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Administrative data

Description of key information

Based on a weight of evidence using studies performed on similar products, di-tert-dodecyl disulfide and di-tert-butyl polysulfide and its raw material and potential metabolite, tert-dodecyl mercaptan, di-tert-dodecyl polysulfide can be considered as a weak sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
Deviations:
yes
Remarks:
no positive control reported in the study report
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study was performed before the implementation of the REACH regulation
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation:
- Weight at study initiation: 300 to 500 g
- Housing: groups of 5 in polystyrene cages
- Diet (ad libitum): guinea pig complete pelleted maintenance food (Pietrement, France)
- Water (ad libitum): softened and filetred drinking water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/-3
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal
Vehicle:
water
Concentration / amount:
1%
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 ml
Day(s)/duration:
Day 9
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 ml
Day(s)/duration:
Days 22
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Treated groups: 10/sex
Control groups: 5/sex
Details on study design:
The applications corresponding to "the induction" were carried out
- By intradermal route: injection of 2 x 0.1 ml
* on one hand, with the test article in a 1 % (W/V) solution in water for, injection ;
* on the other hand, with the 50/50 (V/V) mixing : test article in a 2 % solution (W/V) in water for injection +complete Freund's adjuvant at 50 % (V/V) in isotonic injectable solution, i.e. a final 1 % concentration of thes ample to control. Injection of the test article in a 1 % solution has provoked a weak to moderate irritation.

- By topical occlusive route for 48 hours, with the test article as supplied and at the dose level of 0.5 ml per animal. This application having not provoked any weak to moderate irritation, a skin painting was carried out on Day 8, with 0.5 ml of Sodium Lauryl Sulfate at 10 % in Codex liquid paraffin.

- During the "challenge exposure", the topical occlusive application for 24 hours was carried out with the test article as supplied and at the dose level of 0.5 ml per guinea-pig (Maximum Non-Irritant Concentration : M.N.I.C.).
Positive control substance(s):
not specified
Positive control results:
No data reported
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.5 ml undiluted
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
2 with an erythema score of 2 and 3 with a score of 1
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.5 ml undiluted
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
1 erythema score of 3, 6 scores of 2 and 11 scores of 1
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.5 ml undiluted
No. with + reactions:
3
Total no. in group:
9
Clinical observations:
erythema score of 3
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5 ml undiluted
No. with + reactions:
10
Total no. in group:
13
Clinical observations:
1 erythrema score of 2 and 9 scores of 1

The histological examinations of the skin of 2 control and 8 treated animals presenting an erythema score of 2 or more, didn't show any specific skin reactions in 2 controls and 4 treated animals and a specific reaction of irritation of weak intensity in 4 treated guinea-pigs. This phenomenon can hide possible weak reactions of cutaneous sensitization.

Interpretation of results:
GHS criteria not met
Conclusions:
Classification: not sensitizing
Executive summary:

In an OECD TG 406, guinea pigs (n=30; sex/strain not specified) were tested for skin sensitization to di-t-dodecyl polysulfide using the guinea pig maximization test. The study was conducted according to OECD TG 406. Induction was accomplished by intracutaneous exposure to 1% of di-t-dodecyl polysulfide followed by an epicutaneous occlusive application of undiluted di-t-dodecyl polysulfide. During the challenge exposure at 24 hours, a 0.5 ml topical occlusive application of di-t-dodecyl polysulfide was carried out with the test article as supplied. From the macroscopic and histological results, it was concluded that di-t-dodecyl polysulfide was a weak irritant in four of 20 treated guinea pigs, which could mask possible weak reactions of cutaneous sensitization. No characteristic cutaneous abnormalities were noted in the 10 control guinea pigs.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
02 March 2016 to 09 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/CaOlaHsd mice
Source: Envigo (Formerly: Harlan Laboratories S.r.l.), San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 10 weeks old (age-matched, within one week)
Body weight range at starting: 19.8 – 22.1 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight).
Acclimatization time: 21 days
Note: In the Preliminary Experiment, mice of 10 weeks of age (17.4-18.7 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.4 - 24.3°C
Relative humidity: 22 - 70 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.

