Disulfiram

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity

Key; NOTOX/ES 62/82.5; Ames (OECD 471); GLP; S. typhimurium TA 97, TA 98, TA 100, TA 1535, TA 1537 and TA 1538; 10 – 330 µg/plate (± S9); negative for all strains ±S9

Gene mutations in mammalian cells

Key; no Rep. no.; TK (OECD 490); no GLP, mouse lymphoma L5178Y cells; 0.0006 – 4.0 µg/mL -S9; ambiguous (Mutagenicity was seen only at cytotoxic concentrations)

Key, 88 -05, UDS (similar to OECD 482), GLP, primary hepatocytes, 0.0005 - 0,5 mg/L; negative

Source, RA-A, CAS 137-26-8; HPRT (OECD 476); GLP, V79 cells; 1 - 10 µg/plate (-S9) and 10 - 56 µg/mL (+S9); negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Chosen concentrations were too low as the cell survival did not reach 20%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the conditions of the study for the read across source substance, no statistically significant increase in mutant frequency was observed for any dose level in the absence or presence of metabolic activation. No significant dose-response was observed either. With this data from the read across source substance it is concluded that the target substance is not mutagenic in mammalian cells in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Aug 1985 - 1 May 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopten in 2016

Deviations:

yes

Remarks:

For details on guideline deviations, please refer to "Principles of method if other than guideline".

Principles of method if other than guideline:

Deviations from current OECD guideline 476 (adopted 2016)
- No information on chromosome number and mycoplasma contamination checks
- No information on cell cycle length
- Chosen concentrations were too low as the relative cell survival did not reach 20%
- No Historical Control Data provided

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine guanine phosporibosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Department Toxicology, Agricultural University of Wageningen, The Netherlands
- Suitability of cells: standard cells for the respective assay

For cell lines:
- Methods for maintenance in cell culture: cell density was kept below 4x10^6 cells per 75 cm² flask

- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media: Ham's F-10 medium (without thymidine and hypoxanthine) supplemented with 10% newborn calf serum, L-glutamine (2 mM), and penicillin/streptomycin/cefoxtamin (50 U/mL, 50 µg/mL and 2 µg/mL, respectively)
- Exposure medium: F-10 culture medium buffered with 20 mM Hepes in the absence of serum
- CO2 concentration: 5% CO2
- humidity level: humid atmosphere (95%)
- Temperature: 37 °C

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver homogenate (S9) was prepared previously. The S9/cofactor mixture consisted of

1.02 mg MgCl2, 2.46 mg KCl, 1.7 mg glucose-6-phosphate, 3.4 mg NADP, 4 µmol HEPES and 0.5 mL S9.

0.2 mL of the S9/cofactor mixture was added to 1 mL of medium for cytotoxicity or mutagenicity testing.

Test concentrations with justification for top dose:

1, 3.3, 5.6 or 10 µg /mL without S9 mix
10, 18, 33 or 56 µg/mL in the presence of S9 mix for 2 h

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9-mix: Ethylmethane sulfonate (EMS); 6 mM With S9-mix: Dimethylnitrosamine (DMN); 4 and 8 mM

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 1 culture per dose
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 6 x 10^6 (exposure), 10^6 (expression)
- Test substance added in cell culture medium as described above (serum-free, buffered with Hepes)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 h (with and without metabolic activation)
- Harvest time after the end of treatment (sampling/recovery times): 14 - 17 days

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days, during this time, the cells were subcultured every 2 or 3 days to maintain exponential growth
After the expression period, in total 10^6 cells were plated into 10 petri dishes with selective medium.
- Selective agent: 10 µM 6-thioguanine, 7 - 10 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 10^6 cells in total for mutant selection after the expression period, 2 x 200 cells of each dose were plated in cloning medium directly after exposure to assess cell survival (cloning efficiency)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency (CE)
CE: (Average No. of colonies on cloning plates)/200 *100%

Mutant Frequency per 10^5 survivors: (100/CE)*(Total number of mutant colonies on selective plates/number of seeded cells)

Rationale for test conditions:

A pre-test was performed assessing the cytotoxicity of the cells. 3x10^6 cells were treated in the presence (0.001 - 10 µg/mL) or absence (0.01 - 100 µg/mL) of S9 mix.

