Zinc bis(dibenzyldithiocarbamate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Zinc bis(dibenzyldithiocarbamate) did not induce an increased number of revertants in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 strains, both in with and without metabolic activation (OECD 471, GLP). In a gene mutation study in L5178Y mouse lymphoma cells (OECD 476, GLP), a dose related increase in mutation frequency was observed in both absence and presence of metabolic activation (with a relative total growth of 10% and 16%, respectively). In an in vitro micronucleus test in human lymphocytes (OECD 487, GLP), no clastogenic and/or aneugenic was observed.

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:

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:

S-9 mix

Test concentrations with justification for top dose:

50, 158, 500, 1580 and 5000 µg per plate.

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Positive controls:

yes

Positive control substance:

9-aminoacridine

Positive controls:

yes

Positive control substance:

sodium azide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 2 days at 37°C

NUMBER OF REPLICATIONS: triplicate

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

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:

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity test performed with a series of eight different concentrations of test material from 2.5 ug to 5 mg per plate, inoculated with an overnight culture of strain TA 98.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to ZDBzC. A top exposure level of 5 mg per plate was therefore selected for use in the main assays.

CYTOTOXICITY:
Inhibition of growth, observed as reduction in revertant colony numbers, occurred in all strains on at least one occasion of testing following exposure to ZDBzC at 5000 ug per plate, and in strain TA 100 only with ZDBzC at 1580 ug per plate.

Remarks on result:

other: all strains/cell types tested

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	ZDBzC sterility check	5000	-	0	0	0	0	0	0	0	0
3	ZDBzC	5000	+	22	30	42	20	7	10	3	4
4	ZDBzC	1580	+	36	31	73	50	13	12	6	7
5	ZDBzC	500	+	35	33	108	113	13	13	6	7
6	ZDBzC	158	+	34	34	112	112	14	13	8	6
7	ZDBzC	50	+	35	32	111	110	14	15	6	8
8	DMSO		+	37	32	110	110	13	13	8	7
9	ZDBzC	5000	-	19	14	34	20	14	8	2	2
10	ZDBzC	1580	-	30	29	64	39	14	14	6	6
11	ZDBzC	500	-	32	33	109	91	13	17	6	7
12	ZDBzC	158	-	35	34	110	109	14	15	7	7
13	ZDBzC	50	-	32	33	106	112	13	16	7	6
14	DMSO		-	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

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Range-finding studies:
24 hours, without S9: 63 to 1000 μg/ml and 3 to 50 μg/ml
Main study:
24-hours, without S9: 0, 0.013, 0.025, 0.1, 0.3, 0.6, 1.2, 1.7, 2.4 and 3.4 μg/ml
4 hours, with S9: 0, 0.1, 0.2, 0.4, 0.8, 1.7, 3.4, 4.9, 7.0 μg/ml

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: + S9: Methyl methanesulphonate (MMS); -S9: 3-methylcholanthrene (MCA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 hours without S9, 4 hours with S9
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: single cultures were used for each concentration of the test substance; two cultures treated with DMSO were used as negative controls; one single culture was used for positive controls.

NUMBER OF CELLS EVALUATED: 1,000,000 clonable cells

DETERMINATION OF CYTOTOXICITY
- Method: the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG).

Evaluation criteria:

A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 126 mutants per 1,000,000 clonable cells. A response was considered to be equivocal if the induced mutant frequency was more than 88 mutants (but smaller than 126 mutants) per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity was considered to be an artefact and not indicative of genotoxicity.
The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.
The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test substance concentrations.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In both the absence and presence of S9-mix a dose related significant increase in the mean mutant frequency (MF) was observed. In the absence of S9-mix the mutant frequency was significantly increased by 134 mutants per 1,000,000 clonable cells compared to the negative control at 3.4 μg/ml. The RTG at this concentration was 10%.
In the presence of S9-mix the mutant frequencies at 4.9 and 7.0 μg/ml were increased by 232 and 497 mutants per 1,000,000 clonable cells, compared to the negative control, respectively. The RTG at these concentrations were 16 and 78%

RANGE-FINDING/SCREENING STUDIES:
In the first dose range finding study single cultures were treated for 24 hours in the absence of S9-mix with 5 concentrations Perkacit ZBEC pdr ranging from 63 to 1000 μg/ml. After 4 and 24 hours aliquots were taken to assess the viability and cell yield. After 4 hours treatment at a concentration of 250 μg/ml the viability was about 50% and cell yield was 91%, at 63 μg/ml 80% and 86%, respectively. After 24 hours, at a concentration of 63 μg/ml the cell yield was 8%. Based on these results a second dose range finding assay was performed.
In the second dose range finding assay single cultures were treated for 24 hours in the absence of S9-mix with 5 concentrations Perkacit ZBEC pdr ranging from 3 to 50 μg/ml. After 4 hours treatment at a concentration of 50 μg/ml the viability and cell yield were about 80%. After 24 hours, at a concentration of 6 μg/ml the viability was below 10% and cell yield was 17%.
Based on these results the highest dose used in the main assay was 3.4 μg/ml and 10 μg/ml in the absence and presence of S9-mix, respectively.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix the test substance was toxic to the cells. The initial cell yield and/or relative suspension growth (RSG) and relative total growth (RTG) were reduced at and above 1.7 μg/ml. The highest dose level of the test substance tested and evaluated for mutagenicity was 3.4 μg/ml; the RTG at this dose was 10%.
In the presence of S9-mix the test substance was also toxic to the cells. The initial cell yield and/or RSG and RTG were reduced at and above 4.9 μg/ml. The highest dose level of the test substance tested and evaluated for mutagenicity was 7.0 μg/ml; the RTG at this dose was 16%.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Summary of the results

Dose (μg/ml)	Absence of S9-mix (24 hr)		Dose (μg/ml)	Presence of S9-mix (4 hr)
	Mutation frequency	Relative total growth		Mutation frequency	Relative total growth
3.4	205	10	7.0	563	16
2.4	107	44	4.9	298	78
1.7	72	71	3.4	105	92
1.2	42	97	1.7	83	95
0.6	47	140	0.8	74	94
0.3	76	127	0.4	78	82
0.1	49	115	0.2	57	86
0.025	72	99	0.1	58	103
0.013	83	100
0	71*	100*	0	66*	100*

 

* Mean of duplicate cultures

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May 2018 to 19 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

The cytotoxicity of the highest concentrations selected for evaluation of micronuclei was slightly lower than the aimed 55 +/- 5%.