Food and feeding
Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary).

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH +Co KG).

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 100 % (undiluted) and 50 % (w/v) in AOO.
Based on the observations, the 100 % (undiluted) was selected as top dose for the main test. At the request of the Sponsor additional dose groups (a total of 5) were tested in the main experiment to ensure that data allow the determination of the EC3 value of the test item.
No. of animals per dose:
4 per dose
Details on study design:
Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 100 % (undiluted) and 50 % (w/v) in AOO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 100 % (undiluted).
In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
During the Preliminary Irritation / Toxicity Test, no mortality was observed. Erythema (erythema score of 1) was observed in the 100 % (undiluted) dose group on Days 2-5 and in the 50 % (w/v) group on Days 3-5. The body weight loss was more than 5% for one animal in the 100 % (undiluted) dose group; however the mean body weight change of both groups was acceptable (less than 5%).
The mean ear thickness values and the ear punch weights were within the acceptable range, however slightly increased ear thickness value was detected for the left ear of one animal in the 100 % (undiluted) dose group.
The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in both dose groups (subjective judgement by analogy with observations of former experiments).
Based on these observations, the 100 % (undiluted) was selected as top dose for the main test. At the request of the Sponsor additional dose groups (a total of 5) were tested in the main experiment to ensure that data allow the determination of the EC3 value of the test item.

Topical application
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of mice from each test group were pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a ß-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The ß-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
Since the test item gave a positive response, the EC3 value of the test item (EC3 means the effective chemical concentration required for SI=3) was calculated by linear interpolation according to the equation:
EC3 = c + [(3-d)/(b-d)] x (a-c)
where the data points lying immediately above and below the SI value of 3 on the LLNA dose-response plot have the co-ordinates (a,b) and (c,d) respectively.
Positive control results:
The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (AOO) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 6.6) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Key result
Parameter:
EC3
Remarks:
% (w/v)
Value:
46.7
Parameter:
SI
Value:
0.9
Test group / Remarks:
test group (2%)
Parameter:
SI
Value:
1.4
Test group / Remarks:
test group (10%)
Parameter:
SI
Value:
1.7
Test group / Remarks:
test group (25%)
Parameter:
SI
Value:
3.2
Test group / Remarks:
test group (50%)
Parameter:
SI
Value:
3.1
Test group / Remarks:
test group (100%)
Parameter:
SI
Value:
6.6
Test group / Remarks:
Positive control

Individual Body Weights for all Animals with Group Means

Animal Number

Identity Number

Test Group Name

Initial Body weight (g)

Terminal Body Weight* (g)

Change # (%)

1258

1274

1250

1270

 

1

2

3

4

Negative (vehicle control)

AOO

 

 

Mean

20.1

21.0

21.5

21.3

21.0

19.1

21.7

21.5

22.1

21.1

-5.0

3.3

0.0

3.9

0.5

1252

1232

1253

1267

 

5

6

7

8

DI-TERT-DODECYL DISULPHIDE

100% (undiluted)

 

 

Mean

20.2

21.4

21.2

21.5

21.1

20.9

21.4

20.1

21.0

20.9

3.5

0.0

-5.2

-2.3

-1.0

1255

1234

1261

1272

 

9

10

11

12

DI-TERT-DODECYL DISULPHIDE

50% (w/v)

in AOO

 

Mean

19.9

20.6

20.5

21.4

20.6

20.2

20.9

20.3

21.0

20.6

1.5

1.5

-1.0

-1.9

0.0

1248

1257

1271

1266

 

13

14

15

16

DI-TERT-DODECYL DISULPHIDE

25% (w/v)

in AOO

 

