Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test according to OECD guideline 471 was performed to investigate the potential of Vulkacit P Extra N to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in three independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

Vulkacit P Extra N did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Vulkacit P Extra N is considered to be negative (non-mutagenic) in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

In a not assignable Ames test with zinc ethylphenyldithiocarbamate conducted on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation strain TA 1535 was positive without metabolic activation and strain TA 100 was positive with metabolic activation.

In a further not assignable Ames test rubber additives were tested for mutagenicity on strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. For zinc ethylphenyldithiocarbamate the lowest effective dose for a positive result was 100 µg per plate. Due to methodological deficiencies and insufficient reporting of the published studies, these tests were disregarded and not used for the assessment of the potential mutagenic potential of zinc ethylphenyldithiocarbamate in bacteria.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 22 November 2017. Experimental completion date: 10 January 2018.
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
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Identification: Vulkacit P extra N
Physical State / Appearance: White powder
Expiry Date: 04 December 2017
Storage Conditions: At room temperature
Stability in Solvent: No analytical determination of stability in solvent conducted, but all formulations were prepared freshly before treatment and used within two hours of preparation.
The dose selection was adjusted to purity.
Target gene:
Histidine locus in S. typhimurium and tryptophan locus in E.coli.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
The maximum concentration was 5000 μg/plate (the maximum recommended dose level).

Experiment II:
All strains without S9 mix: 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate
All strains with S9 mix: 3, 10; 33; 100; 333; 1000; and 2500 µg/plate
Since toxic effects were observed in experiment I seven concentrations were tested in experiment II. 5000 µg/plate were chosen as maximal concentration for all strains without S9 mix, respectively 2500 µg/plate for all strains with S9 mix.

Experiment IIa:
Strains TA 1535, TA 1537,
TA 98, and WP2 uvrA with S9 mix: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Since no toxic effects were observed in experiment II in strains TA 1535, TA 1537, TA 98 and WP2 uvrA with S9 mix, this part of experiment II had to be repeated
Vehicle / solvent:
DMSO. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate in strains TA 1535 and TA 100
Positive control substance:
sodium azide
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate in strain TA 98, 50 µg/plate in strain TA 1537
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2.0 µL/plate in strain WP2 uvrA
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2.5 µg/plate for TA 1535, TA 1537, TA 98, TA 100, 10.0 µg/plate in WP2 uvrA.
Positive control substance:
other: 2-aminoanthracene,
Remarks:
With metabolic activation
Details on test system and experimental conditions:
Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria were met:
Evaluable plates (>0 colonies) at five concentrations or more were used in all strains.

Experimental Performance
For each strain and dose level, including the controls, three plates were used.
Experiment I (Plate Incorporation)

The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar

Experiment II and IIa (Pre-Incubation)
In the pre-incubation assay 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 µL of the stock solution, 500 µl S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item Vulkacit P Extra N was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
All strains without S9 mix: 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate
All strains with S9 mix: 3, 10; 33; 100; 333; 1000; and 2500 µg/plate
Experiment IIa:
Strains TA 1535, TA 1537,
TA 98, and WP2 uvrA with S9 mix: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in all experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate, respectively the highest investigated dose in all experiments. The undissolved particles had no influence on the data recording.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Vulkacit P Extra N at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

Results

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Strain

Experiment I

Experiment II

Experiment IIa

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

with S9 mix

TA 1535

/

/

/

/

/

TA 1537

/

/

/

333 – 2500

1000 – 5000

TA 98

/

/

/

1000 – 2500

1000 – 5000

TA 100

/

/

/

333 – 2500

n.p.

WP2 uvrA

/

/

/

/

1000 – 5000

/ = normal background growth; n.p. = not performed

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain

Experiment I

Experiment II

Experiment IIa

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

with S9 mix

TA 1535

/

5000

/

/

2500 – 5000

TA 1537

5000

5000

5000

/

2500 – 5000

TA 98

5000

2500 – 5000

2500 – 5000

/

1000 – 5000

TA 100

5000

1000 – 5000

5000

333 – 2500

n.p.