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors if applicable:
Blood samples were obtained by venapuncture from two young healthy, non-smoking donors (22 and 23 years old for the first and second experiment, respectively) with no known recent exposures to genotoxic chemicals or radiation.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The medium for culturing the human peripheral blood lymphocytes consisted of RPMI 1640 medium (with HEPES and Glutamax-I), supplemented with heat-inactivated (30 min, 56 °C) fetal calf serum (20% v/v), penicillin (100 U/mL medium), streptomycin (100 μg/mL medium) and phytohemagglutinin (2.4 μg/mL). Whole blood (0.5 mL) was added to sterile screw-capped tubes containing 4.5 mL culture medium. The blood cultures were incubated for ca. 48 hours at ca. 37 °C in humidified air containing ca. 5% CO2.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

cytochalasin B (final concentration 6 μg/mL, stock solution 600 μg/mL in DMSO)

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

EXPERIMENT 1
- 4 hours without S9-mix: 12.5, 6.25 and 3.13 µg/mL were selected from a series of the following concentrations: 150, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 and 0.39 μg/mL, because the higher concentrations were too cytotoxic (> 60% cytotoxicity) and the lower concentrations did not show sufficient cytotoxicity.

- 4 hours with S9-mix: 25, 12.5 and 6.25 µg/mL were selected from a series of the following concentrations: 150, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 and 0.39 μg/mL, because the higher concentrations were too cytotoxic (> 60% cytotoxicity) and the lower concentrations did not show sufficient cytotoxicity.

EXPERIMENT 2
- 24 hours without S9-mix: 20, 10 and 2.5 µg/mL were selected from a series of the following concentrations: 100, 80, 60, 50, 40, 30, 20, 10, 5, 2.5 and 1.25 μg/mL, because the higher concentrations were too cytotoxic (> 60% cytotoxicity) and the lower concentrations did not show sufficient cytotoxicity.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Vinblastine Sulphate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Experiment 1:
DURATION
- Exposure duration: 4 hours in the absence and presence of metabolic activation
- Recovery duration: 20 hours in the presence of cytochalasin B (final concentration 6 µg/mL)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

Experiment 2:
DURATION
- Exposure duration: 24 hours in the absence of metabolic activation, but in the presence of cytochalasin B (final concentration 6 µg/mL)
- Recovery duration: 0 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

NUMBER OF REPLICATIONS: duplicate cultures for both experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Each culture was harvested and processed separately. The cells were harvested by low speed centrifugation, treated with a hypotonic solution (0.075 M potassium chloride), fixed three times with a freshly prepared mixture of methanol and acetic acid, spread on clean slides and air dried. All procedures were performed at ambient temperature. Three slides were prepared from the negative (solvent) controls, positive controls and from each selected culture treated with the test substance. The slides were coded by a qualified person not involved in scoring the slides to enable ‘blind’ scoring and thereafter stained with a fluorescence DNA-specific dye (acridine-orange) for analysis. One slide per culture was analysed for Cytokinesis-Block Proliferation Index (CBPI) and two slides were analysed for micronucleus formation.

NUMBER OF CELLS EVALUATED: The CBPI was determined from at least 500 cells per slide (in total 1000 cells per dose level). At least 2000 binucleated cells per concentration (500 cells per slide, two slides per culture, two cultures per concentration) were examined for the presence of micronuclei.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Micronuclei are morphologically identical to but smaller than nuclei. They have the following characteristics:
1. The diameter of micronuclei usually varies between 1/16 and 1/3 of the diameter of the main nuclei.
2. Micronuclei are round or oval in shape.
3. Micronuclei are nonrefractile and can therefore be readily distinguished from artefacts such as staining particles.
4. Micronuclei are not linked or connected to the main nuclei.
5. Micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary.
6. Micronuclei usually have the same staining intensity as the main nuclei but occasionally staining is more intense.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index (CBPI)

Evaluation criteria:

The study is considered valid if
- the positive controls demonstrate a statistically significantly increase in the number of binucleated cells containing micronuclei
- the response of the positive controls is comparable to historical positive control data
- the micronuclei frequency of the negative (solvent) controls is within 95% control limits of the historical data of the test facility and /or comparable to the data presented in the literature
- proliferation criteria in the negative (solvent) controls are fulfilled
- adequate numbers of cells and concentrations are analysable
A response is considered positive if all of the following criteria are met:
- at least one of the test concentrations exhibits a statistically significant increase compared to the concurrent negative control
- the increase is concentration-related in at least in one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical solvent control data
A response is considered negative if all of the following criteria are met:
- none of the test concentrations exhibits a statistically significant increase compared to the concurrent negative control
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical negative control data
In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, justification should be provided or the results should be further evaluated (e.g. scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing).
A test substance is considered equivocal if the response is neither positive nor negative, even after further investigation.

Statistics:

The frequencies of micronuclei were compared with those of the concurrent solvent controls using the Chi-Square test (one sided). In addition, a statistical test for trend was performed. The results were considered statistically significant if the P-value was less than 0.05.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Prior to the conduct of the test, the solubility of the test substance was determined and dimethyl sulfoxide (DMSO) was considered a suitable solvent for the in vitro micronucleus test. The osmolality and pH values were comparable to the negative (solvent) control. Based on these results and due to the precipitation of the test substance observed during the solubility test, a concentration of 150 μg/mL was considered the maximum feasible final concentration of the test substance in the culture medium.

NUMBER OF CELLS WITH MICRONUCLEI
The test substance did not show a statistically significant increase in the number of binucleated cells containing micronuclei at any of the concentrations analysed in any of the experiments when compared to the concurrent solvent control cultures. In addition, no dose related micronuclei induction was observed in treatment groups, except in the pulse treatment group with S9-mix. As the number of binucleated cells containing micronuclei found in the test substance treated cultures in the presence of S9-mix was well within (and even at the lower end of) the historical range of the test facility and was not statistically significantly increased compared to the concurrent solvent control cultures, this observation was considered not biologically relevant.