Evaluation criteria:

A mutant assay was considered acceptable if the following criteria were met:
- The absolute colony forming efficiency of the solvent control is 60%
- At least 3/4 doses of the test substance have an acceptable number of surviving cells (10^6) analysed for HPRT mutations.
- The spontanous mutant frequency in the untreated or solvent treated control is <3 per 10^5 survivors.
- The positive controls induced significant (at least 3-fold) increases in mutant frequencies.

A test substance is considered mutagenic if
- It induces a mutant frequency that is at least 3 x times higher than the spontaneous mutant frequency of the untreated control.
- The results show a dose response relationship and are reproducible in an independently repeated test.

A test substance is considered negative (nonmutagenic) if
- None of the test concentrations induce a mutant frequency that is at least 3 x times higher than the spontaneous mutant frequency of the untreated control.
- The results are reproducible in an independently repeated test.

Statistics:

No statistical evaluation was performed.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Chosen concentrations were too low as the cell survival did not reach 20%

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A concentration of 10 µg/mL,without metabolic activation, reduced cloning efficiency by approximatively 50%. In the presence of S9 mix, cytotoxicity occurred only from 30 µg/mL. Summarized results can be found in Attachment 1 in the attached background material.

STUDY RESULTS
- Genotoxicity: No statistically significant increases in mutant frequency or dose– response were observed.
- Concurrent vehicle negative and positive control data: The solvent and positive control induced satisfactory mutagenicicty rates, indicating the adequacy of the experimental conditions.
- Results from cytotoxicity measurements: the test item was observed to be significantly cytotoxic at the level of 10 µg/mL in the absence of S9 mix (CE was reduced by approcx. 55 - 75%). In the presence of S9 mix, the cloning efficiency was only reduced by 25 - 50%, even though the test item concentrations were higher (up to 56 µg/mL).

For summarized results, please refer to Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA: not provided

Conclusions:

The present study was conducted in compliance with GLP and EPA OPP 84-2, and similar to OECD test guideline 476. Under the conditions of the study, no statistically significant increase in mutant frequency was observed for any dose level in the absence or presence of metabolic activation. No significant dose-response was observed either. Therefore, the test substance is concluded not to be mutagenic in CHO cells in vitro.

Reference 3

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:

other: Comparable to guideline study, published report, minor restrictions, adequate for assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

no test in presence of metabolic activation. The test material was described poorly.

Principles of method if other than guideline:

Method; Other: based upon those described by McGregor et al. (McGregor DB, Martin R, Cattanach P, Edwards I, McBride D, Caspary WJ (1987): Environ Mal Mutagen 9:143-160).

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source: Dr. D. Clive, Burroughs Wellcome Co., Research Triangle Park
- Storage: in liquid nitrogen
- Type and identity of media: Fischer's medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

without

Metabolic activation system:

Aroclor 1254

Test concentrations with justification for top dose:

Trial 1: 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 µg/mL
Trial 2: 0.25, 0.5, 1.0 and 2.0 µg/mL
Trial 3: 0.032, 0.063, 0.125, 0.25 and 0.5 µg/mL
Trial 4: 0.016, 0.032, 0.063, 0.125, 0.25 and 0.5 µg/mL
Trial 5: 0.0006, 0.0013, 0.0025, 0.005, 0.01 and 0.02 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl suiphoxide (DMSO) - analytical grade obtained from BDH Limited, Poole, Dorset, England.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicates (treatment groups and positive controls), quadriplates (vehicle control)
- Number of independent experiments: 2 without S9 mix, if no clear response was observed, 2 further experiments in the presence of S9 mix followed.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 6 x 10^6 cells in a final volume of 10 mL medium
- Test substance added in medium (F5P)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Expression time: 2 days (the cell population density was adjusted to 20 mL of 3 x 10^5 cells/mL after 24 h and after 48 h to 2 x 10^5 cells/mL)


