Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation

In a study similar to OECD TG 471 (Jones et al., 1986), polysulfides, di-tert-dodecyl was tested in a reverse mutation assay withSalmonella typhimuriumstrains, TA98, TA100, TA102, TA1535, and TA1537 exposed to five concentrations in the presence and absence of a metabolic activation system using plate incorporation at doses of 5 to 5,000 µg/plate. Exposure to five graded doses of polysulfides, di-tert-dodecyl in the presence and absence of metabolic activation, did not increase the reversion rates in any of the tester strains and polysulfides, di-tert-dodecyl is not considered to be a mutagen.

The potential of polysulfides, di-tert-dodecyl to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated in a study performed according OECD TG 476 and Good Laboratory Practice (Nakab, 2010). After a preliminary toxicity test, polysulfides, di-tert-dodecyl was tested in two independent experiments, with and without a metabolic activation system, the S9-mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 5 x 10e6 (3-hour treatment) in 10 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9-mix (final concentration of S9 fraction 5%), at 37°C. Cytotoxicity was measured by assessment of plating efficiency, Relative Survival Growth (RSG), and Relative Total Growth (RTG), after treatment (T0) and 48 hours after treatment (PE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. Di-tert-dodecyl polysulfide (TPS 32) was dissolved in DMSO and the positive controls were methylmethane sulfonate (without S9-mix) and Cyclophosphamide (with S9-mix). In the culture medium, no precipitate was noted at the concentration of 350 µg/mL (i. e. when initial solutions are added at 0.5% in the culture medium). At this concentration, the pH and the osmolality values were comparable to those of the vehicle control culture. The cloning efficiencies and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. The selected concentrations were 175, 131.25, 87.5, 43.75, 21.88, 10.94 and 5.47 µg/ml (assay 1) and 175, 153.13, 131.25, 109.38, 87.5, 65.63 and 43.75 (assay 2) without S9 -mix and 350, 175, 87.5, 43.75, 21.88 and 10.94 (assay 1) and 350, 280, 224, 179.2, 143.36, 114.69and 91.75 (assay 2) with S9-mix. Following the 3-hour treatment, cytotoxicity (decreased adjusted RTG) was observed from 153.13 µg/ml without S9-mix and at 350 µg/ml with S9-mix. A biologically significant mutagenic activity was observed either with or without metabolic activation, in the two independent assays. The clear increase in the number of small colonies is in favour of a clastogenic activity.

Chromosomal aberration

A chromosomal aberration test with human lymphocytes was performed according to OECD TG 473 with polysulfides, di-tert-dodecyl at the concentrations of 300, 1000 and 2500 µg/ml (2500 µg/ml being the limit of solubility in the culture medium) (Molinier, 1994). The test was carried out with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254. Without S9 mix, the cultures were incubated with the test or control substances, which remained in the culture medium, until the appropriate harvest times at 24 and 48 hours. With S9 mix, the test or control substances remained in a culture medium containing 15% S9 mix (10% S9/S9 mix) for 2 hours. The cells were then centrifuged, the treatment medium removed, the cells resuspended in fresh culture medium. The cultures were then incubated until the appropriate harvest times at 24 and 48 hours. Two hours before harvesting, the cells were treated with a colcemid solution to block them at the metaphase-stage of mitosis. The chromosomal preparations were stained and screened microscopically for mitotic index and for aberrations, 200 well-spread metaphases per concentration were read, whenever possible. The aberrant cells frequency in the negative and vehicle controls was within the range of the historical data (i. e. 0.5 ± 0.6%, gaps excluded). The aberrant cells frequency in the positive controls was significantly higher (p < 0.001) than that of the negative controls, indicating the sensitivity of the test system. Di-tert-dodecyl polysulfides (TPS 32) did not induce any significant increase in the aberrant cells frequency, with or without S9 mix, for both of the 2 harvest times.

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:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from liver homogenates of rats induced with 500 mg/kg bw Aroclor 1254
Test concentrations with justification for top dose:
0, 5, 150, 500, 150, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9: 2-nitrofluorene (TA98 & TA1538), ENNG (TA100 & TA1535), 9-aminoacridine (TA1537). With S9: 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 2 (3 plates per concentration)

DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was performed to define the concentrations to be used for the mutagenicity study.
Evaluation criteria:
- negative and positive controls within the range of historical controls.
- positive: reproducible and significant dose related increase in revertants and/or reproducible doubling in the number of revertants compared with negative controls for one dose.
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TPS-32 was not toxic towards the tester strains. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with TPS-32 at any dose level, either in the presence or absence of metabolic activation (S-9 mix).