CONTROL DATA
In both experiments, the micronuclei frequency of the solvent controls was within the 95% control limits of historical data of the test facility. Treatment with the positive controls Cyclophosphamide (20 μg/mL) and Vinblastine Sulphate (0.0125 μg/mL) resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the concurrent solvent control cultures in the first and second experiment, respectively. In addition, the micronuclei frequency of the positive controls was comparable to historical data of the test facility. Therefore, both experiments were considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first experiment, i.e. the pulse treatment groups, the test substance showed concentration-related cytotoxicity. In the absence of S9-mix, the highest concentration (150 μg/mL) was severely cytotoxic to the cells. The next three test concentrations (100, 50 and 25 μg/mL) showed a strong cytotoxicity of 96%, 81% and 75%, respectively. Concentrations selected for analysis of micronucleus induction (12.5, 6.25 and 3.13 μg/mL) showed a cytotoxicity of 33%, 12% and 4% when compared to the concurrent solvent control, respectively. It should be noted that the highest concentration selected for micronucleus induction analysis (12.5 μg/mL) showed a slightly lower cytotoxicity than the aimed 55 ± 5% stated in OECD guideline 487 when compared to the concurrent solvent control. However, the next higher concentration tested was strongly cytotoxic to the cells and the concentrations were closely spaced. Furthermore, during CBPI analysis a low cell density (few intact cells) on the slides were observed, which indicates a reduction in total cell amount as a result of the treatment. As a repeat test of this pulse treatment group without S9-mix with even more closely spaced concentrations with the aim to reach the required cytotoxicity was anticipated to be of very limited added value, it was considered justified to select a top concentration with a slightly lower cytotoxicity than the aimed 55 ± 5% cytotoxicity.
In the presence of S9-mix, the highest concentration (150 μg/mL) was severely cytotoxic to the cells. The next two test concentrations (100 and 50 μg/mL) showed a strong cytotoxicity of 93% and 84%, respectively. Concentrations selected (25, 12.5 and 6.25 μg/mL) showed 37%, 22% and 4% cytotoxicity, respectively. It should be noted that the highest concentration selected for micronucleus induction analysis (25 μg/mL) showed a slightly lower cytotoxicity than the aimed 55 ± 5% stated in OECD guideline 487 when compared to the concurrent solvent control. However, the next higher concentration tested was strongly cytotoxic to the cells and the concentrations were closely spaced. Furthermore, during CBPI analysis a low cell density (few intact cells) on the slides were observed, which indicates a reduction in total cell amount as a result of the treatment. As a repeat test of this pulse treatment group without S9-mix with even more closely spaced concentrations with the aim to reach the required cytotoxicity was anticipated to be of very limited added value, it was considered justified to select a top concentration with a slightly lower cytotoxicity than the aimed 55 ± 5% cytotoxicity. Slides of the solvent controls and positive control Cyclophosphamide were analysed for micronuclei induction in parallel.
In the second experiment, i.e. the continuous treatment group in the absence of S9-mix, the highest two concentrations were severely cytotoxic to the cells, as demonstrated by a very low cell density (only very few mononucleated cells) along with damaged cytoplasm (100 and 80 μg/mL). The next lower concentrations (60, 50, 40 and 30 μg/mL) showed 90%, 87%, 65% and 63% cytotoxicity, respectively. The concentrations selected (20, 10 and 2.5 μg/mL) showed 45%, 34% and 3% cytotoxicity, respectively. It should be noted that 45% cytotoxicity is slightly below the aimed 55 ± 5% cytotoxicity stated in the OECD guideline 487. However, the next higher concentration tested was strongly cytotoxic to the cells and the concentrations were closely spaced. Although this experiment did not fully meet the aimed cytotoxicity range of the OECD guideline 487, it was considered justified to select a top concentration with a slightly lower cytotoxicity than the aimed 55 ± 5% cytotoxicity. Slides of the solvent control (2% DMSO), additional solvent control (1% DMSO) and positive control Vinblastine sulphate were analysed for micronuclei induction in parallel.

Table 1: Pulse treatment with metabolic activation

Treatment /recovery time (h)	Dose level (µg/ml)	Cell stage analysis/500 (MO-BN-MU)			BN (%)	CBPI	CBPI (mean)	RI (%)	% Cytotox. (100-RI)	Selected for MN analysis (+/-)	MNBN/ 1000BN	MNBN/ 2000 BN (%)	Statistics1 (p-value)
	1% DMSO	222	274	4	54.80	1.564	1.579	100	0	+	4	8 (0.40)	-
		209	285	6	57.00	1.594					4
	150	*	*	*	-	-	-	-	-	-	-	-	-
		*	*	*	-	-					-
	100	478	22	0	4.40	1.044	1.043	7	93	-	-	-	-
		479	21	0	4.20	1.042					-
4/20	50	452	47	1	9.40	1.098	1.091	16	84	-	-	-	-
(+S9)		458	42	0	8.40	1.084					-
	25	309	190	1	38.00	1.384	1.363	63	37	+	7	13 (0.65)	n.s.
		329	171	0	34.20	1.342					6
	12.5	298	199	3	39.80	1.410	1.450	78	22	+	3	10 (0.50)	n.s.
		256	243	1	48.60	1.490					7
	6.25	235	263	2	52.60	1.534	1.554	96	4	+	2	8	n.s.
		244	296	10	53.82	1.575					6	(0.40)
	3.13	246	251	3	50.20	1.514	1.518	89	11	-	-	-	-
		245	249	6	49.80	1.522					-
	CP20	402	98	0	19.60	1.196	1.205	35	65	+	24	49	****
		393	107	0	21.40	1.214					25	(2.45)	(<0.0001)

The fixed cells of dose levels (1.56 to 0.39 µg/ml) were stored without slide preparation.