WP2 uvrA

5000

2500 – 5000

5000

/

5000

/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

n.p.: not performed

Summary of Experiment 1

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO, dried

 

 

9 ± 2

10 ± 3

25 ± 6

185 ± 15

29 ± 3

Untreated

 

 

13 ± 4

9 ± 4

33 ± 4

187 ± 12

31 ± 9

Vulkacit P

3 µg

 

8 ± 3

9 ± 4

22 ± 6

201 ± 4

29 ± 6

Extra N

10 µg

 

9 ± 1

11 ± 4

26 ± 6

213 ± 10

27 ± 3

 

33 µg

 

10 ± 3

10 ± 6

27 ± 6

202 ± 16

40 ± 5

 

100 µg

 

12 ± 3

11 ± 3

31 ± 5

200 ± 17

31 ± 10

 

333 µg

 

11 ± 0

10 ± 5

32 ± 6

167 ± 14

27 ± 9

 

1000 µg

 

13 ± 2P

12 ± 2P

25 ± 3P

153 ± 16P

27 ± 8P

 

2500 µg

 

7 ± 2P M

6 ± 1P M

13 ± 2P M

108 ± 15P

16 ± 1P M

 

5000 µg

 

5 ± 1P M

3 ± 1P M

3 ± 1P M

40 ± 3P M

6 ± 2P M

NaN3

10 µg

 

1305 ± 27

 

 

2165 ± 89

 

4-NOPD

10 µg

 

 

 

329 ± 33

 

 

4-NOPD

50 µg

 

 

122 ± 5

 

 

 

MMS

2.0 µL

 

 

 

 

 

1005 ± 42

 

 

 

 

 

 

 

 

 

With Activation

DMSO, dried

 

 

14 ± 2

12 ± 4

35 ± 12

139 ± 15

45 ± 7

Untreated

 

 

10 ± 2

19 ± 2

37 ± 7

195 ± 12

45 ± 4

Vulkacit P

3 µg

 

10 ± 3

17 ± 3

38 ± 5

131 ± 7

39 ± 9

Extra N

10 µg

 

12 ± 3

13 ± 2

35 ± 9

120 ± 16

45 ± 7

 

33 µg

 

11 ± 5

19 ± 2

38 ± 2

134 ± 12

50 ± 9

 

100 µg

 

11 ± 4

12 ± 1

33 ± 4

129 ± 13

46 ± 12

 

333 µg

 

13 ± 2

21 ± 2

30 ± 2

96 ± 29

39 ± 8

 

1000 µg

 

12 ± 1P

18 ± 2P

26 ± 1P

60 ± 3P

38 ± 11P

 

2500 µg

 

9 ± 3P

8 ± 2P M

13 ± 4P M

46 ± 4P M

18 ± 1P M

 

5000 µg

 

5 ± 2P M

3 ± 1P M

6 ± 2P M

29 ± 2P M

10 ± 1P M

2-AA

2.5 µg

 

469 ± 13

134 ± 21

4692 ± 474

3713 ± 289

 

2-AA

10.0 µg

 

 

 

 

 

425 ± 13

 

 

 

 

 

 

 

 

 

Summary of Experiment II

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

Without Activation

DMSO, dried

 

10 ± 1

11 ± 1

26 ± 0

161 ± 10

33 ± 4

Untreated

 

9 ± 2

15 ± 5

25 ± 3

198 ± 38

33 ± 10

Vulkacit P

10 µg

8 ± 3

9 ± 2

29 ± 7

176 ± 21

33 ± 8

Extra N

33 µg

9 ± 3

10 ± 3

21 ± 4

146 ± 24

33 ± 6

 

100 µg

11 ± 2

10 ± 3

17 ± 4

187 ± 19

40 ± 6

 

333 µg

9 ± 0

9 ± 2

26 ± 2

140 ± 28

31 ± 9

 

1000 µg

10 ± 3P

10 ± 4P

28 ± 5P

150 ± 12P

30 ± 3P

 

2500 µg

6 ± 1P M

6 ± 1P M

11 ± 2P M

89 ± 10P M

16 ± 2P M

 

5000 µg

6 ± 1P M

2 ± 2P M

2 ± 2P M

30 ± 5P M

6 ± 1P M

NaN3

10 µg

1149 ± 128

 

 

1762 ± 259

 

4-NOPD

10 µg

 

 

354 ± 16

 

 

4-NOPD

50 µg

 

190 ± 9

 

 

 

MMS

2.0 µL

 

 

 

 

859 ± 74

 

 

 

 

 

 

 

 

With Activation

DMSO, dried

 

12 ± 3

12 ± 4

30 ± 2

142 ± 21

37 ± 10

Untreated

 

13 ± 2

14 ± 2

40 ± 7

202 ± 3

43 ± 13

Vulkacit P

3 µg

11 ± 3

14 ± 2

34 ± 6

134 ± 12

45 ± 4

Extra N

10 µg

11 ± 1

13 ± 2

30 ± 7

102 ± 15

47 ± 12

 