- Selection time: 11 - 14 days
- Selection agent: 5 trifluorthymidine (TFT), 3 µg/mL
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 16 days
- Method used: agar or microwell plates for the mouse lymphoma assay.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 10^6 cells, colonies of approx. 0.1 mm in diameter were counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: total growth (measurement of cell population expansion) or cloning efficiency
- Number of cells evaluated for Cloning Efficiency: 200

Evaluation criteria:

Four response categories were defined. Primary judgments were made at the level of individual experiments, but judgment on the mutagenic potential of a chemical was made on a basis of consensus of all valid experimental results.

Calculations:
Relative total growth (RTG): total suspension growth * cloning efficiency) in dosed culture/total suspension growth * cloning efficiency in vehicle control culture
Mutant fraction (MF) = 200 * mutant clones per plate (usually a mean of 3) / total clones per plate (usually a mean of 3) = mutants/10^6 clonable cells (200 as the number of cells seeded in one plate)

Statistics:

The statistical analysis was based upon the mathematical model proposed for this system [Lee YJ, Caspary WJ (1983): Mathematical model of L5178Y mouse lymphoma forward mutation assay. Mutat Res 113:417-430. McGregor DB, Brown AG, Cattanach P, Edwards I, McBride D, Caspary] and consisted of a dose trend test [Barlow RE, Bartholomew CJ, Bremner JM, Brunk HD: Statistical inference under order restrictions: The theory and application of isotonic regression. New York: John Wiley & Sons, 1972, p 215.] and a variance analysis of pair-wise comparisons of each dose against the vehicle control.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Ten-fold differences in test compound concentrations were used in the toxicity test.
Test compound concentrations were primarily two-fold dilutions from the highest testable concentration, as estimated from the toxicity test.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:yes

The LOED was 0.016 µg/mL, where the RTG was about 10%. The maximum mutagenic response was also obtained at this dose level, while at higher dose levels, up to 2 µg/mL, the mutagenic response gradually diminished. In trial 5 (see the Tables in "Any additional information on results incl. tables") there was little evidence of toxicity at dose levels between 0.0006 and 0.01 µg/mL, above which there was a sharp reduction in survival. In other trials (2, 3, and 4) with further increases in doses up to about 1 µg/mL, survival improved and the mutant fraction reduced in parallel with this response to treatment.

Cellular response suggested a close relationship between low survival and high mutagenicity, while the effect of test compound concentration was complex and ill-defined. There appeared to be a biphasic response in toxicity, survival being good both at 0.01 µg/mL and below and again at about 0.25-0.05 µg/mL (trials 3 and 4).

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

 

Without S-9 – Trial 1 Conc. (µg/mL) CE RTG MC MF AVEMF DMSO 0.0 77 99 132 57   82 102 111 45   79 100 115 49   76 100 113 50 50 0.125 52 20 290 184   55 26 229 140 162 0.25 54 25 227 141   59 26 224 127 134 0.5 48 16 237 164   54 21 237 146 155 1.0 58 17 200 114   68 17 182 89 101 2.0 84 25 196 78   77 14 234 102 90 4.0 Lethal Lethal EMS 250.0 88 91 607 231   85 73 534 210 221 MMS 20.0 34 19 243 237   30 18 222 244 241

Without S-9 – Trial 2 Conc. (µg/mL) CE RTG MC MF AVEMF DMSO 0.0 76 126 81 35   75 87 82 36   85 90 106 42   82 98 76 31 36 0.25 62 9 343 184   53 8 359 226 205 0.5 54 5 328 204   69 10 352 171 188 1.0 85 12 378 148   64 16 309 160 154 2.0 Lethal Lethal MMS 20.0 10 4 115 371   9r 4 112 407     Experiment failed to meet quality control r = Rejected when 50% > CE > 120% or RTG < 1%

 

 