Mean

19.9

20.1

21.4

21.4

20.7

20.6

19.6

20.4

21.4

20.5

3.5

-2.5

-4.7

0.0

-0.9

1254

1263

1273

1260

 

17

17

19

20

DI-TERT-DODECYL DISULPHIDE

10% (w/v)

in AOO

 

Mean

19.8

20.0

21.4

21.8

20.8

19.7

20.7

21.2

21.3

20.7

-0.5

3.5

-0.9

-2.3

-0.1

1259

1275

1269

1262

 

21

22

23

24

DI-TERT-DODECYL DISULPHIDE

2% (w/v)

in AOO

 

Mean

19.8

20.7

21.3

21.3

20.8

21.0

20.5

21.4

21.1

21.0

6.1

-1.0

0.5

-0.9

1.2

1279

1264

1277

1268

 

25

26

27

28

Positive control

25 (w/v) % HCA

in AOO

 

Mean

20.0

19.8

20.8

22.1

20.7

20.2

20.2

20.2

21.8

20.6

1.0

2.0

-2.9

-1.4

-0.3

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Measured DPM / group

DPM

Number of lymph nodes

DPN

Stimulation Index

Background

(5% (w/v) TCA)

31

36

-

-

-

-

Negative control

(AOO)

8230

8196.5

8

1024.6

1.0

DI-TERT-DODECYL DISULPHIDE

100% (undiluted)

25149

25115.5

8

3139.4

3.1

DI-TERT-DODECYL DISULPHIDE

50% (w/v) in AOO

26570

26536.5

8

3317.1

3.2

DI-TERT-DODECYL DISULPHIDE

25% (w/v) in AOO

13801

13767.5

8

1720.9

1.7

DI-TERT-DODECYL DISULPHIDE

10% (w/v) in AOO

11785

11751.5

8

1468.9

1.4

DI-TERT-DODECYL DISULPHIDE

2% (w/v) in AOO

7224

7190.5

8

898.8

0.9

Positive control

(25% (w/v) HCA in AOO)

53746

53712.5

8

6714.1

6.6

 

Results of the Preliminary Irritation / Toxicity Test

 

Individual Body Weights for all Animals with Group Means

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change# (%)

988

1001

1

2

100% (undiluted)

100% (undiluted)

Mean

18.1

17.4

17.8

16.9

17.0

17.0

-6.6

-2.3

-4.5

996

991

3

4

50% (w/v)

50% (w/v)

Mean

18.7

17.6

18.2

18.6

17.9

18.3

-0.5

1.7

0.6

*: Terminal body weights are measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals

Identity Number

Animal Number

Test Group Name

Ear Thickness on Day 1 (mm)

Ear Thickness on Day 3 (mm)

Ear Thickness on Day 6 (mm)

Biopsy weight* on Day 6 (mg)

Right

Left

Right

Left

Right

Left

988

1001

996

991

1

2

3

4

100% (undiluted)

100% (undiluted)

50% (w/v)

50% (w/v)

0.21

0.21

0.20

0.21

0.20

0.22

0.20

0.21

0.22

0.22

0.23

0.22

0.23

0.23

0.23

0.22

0.24

0.24

0.24

0.24

0.25

0.24

0.24

0.24

18.81

21.08

18.32

18.37

*: Historical control range: 11.92 – 22.53 mg. Positive response is over 28.16 mg (=25%)

 

Summarized Clinical Observations

Period

Group

Animal No.

Identity No.