33 µg

9 ± 3

11 ± 4

24 ± 3

94 ± 12

41 ± 5

 

100 µg

9 ± 3

15 ± 4

43 ± 9

91 ± 3

38 ± 9

 

333 µg

11 ± 5

14 ± 7R

29 ± 9

40 ± 12R

34 ± 5

 

1000 µg

8 ± 3P

7 ± 1P M R

24 ± 3P R

31 ± 9P R

33 ± 6P

 

2500 µg

7 ± 1P M

7 ± 2P M R

16 ± 2P M R

28 ± 3P M R

22 ± 3P M

2-AA

2.5 µg

476 ± 11

97 ± 8

3413 ± 629

2726 ± 213

 

2-AA

10.0 µg

 

 

 

 

546 ± 40

 

 

 

 

 

 

 

 

Summary of Experiment IIA

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

WP2 uvrA

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO, dried

 

 

11 ± 1

11 ± 1

31 ± 10

35 ± 12

Untreated

 

 

8 ± 2

22 ± 9

36 ± 9

40 ± 5

Vulkacit P

10 µg

 

9 ± 3

12 ± 3

35 ± 7

44 ± 4

Extra N

33 µg

 

12 ± 2

11 ± 5

38 ± 6

34 ± 11

 

100 µg

 

15 ± 5

13 ± 4

30 ± 3

33 ± 12

 

333 µg

 

11 ± 1

13 ± 3

27 ± 1

35 ± 5

 

1000 µg

 

8 ± 2P

11 ± 3R P

14 ± 5R M P

33 ± 5P R

 

2500 µg

 

5 ± 2P M

4 ± 2P M R

10 ± 3P M R

22 ± 5P M R

 

5000 µg

 

1 ± 1P M

2 ± 1P M R

6 ± 1P M R

11 ± 3P M R

2-AA

2.5 µg

 

451 ± 33

122 ± 17

3612 ± 1049

 

2-AA

10.0 µg

 

 

 

 

334 ± 63

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

R

Precipitate

Manual count

Reduced background growth

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item, Vulkacit P Extra N, did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

This study was performed to investigate the potential of Vulkacit P Extra N to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in three independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:         3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

All strains without S9 mix:               10; 33; 100; 333; 1000; 2500 and 5000 µg/plate

All strains with S9 mix:                    3, 10; 33; 100; 333; 1000; and 2500 µg/plate

Experiment IIa:

Strains TA 1535, TA 1537,
TA 98, and WP2 uvrA with S9 mix:  10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in all experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate, respectively the highest investigated dose in all experiments. The undissolved particles had no influence on the data recording.

In experiment I the plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. In experiment II and IIa reduced background growth was observed in all strains used with S9 mix except of strain TA 1535.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Vulkacit P Extra N at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Vulkacit P Extra N is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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

Genetic toxicity in vivo

Description of key information

To investigate the potential of zinc ethylphenyl dithiocarbamate to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse a MNT according to OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test) was conducted. The test article was formulated in corn oil. This vehicle was used as negative control. The volume administered intraperitoneally was 10 ml/kg bw. 16h, 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. The occurrence of micronuclei in ten animals (5 males, 5 females) per test group was evaluated. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. Zinc ethylphenyl dithiocarbamate was negative (non-mutagenic) in this micronucleus assay.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
This study was performed to investigate the potential of zinc ethylphenyl dithiocarbamate to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in corn oil. his vehicle was used as negative control. The volume administered intraperitoneally was 10 ml/kg bw. 16h, 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. The occurrence of micronuclei in ten animals (5 males, 5 females) per test group was evaluated. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay
Specific details on test material used for the study:
Name: Zinc ethylphenyl dithiocarbamate
CAS-No.: 14634-93-6
Aggregate State at RT: solid
Colour: white
Purity: 99.6 %
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Reasons for the Choice of the Experimental Animal Species:
The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Strain: NMRI
Source: Charles River Wiga GmbH
Number of animals: 108 (54 males/54 fe ales)
Initial age at start of acclimatization: minimum 10 weeks
Initial Body eight at start of treatment: 28.2 - 37.2 g

The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum
Environment: temperature 21 ± 3°C, relative humidity 30-70%, artificial light 6.00 a.m. - 6.00 p.m.
Route of administration:
intraperitoneal
Vehicle:
corn oil
Details on exposure:
Six males and six females were assigned to each test group.
The following dose levels of the test article were investigated:
16 h preparation interval: 10 mg/kg b.w.
24 h preparation interval: 1, 3, and 10 mg/kg b.w..
48 h preparation interval: 10 mg/kg b.w..
In pre-experiments the highest dose administered was estimated to be the maximum tolerated dose.