Abbreviations:

Cytotox: cytotoxicity; DMSO: solvent control (1% DMSO); MO: mononucleated cells; BN: binucleated cells; MU: multinucleated cells; CBPI: Cytokinesis-Block Proliferation Index; RI: Replication index; MN: micronuclei; MNBN: micronucleated binucleated cells; CP: Cyclophosphamide; n.s: not significant compared to the concurrent control; *: no cells available on the slides for microscopic analysis,¹Chi-square test (one-sided); **** p<0.0001

Table 2: Pulse treatment without metabolic activation

Treatment /recovery time (h)	Dose level (µg/ml)	Cell stage analysis/500 (MO-BN-MU)			BN (%)	CBPI	CBPI (mean)	RI (%)	% Cytotox. (100-RI)	Selected for MN analysis (+/-)	MNBN/ 1000BN	MNBN/ 2000 BN (%)	Statistics1 (p-value)
	1% DMSO	172	306	22	61.20	1.700	1.656	100	0	+	5	7 (0.35)	-
		207	280	13	56.00	1.612					2
	150	*	*	*	-	-	-	-	-	-	-	-	-
		*	*	*	-	-					-
	100	482	18	0	3.60	1.036	1.029	4	96	-	-	-	-
		489	11	0	2.20	1.022					-
4/20	50	422	78	0	15.60	1.156	1.123	19	81	-	-	-	-
(-S9)		455	45	0	9.00	1.090					-
	25	407	93	0	18.60	1.186	1.164	25	75	-	-	-	-
		429	71	0	14.20	1.142					-
	12.5	284	216	0	43.20	1.432	1.442	67	33	+	1	4 (0.20)	n.s.
		275	224	1	44.80	1.452					3
	6.25	209	287	4	57.40	1.590	1.576	88	12	+	4	9	n.s.
		220	279	1	55.80	1.562					5	(0.45)
	3.13	191	299	10	59.80	1.638	1.627	96	4	+	1	6	n.s.
		201	290	9	58.00	1.616					5	(0.30)

The fixed cells of dose levels (1.56 to 0.39 µg/ml) were stored without slide preparation.

Abbreviations:

Cytotox: cytotoxicity; DMSO: solvent control (1% DMSO); MO: mononucleated cells; BN: binucleated cells; MU: multinucleated cells; CBPI: Cytokinesis-Block Proliferation Index; RI: Replication index; MN: micronuclei; MNBN: micronucleated binucleated cells, n.s: not significant compared to the concurrent control

*: no cells available on the slides for microscopic analysis

Table 3.3: Continuous treatment without metabolic activation                          

Treatment /recovery time (h)	Dose level (µg/ml)	Cell stage analysis/500 (MO-BN-MU)			BN (%)	CBPI	CBPI (mean)	RI (%)	% Cytotox. (100-RI)	Selected for MN analysis (+/-)	MNBN/ 1000BN	MNBN/ 2000 BN (%)	Statistics1 (p-value)
24/0 (-S9)	2% DMSO	184	268	48	53.6	1.728	1.688	100	0	+	9	19 (0.95)	-
		214	248	38	49.6	1.648					10
	Untreated	200	248	52	49.6	1.704	1.706	103	0	+	9	19 (0.95)	n.s.
		206	234	60	46.8	1.708					10
	100	*	*	*	-	-	-	-	-	-	-	-	-
		*	*	*	-	-					-
	80	*	*	*	-	-	-	-	-	-	-	-	-
		*	*	*	-	-					-
	60	463	37	0	7.4	1.074	1.067	10	90	-	-	-	-
		470	30	0	6.0	1.060					-
	50	457	43	0	8.6	1.086	1.088	13	87	-	-	-	-
		455	45	0	9.0	1.090					-
	40	377	120	3	24.0	1.252	1.239	35	65	-	-	-	-
		389	109	2	21.8	1.226					-
	30	366	134	0	26.8	1.268	1.255	37	63	-	-	-	-
		381	117	2	23.4	1.242					-
	20	326	169	5	33.8	1.358	1.377	55	45	+	13	22	n.s.
		304	194	2	38.8	1.396					9	(1.10)
	10	281	209	10	41.8	1.458	1.457	66	34	+	13	22	n.s.
		280	212	8	42.4	1.456					9	(1.10)
	5	230	251	19	50.2	1.578	1.589	86	14	-	-	-	-
		218	264	18	52.8	1.600					-
24/0 (-S9)	2.5	197	256	47	51.2	1.700	1.670	97	3	+	11	22	n.s.
		214	252	34	50.4	1.640					11	(1.10)
	1.25	206	256	38	51.2	1.664	1.662	96	4	-	-	-	-
		213	244	43	48.8	1.660					-
	VB 0.0125	282	190	28	38.0	1.492	1.488	71	29	+	21	50	****
		289	180	31	36.0	1.484					29	(2.50)	(<0.0001)
	VB 0.00625	184	276	40	55.2	1.712	1.742	108	0	-	-	-	-
		180	254	66	50.8	1.772					-

Abbreviations and footnotes:

Cytotox: cytotoxicity; DMSO: solvent control (1% DMSO); Untreated: additional control (1% DMSO); MO: mononucleated cells; BN: binucleated cells; MU: multinucleated cells; CBPI: Cytokinesis-Block Proliferation Index; RI: Replication index; MN: micronuclei; MNBN: micronucleated binucleated cells; VB 0.0125: Vinblastine sulphate (0.0125 µg/ml); VB 0.00625: Vinblastine sulphate (0.00625 µg/ml), *: no binucleated cells available on the slides for microscopic analysis, only very few mononucleated cells present along with damaged cytoplasm; n.s: not significant compared to DMSO.¹Chi-square test (one-sided); **** p≤0.0001.

Conclusions:

From the results obtained in this in vitro micronucleus test, it is concluded that the test substance was not clastogenic and/or aneugenic to cultured human lymphocytes, under the conditions used in this study.

Executive summary:

In this GLP compliant in vitro micronucleus test performed according to OECD 487, the test substance ZBEC (Zinc bis(dibenzyldithiocarbamate)) was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes both in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix).

Two independent experiments were performed. In the first experiment the treatment/recovery time was 4/20 hours (pulse treatment), both in the presence and absence of S9-mix. In the second experiment, the treatment/recovery time was 24/0 hours in the absence of S9-mix (continuous treatment). Dimethyl sulfoxide was used as a solvent for the test substance. In the first experiment, the final test concentrations ranged from 150 to 0.39 μg/mL. In the second experiment the final test concentrations ranged from 100 to 1.25 μg/mL. Duplicate cultures were used. Cytotoxicity was determined from the Cytokinesis-Block Proliferation Index (CBPI). Negative controls (solvent) and positive controls were run simultaneously with the test substance.