Without S-9 – Trial 3 Conc. (µg/mL) CE RTG MC MF AVEMF DMSO 0.0 93 97 197 70   95 108 155 55   88 93 30 11   94 101 118 42 45 0.032 45# 10 680 501   51 11 980 647 574 0.063 57 7 1059 616   35 7 480 457 536 0.125 80 19 595 247   56 8 529 317 282 0.25 85 27 402 158   98 30 375 128 143 0.5 77 16 368 160   88 31 248 94 127 MMS 15.0 38 22 248 216   40 21 253 213 214

Without S-9 – Trial 4 Conc. (µg/mL) CE RTG MC MF AVEMF DMSO 0.0 88 92 113 43   82 106 118 48   89 109 124 46   91 93 165 61 49 0.016 40 10 654 543   40 9 769 649 596 0.032 54 13 517 320   46 12 448 322 321 0.063 54 27 243 149   66 27 299 151 150 0.125 82 55 237 96   72 51 251 118 107 0.25 75 64 179 80   68 48 213 105 93 0.5 68 57 164 81   64 63 136 71 76 MMS 15.0 28 15 240 282   32 7 211 221 252

 

Without S-9 – Trial 5
Conc. (µg/mL)	CE	RTG	MC	MF	AVEMF
DMSO 0.0	88	96	138	52
	103	110	181	59
	106	93	189	59
	114	101	256	75	61
0.0006	99	101	158	53
	73	113	110	50	52
0.0013	97	103	143	49
	83	89	144	58	54
0.0025	88	97	135	51
	87	85	161	62	56
0.005	104	97	163	52
	109	83	233	72	62
0.01	95	84	158	56
	93	93	171	61	59
0.02	82	42	215	87
	60	32	223	124	106
MMS 15.0	54	22	558	348
	42	26	512	406	377

 CE = cloning efficiency (%); RTG = relative total growth; MC = mutant colony count; MF = mutant fraction (mutant colonies per 10E6 clonable cells); AVE MF = group average mutant fraction; underline=P < 5%; DMSO = dimethylsulfoxide; MMS= methyl meth­anesulfonate; EMS= ethyl methanesulfonate; # = Cloning efficiency was determined from two rather than three plates.

Conclusions:

The present study was conducted similar to OECD test guideline 490. Under the conditions of the study, the result was ambiguous without metabolic activation. Mutagenicity was seen only at cytotoxic concentrations.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 - 28 May 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 2020

Deviations:

yes

Remarks:

Strain testing AT mutation site is missing (S. typhimurium TA 102 or E. coli WP2 urvA)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

gene of histidine

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa: deep rough (defective lipopolysaccharide cellcoat). gal: mutation in the galactose metabolism. chl: mutation in nitrate reductase bio: defective biotin synthesis. uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene).

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: rfa: deep rough (defective lipopolysaccharide cellcoat). gal: mutation in the galactose metabolism. chl: mutation in nitrate reductase bio: defective biotin synthesis. uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene).

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

other: rfa: deep rough (defective lipopolysaccharide cellcoat). gal: mutation in the galactose metabolism. chl: mutation in nitrate reductase bio: defective biotin synthesis. uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene).

Metabolic activation:

with and without

Metabolic activation system:

Adult male Wistar rats (3 months old) were injected intraperitoneally with a solution (20% w/v) of Aroclor 1254 (500 mg/kg bw) in corn oil. Five days after injection, rats were killed by decapitation and their livers were removed aseptically. The S9 mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5 mL aqua bidest; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution, and 1 mL S9. S9 mix is prepared immediately before use and kept on ice during the test.

Test concentrations with justification for top dose:

10, 50, 100, 200, 250 and 330 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO and water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation, TA1535, TA100, 1 µg per plate in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

Without metabolic activation, TA1538, TA98, 10 µg per plate in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation, TA 1537, TA 97, 60 µg per plate in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation, all strains, 0.5 µg per plate in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 48h
- Expression time (cells in growth medium): 10E9 cells/ml

NUMBER OF REPLICATIONS: 3

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 330 µg/plate for TA98 in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test substance showed toxicity starting at a dose of 330 µg/plate. Therefore, for the main staudy 330 µg/plate was used as the upper limit dose.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: practically insoluble in water; Soluble in tetrahydrofurane: 60.8% w. at 45°C and ethanol: 7.5% w. at 45°C.

