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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

Referenceopen allclose all

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

 

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

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.
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.

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.