TABLE 1:Dose range finding test on TPS-32 - revertant colony counts obtained- with bacterial strains TA 1535, TA 1537, TA 1538,TA 98 and TA 100

Dose level

(µg/plate)

Metabolic•

activation

Bacterial strain

TA 1535

TA 1537

TA 1538

TA 98

TA 100

 

5000

-

16

16

4

20

107

 

500

-

11

12

7

13

115

 

50

-

20

17

9

12

92

 

5

-

12

19

8

19

111

 

Solvent

-

17

15

7

16

126

 

5000

+

13

15

14

21

120

 

500

+

21

15

10

27

122

 

50

+

19

14

10

27

141

 

5

+

19

18

13

22

127

 

Solvent

+

11

11

11

21

133

 

TABLE 2 Test 1:TPS-32 - revertant colony counts obtained per plate using bacterial strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Strain

Dose level

(µg/plate)

Metabolic•

activation

Mean revertant

colony counts

SD

Individual revertant

colony counts

TA 1535

5000

-

9

2.5

12,9,7

1500

-

10

1.5

8,11,10

500

-

10

1.5

9,10,12

150

-

12

1.5

11,12,14

50

-

12

1.2

11,11,13

0

-

15

3.6

19,14,12

Solvent

-

15

4.0

11,15,19

5000

+

13

2.1

11,12,15

1500

+

16

2.5

16,18,13

500

+

15

3.1

12,14,18

150

+

17

0.6

17,16,17

50

+

16

1.7

17,14,17

0

+

11

2.5

11,14,9

Solvent

+

16

1.5

18,15,16

TA 1537

5000

-

15

1.7

14,14,17

1500

-

16

2.6

14,19,15

500

-

15

1.2

14,16,16

150

-

16

3.5

12,19,16

50

-

15

2.1

17,13,16

0

-

17

1.0

16,17,18

Solvent

-

16

6.4

9,19,21

5000

+

16

2.6

19,14,15

1500

+

19

4.6

14,23,20

500

+

17

4.6

18,12,21

150

+

19

4.6

24,18,15

50

+

15

4.7

19,10,17

0

+

15

1.2

14,16,14

Solvent

+

18

5.0

13,23,18

TA 1538

5000

-

3

1.0

4,2,3

1500

-

4

1.7

3,3,6

500

-

10

1.0

10,9,11

150

-

5

4.2

4,10,2

50

-

6

0.6

6,6,7

0

-

10

2.5

12,7,10

Solvent

-

7

1.0

6,8,7

5000

+

11

2.0

11,13,9

1500

+

11

4.7

9,16,7

500

+

12

1.5

12,13,10

150

+

10

2.1

11,12,8

50

+

13

4.6

18,9,12

0

+

9

4.0

10,13,5

Solvent

+

8

2.6

6,11,7

TABLE 2(continued)

Strain

Dose level

(µg/plate)

Metabolic•

activation

Mean revertant

colony counts

SD

Individual revertant

colony counts

TA 98

5000

-

15

1.2

14,16,14

1500

-

18

2.6

15,20,19

500

-

17

3.5

14,17,21

150

-

21

5.5

17,18,27

50

-

19

4.9

16,25,17

0

-

21

1.5

19,21,22

Solvent

-

21

6.0

27,15,20

5000

+

24

4.9

27,18,26

1500

+

25

3.2

24,29,23

500

+

27

1.5

27,26,29

150

+

25

0.6

25,25,26

_

50

+

23

2.0

25,23,21

0

+

21

1.5

21,23,20

Solvent

+

17

4.5

17,22,13

TA 100

5000

-

98

4.4

103,96,95

1500

-

91

9.9

102,86,84

500

-

95

7.4

92,103,89

150

-

96

3.8

100,93,94

50

-

99

7.0

107,94,96

0

-

97

5.6

103,92,96

Solvent

-

88

5.1

84,94,87

5000

+

102

2.6

103,104,99

1500

+

111

5.1

117,107,110

500

+

116

4.0

112,115,120

150

+

109

15.0

124,94,109

50

+

116

3.2

114,120,115

0

+

114

9.1

115,104,122

Solvent

+

108

9.3

116,111,98

TABLE 3 Test 1 : Mutability and sterility tests with bacterial strains TA 1535,TA 1537, TA 1538, TA 98 and TA 100

