Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test was performed in 2005 according to the OECD 471 guideline ; negative results were showed in this study.
Negative results were observed in the two others in vitro tests : in vitro mammalian cells chromosomal aberrations test (OECD 473) and in vitro mammalian cells gene mutations test (OECD 476, MLA/TK).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16-Sep-2011/ 19-Jan-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully GLP compliant (with GLP certificate)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Whole blood was collected from healthy volunteer donors. The volunteers were non-smokers and were not receiving any medication or radiation exposure prior to the time of sampling.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
male rat S9 liver induced by phenobarbital
Test concentrations with justification for top dose:
First experiment: Dose levels of 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/ml were used both in the absence and presence of S9 metabolism.
As negative results were obtained in the first main experiment, a second experiment was performed in the absence of S9 metabolism with a treatment time of 24 hours and using the same harvest time (24 hours). A continuous treatment until harvest was used.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle:Solubility of the test item
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin-C (without S9) and cyclophosphamide (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:3 hours +/-S9; 24 hours -S9
- Fixation time (start of exposure up to fixation or harvest of cells): The harvest time of 24 hours, corresponding to approximately 1.5 cell cycle, was used.

SPINDLE INHIBITOR (cytogenetic assays):Colcemid
STAIN (for cytogenetic assays):Giemsa
Three air-dried slides were prepared from each culture and stained in 3% Giemsa in tap water.

NUMBER OF REPLICATIONS: Two independent experiments for chromosomal damage were performed.
Two cultures were prepared at each test point.

NUMBER OF CELLS EVALUATED:100/culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy
- Determination of endoreplication
Evaluation criteria:
In this assay, the test item is considered to have clastogenic properties if the following criteria are all fulfilled:
(i) Statistically significant increases in the incidence of cells bearing aberrations are observed at any dose level over the concurrent control.
(ii) The increases are reproduced in both replicate cultures.
(iii) The increases must exceed historical controls. Any significant increase over the concurrent negative controls is therefore compared with the historical control.
The evaluation is based on the set of results which excludes gaps.
Statistics:
For the statistical analysis, Fisher's Exact Test is used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis is performed using sets of data either including or excluding gaps.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility:
A preliminary solubility trial was performed starting from the concentration of 472 mg/ml in DMSO. This concentration, when added to culture medium in the ratio of 1:100, gave a maximum dose level of 4720 µg/ml corresponding to 10.0 mM. Since this concentration is lower than 5000 µg/ml it was chosen as upper limit to be tested as indicated in the Study Protocol.
The test item was found to be soluble at the concentration of 15.0 mg/ml after approximately 10 minutes of vortex mixing.
An aliquot of this solution, added to supplemented RPMI 1640 medium (Dutch modification), in the ratio of 1:100, gave turbidity of the medium. Opacity of the medium was obtained by adding an aliquot at 7.50 mg/ml. A clear solution was obtained by adding an aliquot at 3.75 mg/ml. Based on solubility results, the concentration of 150 µg/ml was selected as the highest dose level to be used in the cytogenetic assay.
Additional solubility trials were performed by using sterile distilled water of injectable grade and ethanol where the test item was found not soluble.

- Osmolarity and pH: Following treatment with the test item, no remarkable variation of osmolality and pH was observed in the absence or presence of S9 metabolism, at any dose level, in any treatment series in any experiment.



Following treatment with the test item, no remarkable increase in the incidence of cells bearing aberrations, including or excluding gaps over the control value, was observed in any experiment.

For the first experiment, following the short treatment in the absence of S9 metabolism, one endoreduplicated cell was observed at the intermediate dose level. One endoreduplicated cell was also observed in the second experiment following the continuous treatment in the absence of S9 metabolism for one replicate culture from the low dose level selected for scoring and for one replicate culture from the solvent control. Since the incidence of cells bearing numerical changes was comparable to historical values observed in our laboratory, these observations were not considered biologically relevant.

Marked increases in the frequency of cells bearing aberrations (including and excluding gaps) were seen in the cultures treated with the positive control substances, Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps over the control values, was observed in both experiments in the absence and presence of S9 metabolism.

On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce chromosomal aberrations in human lymphocytes cultured in vitro.

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that Copper dibutyl dithiocarbamate does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions, in the absence or presence of S9.
Executive summary:

The test item Copper dibutyl dithiocarbamate was assayed for the ability to cause chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation.

Two independent experiments for chromosomal damage were performed, as limited by solubility.