In both experiments, the micronucleus frequency of the solvent controls was within 95% control limits of historical data of the test facility. Treatment with the positive controls Cyclophosphamide and Vinblastine sulphate resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the concurrent solvent control cultures. In addition, the micronuclei frequency of the positive controls was comparable to the historical data of the test facility. Therefore, the test was considered valid.

In the first experiment, i.e. the pulse treatment groups, the test substance showed concentration-related cytotoxicity. In the absence of S9-mix, the highest concentration (150 μg/mL) was severely cytotoxic to the cells. The next three test concentrations (100, 50 and 25 μg/mL) showed a strong cytotoxicity of 96%, 81% and 75%, respectively. Concentrations selected for analysis of micronucleus induction (12.5, 6.25 and 3.13 μg/mL) showed a cytotoxicity of 33%, 12% and 4% when compared to the concurrent solvent control, respectively. It should be noted that the highest concentration selected for micronucleus induction analysis (12.5 μg/mL) showed a slightly lower cytotoxicity than the aimed 55 ± 5% stated in OECD guideline 487 when compared to the concurrent solvent control. However, the next higher concentration tested was strongly cytotoxic to the cells and the concentrations were closely spaced. Furthermore, during CBPI analysis a low cell density (few intact cells) on the slides were observed, which indicates a reduction in total cell amount as a result of the treatment. As a repeat test of this pulse treatment group without S9-mix with even more closely spaced concentrations with the aim to reach the required cytotoxicity was anticipated to be of very limited added value, it was considered justified to select a top concentration with a slightly lower cytotoxicity than the aimed 55 ± 5% cytotoxicity.

In the presence of S9-mix, the highest concentration (150 μg/mL) was severely cytotoxic to the cells. The next two test concentrations (100 and 50 μg/mL) showed a strong cytotoxicity of 93% and 84%, respectively. Concentrations selected (25, 12.5 and 6.25 μg/mL) showed 37%, 22% and 4% cytotoxicity, respectively. It should be noted that the highest concentration selected for micronucleus induction analysis (25 μg/mL) showed a slightly lower cytotoxicity than the aimed 55 ± 5% stated in OECD guideline 487 when compared to the concurrent solvent control.

However, the next higher concentration tested was strongly cytotoxic to the cells and the concentrations were closely spaced. Furthermore, during CBPI analysis a low cell density (few intact cells) on the slides were observed, which indicates a reduction in total cell amount as a result of the treatment. As a repeat test of this pulse treatment group without S9-mix with even more closely spaced concentrations with the aim to reach the required cytotoxicity was anticipated to be of very limited added value, it was considered justified to select a top concentration with a slightly lower cytotoxicity than the aimed 55 ± 5% cytotoxicity. Slides of the solvent controls and positive control Cyclophosphamide were analysed for micronuclei induction in parallel.

In the second experiment, i.e. the continuous treatment group in the absence of S9-mix, the highest two concentrations were severely cytotoxic to the cells, as demonstrated by a very low cell density (only very few mononucleated cells) along with damaged cytoplasm (100 and 80 μg/mL). The next lower concentrations (60, 50, 40 and 30 μg/mL) showed 90%, 87%, 65% and 63% cytotoxicity, respectively. The concentrations selected (20, 10 and 2.5 μg/mL) showed 45%, 34% and 3% cytotoxicity, respectively. It should be noted that 45% cytotoxicity is slightly below the aimed 55 ± 5% cytotoxicity stated in the OECD guideline 487. However, the next higher concentration tested was strongly cytotoxic to the cells and the concentrations were closely spaced. Although this experiment did not fully meet the aimed cytotoxicity range of the OECD guideline 487, it was considered justified to select a top concentration with a slightly lower cytotoxicity than the aimed 55 ± 5% cytotoxicity. Slides of the solvent control (2% DMSO), additional solvent control (1% DMSO) and positive control Vinblastine sulphate were analysed for micronuclei induction in parallel.

In both experiments, the micronuclei frequency of the solvent controls was within the 95% control limits of historical data of the test facility. Treatment with the positive controls Cyclophosphamide (20 μg/mL) and Vinblastine sulphate (0.0125 μg/mL) resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the concurrent solvent control cultures in the first and second experiment, respectively. In addition, the micronuclei frequency of the positive controls was comparable to historical data of the test facility. Therefore, both experiments were considered valid.

The test substance did not show a statistically significant increase in the number of binucleated cells containing micronuclei at any of the concentrations analysed in any of the experiments when compared to the concurrent solvent control cultures. In addition, no dose related micronuclei induction was observed in treatment groups, except in the pulse treatment group with S9-mix. As the number of binucleated cells containing micronuclei found in the test substance treated cultures in the presence of S9-mix was well within (and even at the lower end of) the historical range of the test facility and was not statistically significantly increased compared to the concurrent solvent control cultures, this observation was considered not biologically relevant.

From the results obtained in this in vitro micronucleus test, it is concluded that the test substance was not clastogenic and/or aneugenic to cultured human lymphocytes, under the conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Zinc bis(dibenzyldithiocarbamate) did not induce DNA damage in the glandular stomach and liver cells of male rats after oral administration up to 2000 mg/kg bw/day (OECD 487, GLP). The data for the duodenum are inconclusive.

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Sep 2018 to 20 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Remarks:

Wistar outbred (Crl:WI(Han)) (SPF)

Details on species / strain selection:

For this study, rats were chosen as test system, because this animal species is normally used in toxicity studies of this type.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 201.9 – 266.1 gram
- Assigned to test groups randomly: yes
- Housing: The animals were housed two to five animals per cage. All animals were housed in Makrolon cages with wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) as bedding material and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly. Due to a staggered start, not all animals were dosed at once. To avoid exposure of the animals not yet dosed, the dosed animals were placed in a different cage. As a consequence, the first animals per group that were dosed, were housed individually until the second animals of the same group had been dosed (time between dosing of animals of the same group was ca. 2-3 hours). Also, the last animals per group that were necropsied, were housed individually for a short period of time (ca. 2-3 hours). Animals treated with the positive controls MMS and 2-AAF were housed in filter top cages (two to three animals per cage) after dosing.
- Diet: Feed was provided ad libitum from the arrival of the rats until the end of the study. The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England).
- Water: Drinking water was provided ad libitum from the arrival of the rats until the end of the study. Tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) was supplied by N.V. Vitens.
- Acclimation period: 12-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 – 65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 September 2018 To: 20 September 2018

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 8, 40 and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw/day
- Lot/batch no. (if required): A1701528
- Purity: 100%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For each day of the study and for each test substance group, the appropriate amount of test substance was weighed in a glass bottle. Each dosing day, the corresponding amount of corn oil was added to obtain the final concentration of the test substance in corn oil. Before dosing, the suspension was stirred for at least 30 minutes, until visual homogeneity was obtained. All suspensions were continuously stirred on a magnetic stirrer during the dosing procedure, in order to maintain the homogeneity of the test substance in the vehicle.