Summarized reuslts can be found under "Any other information on results incl. tables".

Toxicity of TETD for strain TA100

Dose (µg/plate)	Colonies	% of control
Control (0.1 ml DMSO)	211;218	100
100	222;243	108
330	123;133	60
1000	63;52	27
3330	- a)	-
5000	- a)	-

a) No bacterial growth

 

Mutagenic response of TETD in the Salmonella/microsome plate test

Dose (µg/plate)	Mean number of revertant colonies / 3 replicate plates (±S.D.) with different strains of S. typhimurium
	TA1537	TA97	TA1538	TA98	TA1535	TA100
	Without S9 mix
10	10±2.1	169±10.7	10±4.4	28±4.0	15±3.0	111±12.5
50	5±1.7	177±18.8	9±2.3	22±1.7	21±2.0	111±9.6
100	11±7.5	166±4.4	8±4.4	21±2.1	17±0.6	106±11.5
200	8±2.6	160±10.4	10±5.7	21±1.5	16±4.5	100±9.8
250	6±2.1	152±10.8	10±2.6	19±1.7	14±2.6	109±17.2
330	9±1.5	140±13.0	10±1.2	12±1.2	16±4.7	92±7.2
Solvent control	8±1.0	156±7.5	12±1.0	23±2.5	17±2.5	135±1.2
Positive control	392±19	1153±11.1	839±22	621±15	179±11	443±9.0
	With S9 mix
10	10±1.2	188±4.6	18;30 a)	31±4.0	13±4.2	134±6.1
50	10±3.2	187±6.7	24±4.0	32±5.9	13±1.5	123±8.0
100	12±2.5	194±3.6	18;29 a)	32±9.5	14±3.0	136±5.0
200	7±1.5	205±29.7	19±2.1	31±3.6	11±1.5	148±8.1
250	7±1.2	162±6.4	22±4.6	32±2.1	14±1.7	126±2.6
330	10±5.5	176±7.4	22±4.2	29±2.5	10±3.0	134±14.9
Solvent control	8±2.6	142±2.0	19±6.7	24±9.5	17±3.8	89±10.1
Positive control	51±3.5	503±51	385±16	356±11	89±15	853±57

a) Duplicate plates only

Conclusions:

The study was conducted similar to OECD guideline 471 and under GLP conditions. Under the conditions of the test, the test substance did not cause mutagenicity in the strains tested.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

other information

Study period:

27 Oct 1988 - 31 May 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-compliant study, available as unpublished report, no restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

adopted in 2014

Deviations:

yes

Remarks:

The test is not recommended anymore for testing of genotoxicity.

Principles of method if other than guideline:

According to the modified procedures of Williams et al. (1977, 1982)

GLP compliance:

yes

Type of assay:

other: DNA repair assay

Target gene:

no specific target gene, as DNA repair was evaluated

Species / strain / cell type:

hepatocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: primary cells, hepatocytes

MEDIA USED
- Type and composition of media: WMES
- CO2 concentration: 5%, humidified
- temperature: 37 °C

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

0.0005; 0.001; 0.005; 0.01; 0.05; 0.1; 0.5 mg/L

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

Fluorene

Positive controls:

yes

Positive control substance:

other: 2-Aminofluorene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicates
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5 * 10^5 cells were seeded onto cover slips in 6-well dishes. 2 h after seeding, the cover slips were washed so that only attached viable cells remained.
- Test substance added in medium, immediatly after washing, together with 10 µCi/mL thymidine ([3H]-TdR), 50 - -80 Ci/mM

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 18 - 20 h
- Harvest procedure: after the treatment period, cover slips were washed with phosphate buffered saline (three times). Then, the cover slips were immersed in 1% sodium citrate (10 min) to allow the nuclei to swell. Finally, the cells were fixed in 3 30-min changes of ethanol-glacial acetic acid (5:1), air dried and mounted up on glass slides. The slides were dipped in total darkness into NTB emulsion, prewarmed for 1 h at approx. 45 °C and afterwards dried overnight. They were then stored at 3 - 10 °C. After 9 days, autoradiographs were developed. Slides were stained in Harris' alum hematoxylin and counterstained with eosin.
- Number of cells counted: 5 - 20 from each quadrant of the slide
-

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Absence of S-phase cells in the autoradiograph and general morphology.