For the first main experiment, the dose levels selected for treatment, as limited by solubility, were: 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/ml, both in the absence and presence of S9 metabolism.

In the first main experiment, the cells were treated for 3 hours both in the presence and absence of S9 metabolism. A harvest time of 24 hours, corresponding to approximately 1.5 cell cycles, was used.

As negative results were obtained in the first main experiment, a second experiment was performed in the absence of S9 metabolism with a treatment time of 24 hours and using the same harvest time (24 hours). A continuous treatment until harvest was used.

Since no cytotoxicity was observed in the first main experiment, dose levels of 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586µg/ml were used.

Solutions of the test item were prepared in dimethylsulfoxide (DMSO).

Each experiment included appropriate negative and positive controls.Two cell cultures were prepared at each test point.

For both experiments, dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments (as determined by the reduction in mitotic index). Since the test item did not induce toxicity at any dose level, the highest treatment level was selected as the highest dose (150 µg/ml) for scoring. Two lower dose levels (37.5 and 75.0 µg/ml) were also selected for scoring.

One hundred metaphase spreads were scored for chromosomal aberrations from each culture.

Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level or any treatment time in the absence or presence of S9 metabolic activation.

Statistically significant increases in the incidence of cells bearing aberrations (both including and excluding gaps) were seen following positive control treatments with Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

It is concluded that Copper dibutyl dithiocarbamate does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 21, 2004 to November 10, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study (OECD 471)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function.
Test concentrations with justification for top dose:
Main test: 62.5, 125, 250, 500 and 1000 µg/plate
Vehicle / solvent:
Acetone.
The test item was dissolved in the vehicle at concentrations of 20 mg/mL for both mutagenicity experiments (main study).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

The direct plate incorporation method was performed as follows: test item solution (0.05 mL),S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.

The preincubation method was performed as follows: test item solution (0.05 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter.

NUMBER OF REPLICATIONS: two independent experiments, using three plates/dose-level
Evaluation criteria:
Acceptance criteria
This study is considered valid if the following criteria are fully met: the number of revertants in the vehicle controls is consistent with the historical data of the testing facility, the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
no
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
A moderate to important precipitate was observed in the Petri plates when scoring the revertants generally at dose-levels = 500 µg/plate or = 250 µg/plate, without and with S9 mix, respectively.
No toxicity was noted towards all the strains used, both with and without S9 mix.
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Tables of results: Ames test with CDBC

 

1/ First experiment / Direct plate incorporation method (with and without S9 mix)

 

strain

compound

Dose level (µg/ plate)

S9-mix

Mean revertant colony counts

SD

Ration treated/

solvent

Individual revertant colony counts

 

 

TA 1535

Acetone

 

-

13

3

 

11

16

11

Test item

62.5

-

14

6

1.1

16

19

8

125

-

12

7

0.9

19

5

11

250

-

11

1

0.9

12

12

10

500

-

11

3

0.8

11Mp

10Mp

8Mp

1000

-

16

4

1.3

12Sp

18Sp

19Sp

NAN3

1

-

590

39

46.6

623

599

547

Acetone

 

+

16

3

 

12

18

18

Test item

62.5

+

13

3

0.8

16

11

13

125

+

15

2

0.9

17

11

14

250

+

11

8

0.7

5Mp

8Mp

20Mp

500

+

14

6

0.9

19Mp

8Mp

14Mp

1000

+

13

8

0.8

17Sp

17Sp

4Sp

2AM

2

+

330

63

20.6

332

267

392

 

 

TA 1537

Acetone

 

-

9

3

 

8

12

6

Test item

62.5

-

5

2

0.6

4

4

8

125

-

7

4

0.8

11

7

4

250

-

5

3

0.5

2

5

7

500

-

8

3

1.0

8Mp

6Mp

11Mp

1000

-

6

0

0.7

6Sp

6Sp

6Sp

9AA

50

-

709

149

81.8

801

517

789

Acetone

 

+

9

4

 

7

11

7

Test item

62.5

+

7

1

0.8

8

7

7

125

+

12

2

1.3

11

11

14

250

+

8

4

0.8

6

5

12

500

+

10

7

1.0

11Mp

2Mp

16Mp

1000

+

9

3

0.9

12Sp

8Sp

6Sp

2AM

2

+

77

7

8.2

81

69

80

 

 

TA 98

Acetone

 

-

27

6

 

