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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Substance-specific information for this endpoint will be submitted by 21 June 2021, as requested by ECHA (Decision number: CCH-D-2114382075-49-01/F).

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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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
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

Genetic toxicity in vivo

Description of key information

This information will be submitted later based on ECHA decision number CCH-D-2114382075-49-01.

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. No further data on genotoxicity of ZBEC are available.

 

Short description of key information:

Zinc bis(dibenzyldithiocarbamate) (ZBEC) gave negative results in a GLP-compliant guideline Ames test with 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, performed according to OECD Guideline 476, it increased mutation frequencies at concentration levels of 3.4 and 7.0 μg/ml (without and with S9 and using 24 and 4 hr exposure duration, respectively). No further information on genotoxicity of ZBEC are available.

 

Endpoint Conclusion: No adverse effect observed (negative), this information will be updated based on ECHA decision number CCH-D-2114382075-49-01.

 

Justification for classification or non-classification

Based on the read-across with a structural analogue of zinc bis(dibenzyldithiocarbamate) (ZBEC), zinc bis(dimethyldithiocarbamate) (ZDMC), it is concluded that classification of ZBEC for genotoxicity 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.

Substance-specific information for this endpoint will be submitted by 21 June 2021, as requested by ECHA (Decision number: CCH-D-2114382075-49-01/F).