Evaluation criteria:

Only cells with swollen nuclei and evenly coated with emulsion were scored. Background was determined by counting 3 nuclear sized areas adjacent to the nucleus. Net nuclear grain counts were calculating by subtracting the highest cytoplasmic count from the nuclear count.

A compound is reported negative if the net nuclear count was not statistically significantly increased at
a) all concentrations up to the highest non-toxic concentration or
b) all concentrations up to the limit of solubility.

A compound is reported positive if the net nuclear grain count exceeded those of the control by at least 2 standard deviations. The evidence was considered stronger if a dose response was observed.

Key result

Species / strain:

hepatocytes: rats

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations ranging from 1 to 5E-3 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: no precipitation was observed up to the highest tested concentration

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Fluorene, the true negative control and DMSO, the vehicle control, had net nuclear grain counts of -14.9 ± 1.7 and -22.4 ± 3.0, respectively. The positive control 2-AF was genotoxic indicating that the cells used were capable of metabolic activation and DNA repair. Net nuclear grain counts of 2-AF were 40.3.

- Genotoxicity results: No genotoxicity was observed.
- Cytotoxicity results: The substance was cytotoxic at and above 5*10^-3 mg/mL. No DNA repair was observed at this concentrations.

Summarized results can be fount in the attached background material.

HISTORICAL CONTROL DATA: No

Conclusions:

The study was not conducted according to any guideline but under GLP conditions. DNA repair was examined in primary hepatocytes from rats. No genotoxicity was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Cytogenetic assays in bone marrow cells
Source, RA-A, CAS 137-26-8, Key; T5558.122; MNT (similar to OECD 474); GLP, mouse bone marrow; 38 - 377 mg/kg bw; negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Jul - 9 Nov 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, NY, USA
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 27.1 - 35.9 g (males), 23.8 - 30.7 g (females)
- Assigned to test groups randomly: yes
- Housing: up to 5 per cage in plastic autoclavable cages with wire lids
- Diet:certified laboratory rodent chow, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26.67
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Jul 1987 1987 To: 15 Oct 1987

Route of administration:

intraperitoneal

Vehicle:

CMC (carboxymethyl cellulose), 1%
- Justification for choice of solvent/vehicle: toxicity profile in animals
- Concentration of test material in vehicle: 3.8, 18.9 and 37.7 for the low, mid and high dose, respectively
- Volume administered: 10 mL/kg bw
- Lot/batch no.: 7-2-87

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared freshly and based on the most recently measured body weight (directly before injection).

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

24, 48 and 72 h

Dose / conc.:

38 mg/kg bw/day

Dose / conc.:

189 mg/kg bw/day

Dose / conc.:

377 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Route of administration: IP injection
- Doses / concentrations: 0.25 mg/kg bw
- Vehicle: water

Tissues and cell types examined:

Bone marrow from femur was prepared.

Details of tissue and slide preparation:

Toxicits test: A toxicity test was conducted with 6 groups of five male and five female rats each. Animals were observed after dose administration and daily thereafter for 7 days for clinical signs of chemical effect. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration

TREATMENT AND SAMPLING TIMES: Animals were observed after dosing for clinical signs. At the scheduled time of sacrifice, 5 mice/sex were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just about the knee and the bone marrow was aspired into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL FBS. The bone marrow cells were pelleted by centrifugation.

DETAILS OF SLIDE PREPARATION: After centrifugation, the pellet was were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS: Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei. The proportion of polychromatic erythrocytes to total erythrocytes and the number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes were also enumerated.