Strain

Compound

Dose level

(µg/plate)

Metabolic•

activation

Mean

revertant

colony

counts

SD

Individual

revertant

colony

counts

TA 1535

ENNG

5

-

226

24.0

216,208,253

TA 1537

9 AC

80

-

2031

10.1

2020,2040,2033

TA 1538

NF

2

-

55

2.3

52,56,56

TA 98

NF

1

-

84

7.4

92,78,81

TA 100

ENNG

3

-

314

11.0

327,309,307

TA 1535

AA

2

+

168

24.2

187,177,141

TA 1537

AA

2

+

98

8.5

101,88,104

TA 1538

AA

0.5

+

113

14.5

120,96,122

TA 98

AA

0.5

+

275

14.6

262,273,291

TA 100

AA

0.5

+

259

24.1

286,239,253

-

S-9 mix

500µl

0

0

-

TPS-32

5000

0

0

Conclusions:
Not mutagenic
Executive summary:

In a study similar to OECD TG 471, a Salmonella typhimurium reverse mutation assay was performed with di-t-dodecyl polysulfides. S. typhimurium strains, TA98, TA100, TA1535, TA1537, and TA1538 were exposed to five concentrations of di-t-dodecyl polysulfides in the presence and absence of a metabolic activation system using plate incorporation at doses of 5 to 5,000 mg/plate. Exposure to five graded doses of di-t-dodecyl polysulfides in the presence and absence of metabolic activation, did not increase the reversion rates in any of the tester strains.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal fraction (S9) of rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
0, 300, 1000, 2500 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C (0.2 µg/ml) without S9, Cyclophosphamide (50µg/ml) with S9.
Details on test system and experimental conditions:
The conditions of treatment were as follows, using 2cultures/experimental point:
. without S9 mix: the cultures were incubated with the test or control substances which remained in the culturemedium, until the appropriate harvest times*: 24 and 48 hours
. with S9 mix: the test or control substances remained inculture medium containing 15% S9 mix (10% S9/S9 mix) for 2hours.
The cells were then centrifuged, the treatment medium removed, the cells resuspended in fresh culture medium. The cultures were then incubated until the appropriate harvest times*: 24 and 48 hours. Two hours before harvesting, the cells were treated with a colcemid solution to block them at themetaphase-stage of mitosis. The chromosomal preparations were stained and screened microscopically for mitotic index and for aberrations: 200 well-spread metaphases perconcentration were read, whenever possible. The concentrations of TPS 32 for scoring were: 300, 1000 and 2500 µg/ml, 2500 µg/ml being the limit of solubility of the test substance in the culture medium.
Evaluation criteria:
The following criteria were used as an aid for determining a positive response:
. a reproducible and statistically significant increase in the aberrant cells frequency for at least one of the tested concentrations.
A test substance was considered as non-clastogenic in this test system if there is no significant increase in aberrant cells frequency at any dose above concurrent control frequencies and in both of the two harvest times.
Both biological and statistical significance was considered together in the evaluation.
Statistics:
For each harvest time, the aberrant cells frequency (exciuding gaps) in treated cultures was compared to that of the vehicle cultures. The comparison was performed using the X² test , in which p = 0.05 was used as the lowest level of significance.
Key result
Species / strain:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Table 1: Clastogenicity test without metabolic activation Hours of treatment: 24 hours Harvest time: 24 hours
Mitotic index

Concentrations

µg/ml

Slides

No.

Mitotic index

(%)

Mean

% of the Control

50M

3.9

3.8

-

43F

3.6

DMSO

18M

4.5

4.1

100

1F

3.6

10

53M

2.4

3.2

78

49F

3.9

30

11M

1.6

2.2

54

24F

2.8

100

59M

1.7

2.9

71

34F

4.0

300

35M

2.5

2.0

49

7F

1.4

1000

58M

2.9

2.7

66

52F

2.5

2500

64M

1.1

1.8

44

44F

2.4

MMC

3M

1.2

1.2

29

(0.2 µg/m1)

25F

1.2

Chromosomal aberrations

Concentrations   µg/ml

Slides

No.