32

20

28

Test item

62.5

-

21

4

0.8

22

17

25

125

-

27

6

1.0

30

30

30

250

-

25

6

0.9

28

18

29

500

-

26

1

1.0

26Mp

26Mp

25Mp

1000

-

30

2

1.1

31Sp

30Sp

28Sp

2NF

0.5

-

294

13

11.0

283

308

292

Acetone

 

+

30

7

 

22

35

32

Test item

62.5

+

37

9

1.2

46

28

36

125

+

41

8

1.4

40

49

34

250

+

21

4

0.7

17

22

25

500

+

36

11

1.2

48Mp

28Mp

32Mp

1000

+

30

5

1.0

35Sp

25Sp

31Sp

2AM

2

+

2018

1038

68.0

3217

1401

1437

 

 

TA 100

Acetone

 

-

127

27

 

141

96

144

Test item

62.5

-

137

9

1.1

144

139

127

125

-

113

34

0.9

151

134

84

250

-

123

7

1.0

125

115

129

500

-

163

15

1.3

156Mp

153Mp

181Mp

1000

-

158

55

1.2

108Sp

149Sp

217Sp

NAN3

1

-

709

25

5.6

683

732

711

Acetone

 

+

158

23

 

132

166

175

Test item

62.5

+

147

5

0.9

152

143

147

125

+

156

38

1.0

125

198

146

250

+

167

18

1.1

186

150

166

500

+

163

17

1.0

182Mp

152Mp

155Mp

1000

+

163

19

1.0

161Sp

181Sp

144Sp

2AM

2

+

1353

61

8.6

1352

1414

1292

 

 

TA 102

Acetone

 

-

407

32

 

380

308

443

Test item

62.5

-

311

27

0.8

357

356

310

125

-

370

56

0.9

429

365

317

250

-

314

63

0.8

374

320

249

500

-

391

19

1.0

375Mp

393Mp

413Mp

1000

-

427

22

1.0

432Sp

446Sp

402Sp

MMC

0.5

-

1886

52

4.6

1835

1863

2939

Acetone

 

+

398

84

 

319

486

389

Test item

62.5

+

346

32

0.9

366

363

309

125

+

403

44

1.0

384

453

372

250

+

440

31

1.1

462

404

453

500

+

398

18

1.0

406Mp

410Mp

377Mp

1000

+

430

52

1.1

432Sp

487Sp

386Sp

2AM

10

+

1381

266

3.5

1077

1491

1571

SD: standard deviation

- : absence S9 ; +: presence S9

Mp : moderate precipitate; Sp: strong precipitate

 

Conclusions:
Under these experimental conditions, the test item Copper dibutyl dithiocarbamate did not show any mutagenic activity in the bacteiral reverse mutation test with S.typhimurium.
Executive summary:

The objective of this study was to evaluate the potential of the tes item Copper dibutyl dithiocarbamate to induce reverse mutation in Salmonella tiphymurium. The study was performed according to the international guidelines (OECD 471) and in compliance with principles of GLP regulations.

The test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mic, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method. Five strains of bacteria S.typhimurium: TA1535, TA1537, TA98, TA100 and TA102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. the evalutation of the toxicity was performed on the basis of the observation of the decease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item Copper dibutyl dithiocarbamate was dissolved in acetone. Positive controls were used.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. The selected treatment-levels were: 62.5, 125, 250, 500 and 1000 µg/plate, for both mutagenicity experiments with and without S9 mix. A moderate to important precipitate was observed in the Petri plates when scoring the revertants generally at dose-levels > 500 µg/plate or > 250 µg/plate, without and with S9 mix, respectively. No toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Under these experimental conditions, the test item Copper dibutyl dithiocarbamate did not show any mutagenic activity in the bacterial reverse mutation test with S.typhimurium.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 September 2011 to 26 March 2012
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH, Topic S2A, Genotoxicity: Specific Aspects of Regulatory Tests, Step 4 (Document, July 1995)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH, Topic S2B, A Standard Battery for Genotoxicity Testing of Pharmaceuticals, Step 4 (Document, July 1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
RPMI 1640 (1X)
L-glutamine (200 mM)
Sodium pyruvate (100 mM)
Non-essential amino acids (100X)
Streptomycin sulphate 50.000 IU/ml + Penicillin G 50.000 units/ml
F 68 Pluronic

Minimal medium B

RPMI 1640 (1X)
L-glutamine (200 mM)
Sodium pyruvate (100 mM)
Non-essential amino acids (100X)
Streptomycin sulphate 50.000 units/ml + Penicillin G 50.000 units/ml

