31 26	184 178	99 103	101
MMS 15 μg/mL	21 16	18 13	128 149	206 271	239

 

 CE = cloning efficiency; RTG = relative total growth (calculated as total suspension growth x CE); MF = mutant fraction (calculated as 200 x mutant colonies per plate / total clones per plate); MC = mutant colony count; AVE MF = group average mutant fraction; underline = P < 5%

Conclusions:

The test material SDEC showed a positive response in this in vitro mouse lymphoma assay performed with mouse lymphoma L5178Y cells.

Executive summary:

In an vitro mouse lymphoma assay performed according to the procedure similar to OECD guideline 476, the ability of SDEC to cause genotoxicity was tested in mouse lymphoma L5178Y cells. The form of the test substance 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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Tested using a standard protocol: Galloway SM, Bloom AD, Resnick MA, Margolin BH, Nakamura F, Archer P, Zeiger E (1985): Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories. Environ Mutagen 7:1-51.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source : Dr. Sheila Galloway (Litton Bionetics, Kensington, MD)
- Type and identity of media: McCoy's 5A (modified) medium (KC Biological, Lenexa, KS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Growth and treatment conditions: Galloway SM, Bloom AD, Resnick MA, Margolin BH, Nakamura F, Archer P, Zeiger E (1985): Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories. Environ Mutagen 7:1-51.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced S9 rat liver microsomal fraction (Microbiological Associates, Kensington, MD)

Test concentrations with justification for top dose:

Without metabolic activation, 1st experiment: 0.0; 0.1; 0.3 and 1.0 µg/mL;
With metabolic activation: 0.0; 15.2; 50.7 and 152 µg/mL

Vehicle / solvent:

- Solvent used: water
- Stock solutions were prepared at 500 mg/mL, or at the limit of solubility, and diluted 1:100 in medium to obtain a high test concentration of 5 mg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Without metabolic activation:
DURATION
- Exposure duration: 8 hours
- Fixation time (start of exposure up to fixation or harvest of cells): After 8 hours of exposure, cells are washed to remove the test chemical, and treated with colcemid (10E-6 M) for 2-2.5 hr before cell harvest.

With metabolic activation:
DURATION
- Exposure duration: 2 hours
- Incubation period: incubated at 37°C with medium for 8 hr.
- Fixation time (start of exposure up to fixation or harvest of cells): After incubation, colcemid was added and the cells were harvested 2 hr later.

STAIN (for cytogenetic assays): slides were stained in 5% Giemsa for 5 min

NUMBER OF REPLICATIONS: one

NUMBER OF CELLS EVALUATED: One hundred cells were scored for each of three concentrations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
Cells were analyzed for the following categories of chromosomal aberrations: "Simple,"defined as chromatid gap, break, fragment, and deletion, chromosome gap and break, double minutes; "complex" rearrangements such as interstitial deletion, triradial, quadriradial, ring, dicentric chromosome; and "other," defined as pulverized chromosome, polyploid cell, or endoreduplicated cell. Chromatid and chromosome gaps were recorded but were not used for evaluation. The frequency of polyploid or endoreduplicated cells was only noted when it seemed excessive; however, these categories were never included in the totals for statistical analysis.

Statistics:

The statistical procedures for evaluation of test data are described in Galloway SM. Armstrong MA, Reuben C, Colman S, Brown C, Cannon AD. Bloom AD, Nakamura F, Ahmed M, Duk S, Rimpo J, Margolin BH, Resnick MA, Anderson B, Zeiger E (1987): Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ Mol Mutagen l0(Suppl 10):1-175.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Results of chromosome aberration test with sodium diethyldithiocarbamate in the absence of metabolic activation.

 

Dose (μg/mL)	Cells	Percentage cells with aberrations
		Total	Simple	Complex
0.0	100	3.00	3.00	0.00
0.1	100	1.00	1.00	0.00
0.3	100	4.00	4.00	0.00
1.0	100	0.00	0.00	0.00
Positive control (MMC) 5.000	50	72.00	46.00	48.00

 

Table 2. Results of chromosome aberration test with sodium diethyldithiocarbamate with metabolic activation.

 

Dose (μg/mL)	Cells	Percentage cells with aberrations
		Total	Simple	Complex
0.0	100	2.00	2.00	0.00
15.2	100	6.00	5.00	1.00
50.7	100	4.00	3.00	1.00
152	100	4.00	4.00	0.00
Positive control (CP) 50.0000	50	42.00	24.00	30.00

Conclusions:

The test substance SDEC did not induce chromosomal damage in this in vitro genotoxicity test with CHO cells, in the presence and absence of metabolic activation.

Executive summary:

In the present study, the ability of sodium diethyldithiocarbamate (SDEC) to induce chromosome aberrations and sister chromatid exchange in Chinese hamster ovary (CHO) cells was tested. 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Several in vitro tests with sodium diethyldithiocarbamate (SDEC) were available for assessment. Its ability to induce gene mutation in bacteria was studied by Mortelmans et al., 1985, in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at dose levels of 0.0, 33.0, 100.0, 333.0, 1000.0, 3333.0 and 10000.0 µg/plate, with and without metabolic activation. The study unfortunately does not specify which form of the substance (anhydrous, trihydrate or aqueous solution) was tested, although the purity is stated to be 99%; nevertheless, as the substance is manufactured and marketed solely as 26% aqueous solution, it is considered to be acceptable to use the results of this study for risk characterization. No increased number of revertants was found, indicating that the substance is not mutagenic in Ames test.

Loveday et al., 1989, studied the ability of sodium diethyldithiocarbamate (SDEC) to induce chromosome aberrations and sister chromatid exchange in Chinese hamster ovary (CHO) cells. 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, 0.0; 15.2; 50.7 and 152.0 µg/mL. Also in this case, it is not clear which form of the substance was tested, although the purity is stated to exceed 99%. The substance was tested to its toxic level and the results were negative.

In the sister chromatid exchange assay, two experiments without and one with metabolic activation 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.

Also an in vitro mouse lymphoma assay (McGregor et al., 1991) was available for assessment, performed according to the procedure similar to OECD guideline 476. Also in this case the form of the substance 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.

Usually, when positive results are observed in the in vitro studies, the performance of in vivo studies needs to be considered by the registrant in accordance with Column 2 of REACH Annex VIII. No in vivo genotoxicity studies with sodium diethyldithiocarbamate (SDEC) were available. However, a reliable negative carcinogenicity study with two species, rats and mice, was available (see Section 7.7 for further details), suggesting that the substance does not induce genetic damage in vivo. Therefore, based on the overall evidence, sodium diethyldithiocarbamate (SDEC) is concluded to be non-genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the negative carcinogenicity study with rats and mice, classification of sodium diethyldithiocarbamate (SDEC) in its manufactured and marketed form (as 26% aqueous solution) for genotoxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. These results are also considered to be applicable to the anhydrous substance.