Chromatid   Chromosome

Nb. of st aberrations

% aberrantcells

G D E

D E MA

PU

NuAb

+G

-G

+G

-G

-

50M

1 0 0

0 0 0

0

0

2

1

1.0

0.5

43F

0 1 0

0 0 0

0

0

DMSO

18M

1 0 0

0 0 0

0

0

1

0

0.5

0.0

1F

0 0 0

0 0 0

0

0

300

35M

1 0 0

0 0 0

0

0

1

0

0.5

0.0

7F

0 0 0

0 0 0

0

0

1000

58M

0 1 0

0 0 0

0

0

3

1

1.5

0.5

52F

2 0 0

0 0 0

0

0

2500

64M

0 0 0

0 0 0

0

0

1

0

0.6

0.0

44F

1 0 0

0 0 0

0

0

MMC

3M

2 5 11

0 0 0

0

0

36

33

20.5

20.5***

(0.2µg/ml)

25F

1 4 11

2 0 0

0

0

G: gap                      200 metaphases/concentration were scored except:

D: deletion                 . concentration 2500 pg/ml: 162 metaphases

E: exchange                 . positive control (MMC): 122 metaphases.MA: multiple aberrations

PU: pulverisation          Statistical test: X2test, ***: p < 0.001

M: male

F: female

NuAb: numerical aberrations (included polyploidy, hyperdiploidy and endoreduplication)

Nb. of st aberrations: number of structural aberrations (numerical aberrations excluded of this total)

Table 2: Clastogenicity test without metabolic activation Hours of treatment: 48 hours Harvest tinte: 48 hours


Mitotic index

Concentrations

µg/m1

Slides

No.

Mitotic index

(%)

Mean

% of the Control

-

56M

3.9

4.0

-

29F

4.0

DMSO

30M

5.1

4.5

100

62F

3.8

10

13M

4.1

4.7

104

48F

5.2

30

61M

3.5

4.3

96

4F

5.1

100

47M

5.9

6.3

140

39F

6.6

300

65M

4.0

4.1

91

12F

4.1

1000

31M

4.5

4.4

98

63F

4.2

2500

57M

2.6

3.8

84

21F

4.9

Chromosomal aberrations

Concentrations Slides

µg/m1

No.

Chromatid Chromosome

G D E  D E MA PU NuAb

Nb. of st

aberrations

% aberrant

cells

+G

-G

+G

-G

56M

1 0 0

0 0 0

0

0

3

0

1.5

0.0

29F

2 0 0

0 0 0

0

0

DMSO

30M

2 1 0

0 0 0

0

0

4

2

2.0

1.0

62F

0 1 0

0 0 0

0

0

300

65M

1 1 0

0 0 0

0

0

2

1

1.0

0.5

12F

0 0 0

0 0 0

0

0

1000

31M

1 0 0

0 0 0

0

0

1

0

0.5

0.0

63F

0 0 0

0 0 0

0

0

2500

57M

0 3 0

0 0 0

0

0

6

3

3.0

1.5

21F

3 0 0

0 0 0

0

0

G: gap                      200 metaphases/concentration were scored:

D: deletion

E: exchange

MA:multiple aberrations

PU: pulverisation          Statistical test: X2test

M: male

F: female

NuAb: numerical aberrations (included polyploidy, hyperdiploidy and endoreduplication)

Nb. of st aberrations: number of structural aberrations (numerical aberrations excluded of this total)
Conclusions:
TPS 32 did not show clastogenic activity in this chromosomal aberration test performed in cultured human lymphocytes.
Executive summary:

A chromosomal aberration test with human lymphocytes was performed according to OECD guideline #473 with di-t-dodecyl polysulfides (TPS 32) at the concentrations of 300, 1000 and 2500 µg/ml (2500 µg/ml being the limit of solubility in the culture medium). The test was carried out with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254. Without S9 mix, the cultures were incubated with the test or control substances, which remained in the culture medium, until the appropriate harvest times at 24 and 48 hours. With S9 mix, the test or control substances remained in a culture medium containing 15% S9 mix (10% S9/S9 mix) for 2 hours. The cells were then centrifuged, the treatment medium removed, the cells resuspended in fresh culture medium. The cultures were then incubated until the appropriate harvest times at 24 and 48 hours. Two hours before harvesting, the cells were treated with a colcemid solution to block them at the metaphase-stage of mitosis. The chromosomal preparations were stained and screened microscopically for mitotic index and for aberrations, 200 well-spread metaphases per concentration were read, whenever possible. The aberrant cells frequency in the negative and vehicle controls was within the range of the historical data (i.e. 0.5 ± 0.6%, gaps excluded). The aberrant cells frequency in the positive controls was significantly higher (p < 0.001) than that of the negative controls, indicating the sensitivity of the test system. Di-t-dodecyl polysulfides(TPS 32) did not induce any significant increase in the aberrant cells frequency, with or without S9 mix, for both of the 2 harvest times.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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)
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):
L5178Y TK+/- mouse lymphoma cells were obtained from ATCC (American Type Culture Collection - Rockville, MD 20852 - USA). A stock of these cells is maintained and stored frozen in liquid nitrogen.
Contamination by mycoplasma is checked using Mycoalert Mycoplasma Detection kit (Cambrex Bio Science Rockland, inc) for each batch of the cells. Only the batches, which contain no mycoplasma, are used in the mutagenicity test.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of rat liver induced by Aroclor 1254
Test concentrations with justification for top dose:
Without S9 mix : 175, 131.25, 87.5, 43.75, 21.88, 10.94 and 5.47 (assay 1); 175, 153.13, 131.25, 109.38, 87.5, 65.63 and 43.75 (assay 2)
With S9 mix : 350, 175, 87.5, 43.75, 21.88 and 10.94 (assay 1); 350, 280, 224, 179.2, 143.36, 114.69 and 91.75 (assay 2)
Vehicle / solvent:
The di-tert-dodecyl polysulfides (TPS 32) was dissolved in dimethylsulfoxide (DMSO) at the maximum initial concentration of 70 mg/mL, corresponding to the maximum of solubility and used at 0.5% in culture medium, giving a final concentration of 350 µg/mL. At this concentration, the solution was clear. Furthermore, when added at 0.5% in the culture medium, no precipitate was noted.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9-mix : methyl methanesulfonate 10 µg/mL (3-h treatment). With S9-mix : cyclophosphamide, 2 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration:
Without S9-mix : 3 hours (short treatment) (as a positive response was obtained in the first assay using a 3-h treatment, the second assay was performed under the same experimental conditions)
With S9-mix : 3 hours
- Expression time (cells in growth medium): 2 days after treatment
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): TFT (3 µg/mL)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Concentrations tested expressed as µg/mL di-tert-dodecyl polysulfides (TPS 32) as supplied
• Without S9-mix : 350 – 175 – 87.5 – 43.75 – 21.88 – 10.94 – 5.47 (3h treatment)
175 – 87.5 – 43.75 – 21.88 – 10.94 – 5.47 – 2.73 – 1.37 – 0.68 (24h treatment)
• With S9-mix : 350 – 175 – 87.5 – 43.75 – 21.88 – 10.94 – 5.47
Evaluation criteria:
A test item is considered as mutagenic in this system if the following conditions are fulfilled:
1. The induced mutation frequency for at least one tested concentration is higher than the mutation frequency in the vehicle control cultures by at least the global evaluation factor of 126 x10-6 (Moore et al., 2006).
2. A statistical trend test demonstrates a positive dose related increase in the mutation frequency (Moore et al., 2006).
3. The results have to be reproducible in an independent study, at least from a qualitative point of view.
If none of the three criteria mentioned above is fulfilled, the tested test item is considered as not mutagenic in this study system.
In all other cases, the results are discussed case by case, and the results obtained on other study systems are taken into account.
All these criteria are not absolute: however, they give help when a decision has to be taken, making a conclusion possible in the majority of the cases.
Statistics:
Statistical evaluation of data for the total number of mutants and for small colony mutants is performed using the method proposed by Robinson et al. (Statistical evaluation of bacterial/mammalian fluctuation tests. In Statistical evaluation of mutagenicity test data. KIRKLAND D.J. (Ed). Cambridge University Press, Cambridge- New York, (1990) 102-140)
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH was comprised in the acceptable range of 6-8 at the highest concentrations tested from 350 to 87.5 µg/mL.
- Effects of osmolality: The concentrations of 350, 175 and 87.5 µg/mL induced no variation in osmolarity higher than 3 mOsmol when compared to the solvent control.
- Precipitation: no precipitate was noted.
Conclusions:
Di-tert-dodecyl polysulfides (TPS 32) induced a biologically significant mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture either with or without metabolic activation, in two independent assays. The clear increase in the number of small colonies is in favour of a clastogenic activity.
Executive summary:

The potential of Di-tertio-decyl polysulfide (TPS 32) to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated in a study performed according to the international guidelines OECD No. 476 and Good Laboratory Practice. After a preliminary toxicity test, Di-tertio-decyl polysulfide (TPS 32) was tested in two independent experiments, with and without a metabolic activation system, the S9-mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 5 x 10e6 (3-hour treatment) in 10 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9-mix (final concentration of S9 fraction 5%), at 37°C. Cytotoxicity was measured by assessment of plating efficiency, Relative Survival Growth (RSG), and Relative Total Growth (RTG), after treatment (T0) and 48 hours after treatment (PE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. Di-tertio-decyl polysulfide (TPS 32) was dissolved in DMSO and the positive controls were methylmethane sulfonate (without S9-mix) and Cyclophosphamide (with S9-mix). In the culture medium, no precipitate was noted at the concentration of 350 µg/mL (i.e. when initial solutions are added at 0.5% in the culture medium). At this concentration, the pH and the osmolality values were comparable to those of the vehicle control culture. The cloning efficiencies and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. The selected concentrations were 175, 131.25, 87.5, 43.75, 21.88, 10.94 and 5.47 µg/ml (assay 1) and 175, 153.13, 131.25, 109.38, 87.5, 65.63 and 43.75 (assay 2) without S9 -mix and 350, 175, 87.5, 43.75, 21.88 and 10.94 (assay 1) and 350, 280, 224, 179.2, 143.36, 114.69and 91.75 (assay 2) with S9-mix. Following the 3-hour treatment, cytotoxicity (decreased adjusted RTG) was observed from 153.13 µg/ml without S9-mix and at 350 µg/ml with S9-mix. A biologically significant mutagenic activity was observed either with or without metabolic activation, in the two independent assays. The clear increase in the number of small colonies is in favour of a clastogenic activity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The potential clastogenic activity of polysulfides, di-tert-dodecyl was tested using the in vivo micronucleus test in male OFA Sprague Dawley rats, in compliance with OECD TG 474, by oral route, using 2 successive daily treatments at the maximum dose recommended by OECD guidelines, i. e. 2000 mg/kg, followed by one sampling time 24 hours after the last treatment (Simar, 2010). The two lower doses of 1000 and 500 mg/kg/day (x2) were also analysed. The validity criteria for the results were fulfilled. The study was thus considered as valid. Di-tert-dodecyl polysulfides (TPS 32) induced no genotoxic activity under these experimental conditions.