Evaluation criteria:

The test article is considered to induce a positive response if a treatment—related increase in micronucleated polychromatic erythrocytes is observed relative to the vehicle control (P<0.05, Kastenbaum—Bowman Tables) . The positive response must be dose—dependent or must be observed at a single dose level at adjacent sacrifice times. If a single treatment group is significantly elevated at one sacrifice time, the assay is considered a suspect or unconfirmed positive and a repeat assay will be recommended.

The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes in the negative (vehicle) control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be statistically significantly increased relative to the negative control (P<0.05, Kastenbaum—Bowman Tables) .

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was presented for each animal and treatment group.

Statistical significance was determined using the Kastenbaum—Bowman tables which were based on the binomial distribution.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF TOXICITY STUDY
- Dose range: 200 - 1000 mg/kg bw
- Results: Deaths were observed in all test article—treated groups, with 1, 6, 9, 7 and 9 deaths occurring at dose groups of 200, 400, 600, 800 and 1000 mg/kg bw, respectively. Clinical observations for the above groups included lethargy, paralysis, and piloerection. The LD50 was calculated by probit analysis to be approximately 471 mg/kg bw. 377 mg/kg bw was estimated to be 80% of the LD50 and was selected as the high dose for the micronucleus test.

RESULTS OF DEFINITIVE STUDY
At the highest dose level, mortality (15/38 animals), clinical signs and bone marrow toxicity (reduced ratio PCE/Total E) were observed. These effects were also apparent in some animals in the mid-dose group. No increase in the number of micronucleated polychromatic erythrocytes was detected at any of the sampling times. Experimental conditions as well as the criteria for the determination of test responses are well defined and appropriate. Positive controls gave the expected response.

Summarized results can be found in Attachment 1 in the attached background material.

Conclusions:

The study was conducted similar to OECD guideline 474 and under GLP conditions.

Under the conditions of the test, the test substance did not increase the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female CD-1 mice.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

With data from the read across source substance it is concluded that the target substance is not genotoxic in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Please refer to the respective endpoint summary.

Additional information

As the required test battery for evaluating possible mutagenic/genotoxic effects of disulfiram in vitro and in vivo is not complete, read-across was performed using the surrogate substance thiram (CAS 137-26-8) (For detailed information on the justification of read-across, please refer to the analogue justification document attached in IUCLID section 13).

 

The mutagenicity of disulfiram in bacteria was assessed using bacterial reverse mutation assays (Ames test) with S. typhimurium strains TA 97, TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (NOTOX/ES 62/82.5, 20838, 88-05, 20998, Hedenstedt et al 1979, Rannung et al. 1984). In all except for one study (Rannug et al., 1984), which is not assignable, disulfiram was negative, with and without mutagenic activation. A mouse lymphoma assay performed without metabolic activation indicated that disulfiram causes gene mutations in mammalian cells in vitro, but only in concentrations which cause cytotoxicity (NIH/NIEHS). Moreover, a HPRT test performed with the analogue substance thiram (CAS 137-26-8) failed to show genotoxicity in the absence of cytotoxicity (0174/EV 1). In addition, disulfiram was tested in an unscheduled DNA synthesis assay (0174/ER156). In this assay, no effects on the DNA repair in primary hepatocytes following exposure to disulfiram were reported.

In a GLP-guideline study, Thiram did not induce micronuclei in bone marrow polychromatic erythrocytes in male and female CD-1 mice (T5558.122). Furthermore, in an in vivo gene mutation test (mouse spot test) and a chromosome aberration test with mouse germ cells (spermatogonia) performed in mice (CCR200902, CCR175127), Thiram revealed no mutagenic capacities.

 

Classification for mutagenicity: None.

 

Short description of key information:

In vitro:

Gene mutation (Bacterial reverse mutation assay, comparable to OECD 471): negative with and without metabolic activation.

Mammalian cell gene mutation test (OECD 476): ambiguous; positive at cytotoxic concentrations only.

 

In vivo:

Mammalian cell chromosome aberration test (EPA OPP 84-2): negative.

 

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity is conclusive but not sufficient for classification according to Regulation (EC) No 1272/2008.

.