Complete medium (5%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium (10%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium A (20%)

Minimal medium A
Horse serum (heat-inactivated)

Complete medium B (20%)

Minimal medium B
Horse serum (heat-inactivated)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix:
150, 75.0, 37.5, 18.8, 9.38, 4.69 and 2.34 ug/mL for the first experiment (3-hour treatment)
2.00, 1.67, 1.39, 1.16, 0.965, 0.804 and 0.558 ug/mL for the second experiment (24-hour treatment)

Experiments with S9 mix:
150, 75.0, 37.5, 18.8, 9.38, 4.69 and 2.34 ug/mL for the first experiment (3-hour treatment)
9.00, 6.00, 4.00, 2.67, 1.78 and 1.19 ug/mL for the second experiment (3-hour treatment)
Vehicle / solvent:
Vehicle used: dimethylsulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: fluctuation method in medium

DURATION
- Exposure duration: 3 and 24 hours in the absence of S9 metabolism; 3 hours in the presence of S9 metabolis
- Expression time (cells in growth medium): Two days after treatment (48 hours)
- Selection time (if incubation with a selection agent): 13/14 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF CELLS EVALUATED: 1.6 cells/well plated in each of 96-well plates (two)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

DETERMINATION OF MUTATION
- Method: Induced mutant frequency (IMF)
Evaluation criteria:
For a test item to be considered mutagenic in this assay, it is required that:

(i) The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell
method (126 x 10-6) at one or more doses.

(ii) There is a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive
responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological
significance. Any increase in mutant frequency should lie outside the historical control range to have biological
relevance.

At low survival levels the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder
effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and
not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason it is
generally recommended that such data are treated with caution or excluded from consideration.
Statistics:
Dunnett test. Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
In the first main assay, precipitation of the test item and/or opacity of the final treatment medium were noted at the end of the treatment incubation period at the highest concentration tested (150 ug/mL) in the presence of S9 metabolic activation. In the absence of S9 metabolism, only opacity of the treated medium was noted at 150 ug/mL.
In the second main assay, using the short (with S9) and long (without S9) treatment time, no precipitation of the test item was seen at the beginning or at the end of treatment at any concentration tested.

RANGE-FINDING/SCREENING STUDIES (Cytotoxicity test):
On the basisi of solubility results, a citotoxicity assay was performed in the absence and presence of S9 metabolic activation, at a maximum dose level of 150 µg/mL and at a wide range of lower dose levels: 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/mL.

In the absence of S9 metabolic activation, using the 3 hour treatment time, mild toxicity was observed at the five higher dose levels reducing relative survival (RS) in the range of 39-58% of the concurrent negative control. No relevant toxicity was observed over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived to treatment or insufficient number of cells recovered after treatment at the six higher dose levels. Slight toxicity (RS=78%) was observed at a concentration of 2.34 µg/mL, while no relevant toxicity was observed over the remaining two dose levels.
Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), moderate toxicity was seen at the four higher dose levels reducing survival in the range of 17-25% of the concurrent negative control value. Slight toxicity was noted at the next lower concentrations of 9.38 and 4.69 ug/mL reducing RS to 56% and 50% of the concurrent negative control value, respectively. No relevant toxicity was observed over the remaining dose levels.

COMPARISON WITH HISTORICAL CONTROL DATA:
Solvent and positive control treatments were included in the mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

ADDITIONAL INFORMATION ON MUTAGENICITY:
Based on the cytotoxicity results obtained in the preliminary assay, two independent assays for mutation to trifluorothymidine resistance were performed using the dose levels described in the following table:

First experiment:
150, 75.0, 37.5, 18.8, 9.38, 4.69 and 2.34 ug/mL (3-hour treatment time in the absence and presence of S9 mix)

Second experiment:
2.00, 1.67, 1.39, 1.16, 0.965, 0.804 and 0.558 ug/mL (24-hour treatment time in the absence of S9 mix)
9.00, 6.00, 4.00, 2.67, 1.78 and 1.19 ug/mL (3-hour treatment time in the presence of S9 mix)

In the absence or presence of S9 metabolic activation, no increases in mutant frequency were observed at any treatment time.
Conclusions:
It is concluded that Copper dibutyl dithiocarbamate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Executive summary:

 The test item, Copper dibutyl dithiocarbamate,was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

 The study was designed to comply with the experimental methods indicated in  OECD Guideline for the testing of chemicals No. 476 (adopted July 1997) and in compliance with the Principles of Good Laboratory Practice.