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
Study period:
17/05/2010 – 06/07/2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-surl’Arbresle; FRANCE
- Age at study initiation: 5 to 10 weeks old
- Weight at study initiation: approximately 200 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: animals were housed in polypropylene cages measuring 42.5 x 26.6 x 15 cm, covered by a stainless steel netted lid, in which they will be placed in groups of 3 or 2
- Diet (ad libitum): 801175 RM1(P)DU IRR 9Kgy irradiated from Special Diets Services
(ENGLAND).
- Water (ad libitum): softened by reverse osmosis and filtered on 0.2 µm membrane
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 20 times per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC at 0.5% in distilled water
Details on exposure:
Dose volume : 10 mL/kg b.w.
Duration of treatment / exposure:
2 treatments
Frequency of treatment:
daily
Post exposure period:
Number of sampling times : for the negative control and the 3 treated groups: 24 hours after the second treatment for the positive control group: 24 hours after the single treatment
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5 (excepted 7 for the high dose level)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (25 mg/kg i.p.)
Tissues and cell types examined:
Bone Marrow
Details of tissue and slide preparation:
At the sampling time, the 5 animals per group were sacrificed by CO2 asphyxia; the femurs were removed, and the bone marrow was extracted with foetal calf serum (1 mL per animal). The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatant was removed. The centrifugate was spread on slides. The smears were stained using a technique, derived from the May Grunwald Giemsa technique (Schmid, 1975), which makes it possible to distinguish between polychromatic (PCE) and normochromatic erythrocytes (NCE): PCE are purple whereas NCE are red.
After coding the slides by a person not involved in the study, two slides per animal were read by two independent operators; for each animal, the number of polychromatic erythrocytes having one or more Howell-Jolly bodies (micronuclei) was determined for at least 2000 polychromatic erythrocytes. (In case of divergence, 2000 new polychromatic erythrocytes were examined; the retained value was the mean of all readings).
The polychromatic/normochromatic erythrocyte ratio will be determined by analyzing at least 1000 erythrocytes per animal.
Evaluation criteria:
For a test item to be considered negative in the micronucleus test, there must be no statistically significant increase in the number of MNPCE observed compared with negative control animals.
For a test item to be considered positive in the micronucleus test, there must be seen a statistically significant increase in MNPCE for at least one dose group and/or a statistically significant dose-related increase in MNPCE, compared to the negative control animals. Statistical significance will not be the only determinant of a positive response, and the Study Director will consider the biological relevance of the results in the final evaluation.
Statistics:
Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney U rank test. An analysis of a large number of control results has shown that the distribution of the numbers of micronuclei does not correspond to a Gaussian distribution, but to a Poisson-type distribution. This makes it necessary to use a non-parametric statistical test, and the Mann Whitney U rank test is recommended by UKEMS (LOVELL et al., 1989).
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
but tested up to the limit dose of 2000 mg/kg
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Clinical signs of toxicity in test animals: No clinical signs were observed after up to 24 hours after the second treatment in male and female rats. As no difference between both sexes were noted, the main assay was performed in male rats only.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The results obtained on negative control animals and those treated with the positive reference
substance were similar to those generally obtained in the laboratory. A statistically significant increase in the frequency of micronuclei was noted in the group treated with Cyclophosphamide, demonstrating the sensitivity of the animal strain used to a clastogenic agent. The validity criteria for the test were fulfilled and the test was validated. Regarding the frequency of micronuclei, no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with di-tert-dodecyl polysulfides (TPS 32) at any dose.
- Ratio of PCE/NCE (for Micronucleus assay): No statistically significant decrease in the ratio PCE to NCE was noted in the 3 di-tertdodecyl polysulfides (TPS 32) treatment groups when compared to the negative control group. In consequence, no proof of systemic exposure was evidenced.
Conclusions:
The validity criteria for the results were fulfilled. The study was thus considered as valid. The test item di-tert-dodecyl polysulfides (TPS 32) was investigated by means of the in vivo micronucleus test, in male OFA Sprague Dawley rats treated orally twice with 2000 – 1000 and 500 mg/kg/day, followed by one sampling time 24 hours after the last treatment. As a conclusion, di-tert-dodecyl polysulfides (TPS 32) induced no genotoxic activity under these experimental conditions.
Executive summary:

The potential clastogenic activity of di-tert-dodecyl polysulfides (TPS 32) was tested using the in vivo micronucleus test in male OFA Sprague Dawley rats, in compliance with the Commission Regulation (EC) No. 440/2008 and the OECD Guideline 474, by oral route, using 2 successive daily treatments at the maximum dose recommended by OECD guidelines, i.e. 2000 mg/kg, followed by one sampling time 24 hours after the last treatment. The two lower doses of 1000 and 500 mg/kg/day (x2) were also analysed. The validity criteria for the results were fulfilled. The study was thus considered as valid. Di-tert-dodecyl polysulfides (TPS 32) induced no genotoxic activity under these experimental conditions.

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

Additional information

Polysulfides, di-tert-dodecyl was not mutagenic in an in vitro bacterial reverse mutation assay (similar to OECD TG 471) or in an in vitro mammalian chromosomal aberration assay (OECD TG 473) with or without metabolic activation. In a mouse lymphoma gene mutation assay with L5178Y cells (OECD TG 46), a biologically significant mutagenic activity was observed either with or without metabolic activation. The clear increase in the number of small colonies was in favour of a clastogenic activity. However, polysulfides, di-tert-dodecyl was not clastogenic in an in vivo micronucleus test (OECD TG 474), in male OFA Sprague Dawley rats treated twice orally with up to 2000 mg/kg/day.

Justification for classification or non-classification

On a weight-of-evidence basis, polysulfides, di-tert-dodecyl is not considered as a genotoxic substance, no classification is warranted according to Regulation no. 1272/2008.