Duration of treatment / exposure:

Two consecutive days

Frequency of treatment:

Twice, with an interval between the first and second dose of ca. 20 hours

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per dose. Surplus animals were included in group 1 and 2 to replace the duodenum collected from animals 2 (group 1) and 12 (group 2).

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene (liver) and methylmethanesulfonate (glandular stomach & duodenum)
- Route of administration: gavage (2-acetylaminofluorene and methylmethanesulfonate)
- Doses / concentrations: dosed once with methylmethanesulfonate: 40 mg/kg bw (nominal); 2-acetylaminofluorene: 50 mg/kg bw (nominal)

Tissues and cell types examined:

glandular stomach, duodenum and liver

Details of tissue and slide preparation:

ISOLATION OF GLANDULAR STOMACH CELLS
All steps were performed on ice as much as possible. The forestomach was removed and preserved in formaldehyde and the glandular stomach was cut open and washed free from food and debris using ice-cold mincing buffer. The glandular stomach was immersed in fresh ice-cold mincing buffer again and incubated for 15-30 minutes. After incubation, the surface epithelia was gently scraped two times using a Teflon scrapper. This layer was discarded and the gastric mucosa was rinsed with the ice-cold mincing buffer. The stomach epithelia was carefully scraped 4-5 times (or more, if necessary) with a scrapper to release the cells. The cell suspension was strained with a 40 μm cell strainer to remove clumps and the remaining suspension was centrifuged (3 min, 500 rpm, ca. 4°C). The supernatant was discarded except for a small volume to re-suspend the cells, followed by preparation of comet slides. The cell density and viability of the cells was determined by trypan blue exclusion.

ISOLATION OF DUODENUM CELLS
All steps were performed on ice as much as possible. The duodenum was cut open and washed free from food and debris using ice-cold mincing buffer. Subsequently, the duodenum was minced with a pair of fine scissors to release the cells. The cell suspension was strained with a 500 μm netwell filter, followed by a 40 μm cell strainer to remove clumps. The remaining suspension was centrifuged (3 min, 500 rpm, ca. 4°C). The supernatant was discarded except for a small volume to re-suspend the cells, followed by preparation of comet slides. The cell density and viability of the cells was determined by trypan blue exclusion.

ISOLATION OF LIVER CELLS
All steps were performed on ice as much as possible. The collected portion of the left lateral liver lobe was washed in ice-cold mincing buffer until as much blood as possible had been removed. Subsequently, the portion was minced with a pair of fine scissors to release the cells. The cell suspension was strained with a 500 μm netwell filter, followed by a 40 μm cell strainer to remove clumps. The remaining suspension was centrifuged (3 min, 500 rpm, ca. 4°C). The supernatant was discarded except for a small volume to re-suspend the cells, followed by preparation of comet slides. The cell density and viability of the cells was determined by trypan blue exclusion.

PREPARATION OF COMET SLIDES
Microscopic slides were prepared by mixing an aliquot of the cell suspension with a low-melting agarose solution (0.5 % (w/v) in Phosphate Buffered Saline). Subsequently, this mixture was loaded on a glass slide, pre-coated with normal-melting agarose (1.5 % (w/v) in PBS), and mounted with a coverslip. Three slides per animal were prepared (one slide was kept in reserve). The slides were stored on a cold plate until the agarose had solidified. Subsequently, the coverslip was removed and the slide was incubated in lysis buffer (2.5 M NaCl, 0.1 M Na2EDTA, 0.175 M NaOH, 0.01 M Tris in Milli-Q water, supplemented with 10% DMSO (v/v) and 1 % Triton X-100 (v/v), pH 10) overnight at 2-10 ºC. After incubation in lysis buffer, the slides were shortly rinsed in ice-cold electrophoreses buffer (0.3 M NaOH, 0.001 M Na2EDTA in Milli-Q water, pH >13). Subsequently, slides were incubated in ice-cold electrophoresis buffer for 20 ± 1 min (unwinding), followed by electrophoresis (28 V and ca. 300 mA) for 30 ± 1 min in ice-cold electrophoresis buffer, while cooled on ice. The temperature of the electrophoresis buffer was measured at the start of unwinding, the start of electrophoresis and the end of electrophoresis. After incubation in neutralization buffer (0.4 M Tris in Milli-Q water, pH 7.5) for at least 5 min, slides were dehydrated by incubating in ethanol at room temperature and air-dried.

SLIDE ANALYSIS AND COUNTING
Slides were coded by a qualified person not involved in analyzing the slides to enable ‘blind’ scoring. Slides were stained with SYBR Gold, which was 10.000 times diluted with TE buffer (10 mM Tris and 1 mM Na2EDTA, pH ca. 7.0-7.5) and covered with a coverslip just before analysis. A fluorescent microscope connected to a camera and Comet Assay IV software was used for the analysis of the slides. Seventy-five cells (randomly selected starting from the center of the slide) per slide and two slides per animal were analyzed to yield a total number of 150 cells per animal. Ghost cells, with a small head and a diffuse and large tail, were excluded from analysis, but their presence was recorded.