 

The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.

In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours) with and without S9. Since negative results were obtained in the first experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a longer treatment time (24 hours). With S9, a second experiment was performed for a short treatment time (3h).

 

Solvent and positive control cultures were included in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range (50-200 x 10-6viable cells). The positive control items induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). The cloning efficiencies at Day 2 in the negative control cultures and the control growth factor over 2 days fell within the range. The study was accepted as valid.

 

In the first experiment, in the absence of S9 metabolic activation, severe toxicity was observed at the three higher dose levels tested (150, 75.0 and 37.5mg/mL) reducing relative total growth (%RTG) in a range of 6-8% of the concurrent negative control value. At the next two lower concentrations (18.8 and 9.38mg/mL), moderate toxicity was seen reducing RTG to 14% and 27%, respectively. At 4.69mg/mL, mild toxicity was observed (RTG 38%) while no relevant toxicity was noted at the lowest concentration tested (2.34mg/mL).

In the presence of S9 metabolic activation, concentrations of 75.0, 37.5, 18.8mg/mL yielded less than 10% relative survival, while at the next lower dose level (9.38mg/mL) and also at 150µg/mL in which precipitation was noted, marked toxicity was observed reducing survival to 13% and 15%, respectively, of the concurrent negative control value. Mild toxicity was seen at the remaining two dose levels tested (4.69 and 2.34mg/mL) which yielded 32% and 50% RTG, respectively.

In the second experiment, in the absence of S9 metabolic activation using a long treatment time, a steep decline in survival was observed between the highest and the next lower dose level; in fact no cells survived to treatment at 2.00mg/mL, while at 1.67mg/mL, slight toxicity was observed reducing the relative total growth to 66% of the concurrent negative control value. No toxicity was observed over the remaining dose levels tested.

In the presence of S9 metabolism, severe toxicity was observed at the two higher dose levels tested (9.00 and 6.00mg/mL). Dose-related toxicity was seen over the remaining concentrations.

 

At low survival levels the mutation data are prone to a variety of artefacts (selection effects, sampling errors, founder effects). Mechanisms other than direct genotoxicityper sec an lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason it is generally recommended that such data are treated with caution or excluded from consideration (Coleet al., 1990; Cole and Arlett, 1984; ICH S2A). Accordingly, we have excluded from the statistical analyses mutation data obtained in the absence of S9 metabolism at 150, 75.0, 37.5 µg/mL (Main assay I) and data obtained in the presence of S9 metabolism at 150, 75.0, 37.5, 18.8 (Main assay I), 9.00 and 6.00 µg/mL (assay II).

In the absence or presence of S9 metabolic activation, no increases in mutant frequency were observed at any treatment time.

In the first main assay, using the short treatment time in the absence of S9 metabolism and in the second experiment in the presence of S9 metabolism, a linear trend was indicated (p<5%). However, no statistically significant increase in mutant frequency was observed at any dose level. The IMF values were lower than the global evaluation factor and the mutant frequencies observed at all dose levels were within the historical control range at RTC. Hence, the observed linear trend was considered to be attributable to a chance event not related to the action of the test item and of no biological significance

 

 It is concluded that Copper dibutyl dithiocarbamate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test - OECD 471 (Haddouk 2005)

The objective of this study was to evaluate the potential of the tes item Copper dibutyl dithiocarbamate to induce reverse mutation in Salmonella typhimurium. The study was performed according to the international guidelines (OECD 471) and in compliance with principles of GLP regulations.

The test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mic, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method. Five strains of bacteria S.typhimurium: TA1535, TA1537, TA98, TA100 and TA102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. the evalutation of the toxicity was performed on the basis of the observation of the decease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item Copper dibutyl dithiocarbamate was dissolved in acetone. Positive controls were used.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. The selected treatment-levels were: 62.5, 125, 250, 500 and 1000 µg/plate, for both mutagenicity experiments with and without S9 mix. A moderate to important precipitate was observed in the Petri plates when scoring the revertants generally at dose-levels > 500 µg/plate or > 250 µg/plate, without and with S9 mix, respectively. No toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Under these experimental conditions, the test item Copper dibutyl dithiocarbamate did not show any mutagenic activity in the bacteiral reverse mutation test with S.typhimurium.