At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow from animals treated with the highest dose was done 16, 24 and 48 hours after treatment. Bone marrow samples fro animals treated with the low and medium dose were taken only at preparation interval 24 hours.
Duration of treatment / exposure:
16h, 24 h, or 48 hour.
Frequency of treatment:
Single treatment.
Dose / conc.:
10 other: mg/kg bw
Remarks:
16 h preparation interval: 10 mg/kg b.w.
24 h preparation interval: 10 mg/kg b.w..
48 h preparation interval: 10 mg/kg b.w..
Dose / conc.:
3 other: mg/kg bw
Remarks:
24 h preparation interval: 3 mg/kg b.w..
Dose / conc.:
1 other: mg/kg bw
Remarks:
24 h preparation interval: 1 b.w..
No. of animals per sex per dose:
6 male and 6 female animals/dose/interval
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
Tissues and cell types examined:
1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.
Details of tissue and slide preparation:
Study Procedure
Test Groups:
Six males and six females were assigned to each test group.
The following dose levels of the test article were investigated:
16 h preparation interval: 10 mg/kg bw.
24 h preparation interval: 1, 3, and 10 mg/kg bw
48 h preparation interval: 10 mg/kg bw

Treatment:
During the study period the animals received feed and water ad libitum . At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow from animals treated with the highest dose was done 16, 24 and 48 hours after treatment. Bone marrow samples from animals treated with the low and medium dose were taken only at preparation interval 24 hours.

Preparation of the Animals:
The survived animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted. At least one slide was made from each bone marrow sample.

Analysis of Cells:
Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated. The
microscopic slides of the remaining animals were scored if an animal died in a test group (same time and dose group, same sex).
Evaluation criteria:
A test article is considered positive if, at any of the intervals, there is a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparisonn to the negative control.
A test article is considered negative if there is no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time interval. A test is also considered negative if there is a significant increase in that rate which, according to the laboratory s experience is within the range of negative controls.
The biometric evaluation can be performed by means of the nonparametric Mann-Whitney test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
In pre-experiments the highest dose administered was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. Reduction of spontaneous activity and eyelid closure followed by apathy were observed.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Under the experimental conditions the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

In comparison to the corresponding negative controls there was no enhancement in the frequency of micronuclei at any preparation interval after ap lication of the test article and with any dose

level used.

30 mg/kg b.w. cyclophosphamide administered intraperitoneally was used as positive control which induced a distinct increase of the micronucleus frequency.

Conclusions:
Zinc ethylphenyl dithiocarbamate was negative (non-mutagenic) in this micronucleus assay.
Executive summary:

This study was performed to investigate the potential of Zinc ethylphenyl dithiocarbamate to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in corn oil. This vehicle was used as negative control. The volume administered intraperitoneally was 10 ml/kg bw. 16h, 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. The occurrence of micronuclei in ten animals (5 males, 5 females) per test group was evaluated. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

After treatment with the test article the number of NCEs was not substantially increased as compared to the corresponding negative controls thus indicating that Zinc ethylphenyl dithiocarbamate had no cytotoxic effect.

In comparison to the corresponding negative controls there as no enhancement in the frequency of micronuclei at any preparation interval after ap lication of the test article and with any dose level used.

In conclusion, it can be stated that during the study described and under the experimental conditions reported the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, Zinc ethylphenyl dithiocarbamate is considered to be negative (non-mutagenic) in this micronucleus assay.

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

Additional information

Justification for classification or non-classification

The Ames test according to OECD guideline 471 to investigate the potential of Vulkacit P Extra N to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA was negative with and without metabolic activation.

In a not assignable Ames test with zinc ethylphenyldithiocarbamate conducted on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation strain TA 1535 was positive without metabolic activation and strain TA 100 was positive with metabolic activation.

In a further not assignable Ames test rubber additives were tested for mutagenicity on strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. For zinc ethylphenyldithiocarbamate the lowest effective dose for a positive result was 100 µg per plate. Due to methodological deficiencies and insufficient reporting of the published studies, these tests were disregarded and not used for the assessment of the potential mutagenic potential of zinc ethylphenyldithiocarbamate in bacteria.

In a MNT according to OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test) zinc ethylphenyl dithiocarbamate was negative (non-mutagenic).

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.