Evaluation criteria:

ACCEPTANCE CRITERIA
The study was considered valid for the tissue if:
- the mean tail intensity of the negative control group was ≤ 20% (glandular stomach), ≤ 10% (duodenum) and ≤ 6% (liver).
- the group mean tail intensity of the group 5 (for MMS) or group 6 (for 2-AAF) demonstrated a statistically significant increase compared to the group mean of the negative control group (group 1)
- at least 150 cells from at least 2 slides were analyzed for all animals included in the group

EVALUATION AND INTERPRETATION OF THE RESULTS
A test substance is considered to be positive if:
- at least one dose level demonstrated a statistically significant increase compared to the negative control (group 1)
- the increase was dose-related when evaluated with a test for a linear trend

When both criteria were met, the test substance was considered to be able to induce DNA strand breakage in the tissue evaluated, under the conditions used in this study.

A test substance was considered to be negative if:
- none of the dose levels demonstrated a statistically significant increase compared to the negative control (group 1)
- there was no dose-related increase when evaluated with a test for a linear trend
- direct or indirect evidence of exposure of, or toxicity to, the target tissue was demonstrated
When all of these criteria were met, the test substance was considered not to be able to induce DNA strand breakage in the tissue evaluated, under the conditions used in this study.

There is no requirement for verification of a clearly positive or negative response. In case the response was neither clearly negative nor clearly positive (i.e. not all the criteria listed above are met) the results will be evaluated by expert judgement and/or further investigations will be conducted, if scientifically justified (in consultation with the Sponsor).

Statistics:

see "Any other information on materials and methods incl. tables"

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: applicable for glandular stomach and liver

Sex:

male

Genotoxicity:

other: inconclusive

Toxicity:

no effects

Vehicle controls validity:

not valid

Negative controls validity:

not examined

Positive controls validity:

not valid

Remarks on result:

other: applicable for duodenum

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Clinical signs and mortality
No mortality was observed and no treatment-related clinical signs were observed in the animals during the study period.

Body weights
Group mean body weights in all groups were considered within the normal range as expected for healthy rats of this age and strain. Possible effects on mean body weight following treatment with the test substance were not determined.

Comet assay
- Viability of isolated cells:
After necropsy, glandular stomach, duodenum and liver cells were isolated by scraping or mincing the cells. Subsequently, comet slides were prepared. The percentage viability of the isolated glandular stomach cells was 77%, 72%, 78% and 76% for the negative control (corn oil), low, mid and high concentration of the test substance respectively, whereas the percentage viability of the isolated liver cells was 80%, 76%, 85% and 77%, for the negative control, low, mid and high concentration of the test substance, respectively. As the percentage viability of the isolated duodenum cells in two animals was extremely low (26% and 12% for animal no. 2 and 12, respectively), the two surplus animals were included in the study to replace the duodenum of these animals. Data for duodenum from animals 2 and 12 were excluded from calculations of the group means and from further evaluation. The percentage viability of the isolated duodenum cells was 93%, 94%, 90% and 94% for the negative control, low, mid and high concentration of the test substance, respectively. Based on the observed viability, all cell suspensions were considered suitable for the comet assay.

- DNA damage:
For each animal, 75 cells per slide and two slides per animal were analysed (i.e. 150 cells per animal per tissue in total). For glandular stomach and liver, all acceptance criteria for a valid test were met. The positive control substances MMS (group 5) and 2-AAF (group 6) demonstrated a statistically significant increase in tail intensity compared to the negative control (group 1, corn oil), respectively (p-value: <0.0001 for both tissues) and the group mean tail intensity of the negative control (corn oil) was <20% and <6% for glandular stomach and liver, respectively. For duodenum, the acceptance criteria were not met. The group mean tail intensity of the negative control was >10% (28.69%). The higher tail intensity observed in the negative control and some of the treatment groups seemed to be associated with an increased number of hedgehog cells. This suggests that the higher tail intensity may be caused by cytotoxicity, which in turn may have been induced during processing of the cells. The positive control substance MMS (group 5) did not demonstrate a statistically significant increase in tail intensity compared to the negative control group (group 1, corn oil) for duodenum. This means that the data obtained for duodenum are not valid and therefore no conclusions can be drawn for this organ.

Tail intensity of the test substance was comparable to the negative control (corn oil) and did not demonstrate a statistically significant increase in tail intensity in the glandular stomach and liver at any of the concentrations tested.

In the current comet assay the liver was evaluated to detect the genotoxic potential of any systemically available fraction of the test substance and its metabolites, whereas the glandular stomach and the duodenum was evaluated to detect the genotoxic potential of the test substance at the ‘site of first contact’. Since the data for duodenum are not valid, the ‘site of first contact’ is covered only by the glandular stomach.

Details on tail intensities measure are presented in 'Any other information on results incl. tables'.

Group	Test substance and concentration	Tail intensity glandular stomach cells	Tail intensity duodenum cells	Tail intensity liver cells
1	Negative control (corn oil)	12.57 ± 5.54	28.69 ± 19.24	0.21 ± 0.25
2	80 mg/kg-bw test substance (low)	9.24 ± 1.37	24.82 ± 19.41	0.21 ± 0.28
3	400 mg/kg-bw test substance (mid)	11.34 ± 1.75	10.98 ± 3.73	0.26 ± 0.33
4	2000 mg/kg-bw test substance (high)	12.47 ± 2.73	16.96 ± 16.37	0.09 ± 0.10
5	Positive control (stomach, duodenum, MMS)	34.15 ± 1.88a	25.31 ± 1.90b	N.D.
6	Positive control (liver, 2-AAF)	N.D.	N.D.	17.46 ± 3.72c

 

a Statistically significantly increased compared to concurrent negative control, Students t-test (unpaired): p<0.0001

b Not statistically significantly increased compared to concurrent negative control, Mann Whitney’s (nonparametric) p-value: 0.6905

c Statistically significantly increased compared to concurrent negative control, Students t-test (unpaired) with transformed data: p<0.0001

N.D. Not determined

 

Conclusions:

Although the data obtained from the duodenum did not fulfill the validity criteria, data were obtained from glandular stomach covering the ‘site of first contact’ and liver as the metabolizing organ, for evaluation of the genotoxic potential of the test substance. Hence, it can be concluded that, under the conditions used in this study, the test substance did not show any indication of induction of primary DNA damage in glandular stomach and liver cells of male rats after oral administration up to 2000 mg/kg bw/day. The data for the duodenum are inconclusive. However, considering all available information in a separate expert statement (included as attachment to this RSS), it can be concluded that ZBEC is not genotoxic.