In vitro mammalian cells gene mutation test - OECD 476 (Salvador 2012) :

The test item, Copper dibutyl dithiocarbamate,was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. 

The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.

In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours) with and without S9. Since negative results were obtained in the first experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a longer treatment time (24 hours). With S9, a second experiment was performed for a short treatment time (3h). Solvent and positive control cultures were included in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range (50-200 x 10^-6 viable cells). The positive control items induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). The cloning efficiencies at Day 2 in the negative control cultures and the control growth factor over 2 days fell within the range. The study was accepted as valid.

In the first experiment, in the absence of S9 metabolic activation, severe toxicity was observed at the three higher dose levels tested (150, 75.0 and 37.5mg/mL) reducing relative total growth (%RTG) in a range of 6-8% of the concurrent negative control value. At the next two lower concentrations (18.8 and 9.38mg/mL), moderate toxicity was seen reducing RTG to 14% and 27%, respectively. At 4.69mg/mL, mild toxicity was observed (RTG 38%) while no relevant toxicity was noted at the lowest concentration tested (2.34mg/mL).

In the presence of S9 metabolic activation, concentrations of 75.0, 37.5, 18.8mg/mL yielded less than 10% relative survival, while at the next lower dose level (9.38mg/mL) and also at 150µg/mL in which precipitation was noted, marked toxicity was observed reducing survival to 13% and 15%, respectively, of the concurrent negative control value. Mild toxicity was seen at the remaining two dose levels tested (4.69 and 2.34mg/mL) which yielded 32% and 50% RTG, respectively.

In the second experiment, in the absence of S9 metabolic activation using a long treatment time, a steep decline in survival was observed between the highest and the next lower dose level; in fact no cells survived to treatment at 2.00mg/mL, while at 1.67mg/mL, slight toxicity was observed reducing the relative total growth to 66% of the concurrent negative control value. No toxicity was observed over the remaining dose levels tested.

In the presence of S9 metabolism, severe toxicity was observed at the two higher dose levels tested (9.00 and 6.00mg/mL). Dose-related toxicity was seen over the remaining concentrations.

In the absence or presence of S9 metabolic activation, no increases in mutant frequency were observed at any treatment time.

In the first main assay, using the short treatment time in the absence of S9 metabolism and in the second experiment in the presence of S9 metabolism, a linear trend was indicated (p<5%). However, no statistically significant increase in mutant frequency was observed at any dose level. The IMF values were lower than the global evaluation factor and the mutant frequencies observed at all dose levels were within the historical control range at RTC. Hence, the observed linear trend was considered to be attributable to a chance event not related to the action of the test item and of no biological significance.

It is concluded that Copper dibutyl dithiocarbamate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

In vitro mammalian cells chromosomal aberrations test - OECD 473 (Ciliutti 2012) :

The test item Copper dibutyl dithiocarbamate was assayed for the ability to cause chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation.

Two independent experiments for chromosomal damage were performed, as limited by solubility.

For the first main experiment, the dose levels selected for treatment, as limited by solubility, were: 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586µg/ml, both in the absence and presence of S9 metabolism.

In the first main experiment, the cells were treated for 3 hours both in the presence and absence of S9 metabolism. A harvest time of 24 hours, corresponding to approximately 1.5 cell cycles, was used.

As negative results were obtained in the first main experiment, a second experiment was performed in the absence of S9 metabolism with a treatment time of 24 hours and using the same harvest time (24 hours). A continuous treatment until harvest was used.

Since no cytotoxicity was observed in the first main experiment, dose levels of 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34, 1.17 and 0.586 µg/ml were used.

Solutions of the test item were prepared in dimethylsulfoxide (DMSO).

Each experiment included appropriate negative and positive controls.Two cell cultures were prepared at each test point.

For both experiments, dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments (as determined by the reduction in mitotic index). Since the test item did not induce toxicity at any dose level, the highest treatment level was selected as the highest dose (150 µg/ml) for scoring. Two lower dose levels (37.5 and 75.0 µg/ml) were also selected for scoring.

One hundred metaphase spreads were scored for chromosomal aberrations from each culture.

Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level or any treatment time in the absence or presence of S9 metabolic activation.

Statistically significant increases in the incidence of cells bearing aberrations (both including and excluding gaps) were seen following positive control treatments with Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

It is concluded that Copper dibutyl dithiocarbamate does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Justification for classification or non-classification

Proposed self-classification according to the Regulation EC No.1272/2008 : not classified