Executive summary:

In this GLP compliant in vivo comet assay performed according to OECD guideline 489, the test substance was examined for its potential to cause primary DNA damage (such as single and double strand DNA breaks, alkali labile sites and incomplete repair sites) in glandular stomach, duodenum and liver cells of rats, following oral (gavage) administration of the test substance.

Male rats (n=5) were orally administered (by gavage, dosing volume 10 mL/kg bw/day) three concentrations (80, 400 and 2000 mg/kg bw/day) of the test substance or vehicle (corn oil) on two successive days, with ca. 20 h interval between the first and second dose. The second dose was administered ca. 3 h before scheduled sacrifice. This exposure regimen meets the OECD guideline 489 requirements to sample 2-6 h and 16-26 h after administration of the test substance in the same animal. Maximum dose levels were based on an oral (gavage) dose range finding study (not part of this study) and read-across data on acute toxicity provided by the sponsor. Positive control animals for glandular stomach and duodenum were orally administered (by gavage) once with methyl methanesulfonate (MMS, 40 mg/kg bw) 2-6 h prior to sacrifice. Positive control animals for liver were orally administered (by gavage) once with 2-acetylaminofluorene (2-AAF, 50 mg/kg bw) 12-16 h prior to sacrifice, respectively.

All animals survived until scheduled sacrifice. No treatment-related clinical abnormalities were observed. There were no statistically significant effects on mean body weight following treatment.

Approximately 3 h after the last oral dose, the glandular stomach, duodenum and part of the left lateral liver lobe were collected, followed by mincing to obtain single cell suspensions and preparation of comet slides. Based on the observed viability, all cell suspensions were considered suitable for the comet assay, except for the duodenum in two animals (animal no. 2 and 12). Therefore, the two surplus animals were included in the study to replace the duodenum of animals no. 2 and 12.

Tail intensity (i.e. the percentage DNA in the ‘tail’ of the comet) was used as a measure for primary DNA damage in the comet assay. Seventy-five cells per slide and two slides per animal were analyzed (i.e. in total 150 cells per animal per tissue). The median of each slide was calculated and the mean of the two medians was calculated per animal. Finally, the group mean of the individual animal values was calculated.

For glandular stomach and liver, all acceptance criteria for a valid test were met. The positive control substances MMS (group 5) and 2-AAF (group 6) demonstrated a statistically significant increase in tail intensity compared to the negative control (group 1, corn oil), respectively (p-value: <0.0001 for both tissues) and the group mean tail intensity of the negative control (corn oil) was <20% and <6% for glandular stomach and liver, respectively. For duodenum, the acceptance criteria were not met. The group mean tail intensity of the negative control was >10% (28.69%). The higher tail intensity observed in the negative control and some of the treatment groups seemed to be associated with an increased number of hedgehog cells. This suggests that the higher tail intensity may be caused by cytotoxicity, which in turn may have been induced during processing of the cells. The positive control substance MMS (group 5) did not demonstrate a statistically significant increase in tail intensity compared to the negative control group (group 1, corn oil) for duodenum. This means that the data obtained for duodenum are not valid and therefore no conclusions can be drawn for this organ.

Tail intensity of the test substance was comparable to the negative control (corn oil) and did not demonstrate a statistically significant increase in tail intensity in the glandular stomach and liver at any of the concentrations tested.

In the current comet assay the liver was evaluated to detect the genotoxic potential of any systemically available fraction of the test substance and its metabolites, whereas the glandular stomach and the duodenum was evaluated to detect the genotoxic potential of the test substance at the ‘site of first contact’. Since the data for duodenum are not valid, the ‘site of first contact’ is covered only by the glandular stomach.

It can be concluded that, under the conditions used in this study, the test substance did not show any indication of induction of primary DNA damage in glandular stomach and liver cells of male rats after oral administration up to 2000 mg/kg bw/day. The data for the duodenum are inconclusive.

In the attached expert statement all available information is considered and it is concluded that it is highly unlikely that ZBEC will be mutagenic, either via systemic toxicity or via site of contact. From the available information it can be concluded that due to the size of the substance, the potential to cross biological membranes is limited, limiting the potential to damage DNA. In addition, no mutagenicity was observed in the other two organs examined in the in vivo Comet assay, neither via site of contact or via systemic toxicity. The absence of (eco)toxicity in available (eco)toxicity studies supports this, as well as information from the structurally similar compound ZDBC. Therefore, the available information is sufficient to conclude that ZBEC is not genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genotoxicity of zinc bis(dibenzyldithiocarbamate) (ZBEC) was assessed in a GLP-compliant guideline Ames test and in vitro mouse lymphoma assay. In the Ames test, ZBEC did not induce increased mutation frequency in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activations, up to limit concentrations of 5000 μg/mL, using DMSO as a vehicle. In the in vitro mouse lymphoma assay, it induced increased mutation frequencies at cytotoxic concentrations of 3.4 and 7.0 μg/mL, without and with S9 and using 24 and 4 hr exposure duration, respectively. From the results obtained in this in vitro micronucleus test, it is concluded that the test substance was not clastogenic and/or aneugenic to cultured human lymphocytes, under the conditions used in this study.

An in vivo comet assay was performed to conclude on the effects observed in the in vitro gene mutation assay. Tail intensity of the test substance was comparable to the negative control (corn oil) and did not demonstrate a statistically significant increase in tail intensity in the glandular stomach and liver at any of the concentrations tested.

In the current comet assay the liver was evaluated to detect the genotoxic potential of any systemically available fraction of the test substance and its metabolites, whereas the glandular stomach and the duodenum was evaluated to detect the genotoxic potential of the test substance at the ‘site of first contact’. Since the data for duodenum are not valid, the ‘site of first contact’ is covered only by the glandular stomach.

It can be concluded that, under the conditions used in this study, the test substance did not show any indication of induction of primary DNA damage in glandular stomach and liver cells of male rats after oral administration up to 2000 mg/kg bw/day. The data for the duodenum are inconclusive.

Justification for classification or non-classification

Taking into account the overall evidence from available genotoxicity studies, classification of Zinc bis(dibenzyldithiocarbamate) (ZBEC) as genotoxic is not warranted in accordance with EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.