Clinical observations

DAY 1

100% (undiluted)

1

988

Before treatment: symptom free, ES: 0

After treatment: symptom free, ES: 0

2

1001

Before treatment: symptom free, ES: 0

After treatment: symptom free, ES: 0

50% (w/v)

3

996

Before treatment: symptom free, ES: 0

After treatment: symptom free, ES: 0

4

991

Before treatment: symptom free, ES: 0

After treatment: symptom free, ES: 0

DAY 2

100% (undiluted)

1

988

Before treatment: symptom free, ES: 0

After treatment: ES: 1

2

1001

Before treatment: symptom free, ES: 0

After treatment: ES: 1

50% (w/v)

3

996

Before treatment: symptom free, ES: 0

After treatment: symptom free, ES: 0

4

991

Before treatment: symptom free, ES: 0

After treatment: symptom free, ES: 0

DAY 3

100% (undiluted)

1

988

Before treatment: symptom free, ES: 0

After treatment: ES: 1

2

1001

Before treatment: symptom free, ES: 0

After treatment: ES: 1

50% (w/v)

3

996

Before treatment: symptom free, ES: 0

After treatment: ES: 1

4

991

Before treatment: symptom free, ES: 0

After treatment: ES: 1

DAY 4

100% (undiluted)

1

988

ES: 1

2

1001

ES: 1

50% (w/v)

3

996

ES: 1

4

991

ES: 1

DAY 5

100% (undiluted)

1

988

ES: 1

2

1001

ES: 1

50% (w/v)

3

996

ES: 1

4

991

ES: 1

DAY 6

100% (undiluted)

1

988

Symptom free, ES: 0

2

1001

Symptom free, ES: 0

50% (w/v)

3

996

Symptom free, ES: 0

4

991

Symptom free, ES; 0

The clinical observation of animals on the first day was performed simultaneously with the body weight measurements.

ES: Erythema score.

 

Summarized Clinical Observations – Main Test

Group

Animal No.

Identity No.

CLINICAL OBSERVATIONS

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control (AOO)

1

 

2

 

3

 

4

1258

 

1274

 

1250

 

1270

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

DI-TERT-DODECYL DISULPHIDE

100% (undiluted)

5

 

6

 

7

 

8

1252

 

1232

 

1253

 

1267

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

DI-TERT-DODECYL DISULPHIDE

50% (w/v) in AOO

9

 

10

 

11

 

12

1255

 

1234

 

1261

 

1272

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

DI-TERT-DODECYL DISULPHIDE

25% (w/v) in AOO

13

 

14

 

15

 

16

1248

 

1257

 

1271

 

1266

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

DI-TERT-DODECYL DISULPHIDE

10% (w/v) in AOO

17

 

18

 

19

 

20

1254

 

1263

 

1273

 

1260

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

DI-TERT-DODECYL DISULPHIDE

2% (w/v) in AOO

21

 

22

 

23

 

24

1259

 

1275

 

1269

 

1262

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Positive control (25% (w/v) HCA in AOO)

25

 

26

 

27

 

28

1279

 

1264

 

1277

 

1268

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

BT: before treatment, AT: after treatment

 

Historical Control Data

Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsd mice (2014-2015)

CBA/CaOlaHsd mice

 

Vehicles

Acetone: Olive oil 4:1 (AOO)

1% Pluronic PE9200 in water (1% Plu)

DPN values

SI value

DPN values

SI value

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

415.2

2922.6

7.5

197.7

1825.3

10.0

Range: min

               max

111.3

847.8

890.3

7674.5

3.3

15.5

23.0

680.8

154.0

6755.8

3.0

33.1

Number of cases

32

32

30

134

234

128

 

 

Vehicles

N,N-Dimethylformanmide (DMF)

Dimethyl sulfoxide (DMSO)

DPN values

SI values

DPN values

SI values

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

244.6

2522.6

10.8

488.7

3212.1

7.8

Range:  min

               max

140.8

505.8

1201.3

4804.6

6.3

21.3

238.5

934.6

2017.2

4877.5

3.1

14.5

Number of cases

21

21

21

13

13

12

 

 

Vehicles

Propylene glycol (PG)

Methyl ethyl ketone (MEK)

DPN values

SI value

DPN values

SI value

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

235.4

2371.8

10.0

260.2

4888.8

19.5

Range:  min

               max

63.3

506.0

817.3

4978.0

6.5

14.4

183.5

383.3

3465.3

8682.5

8.9

36.3

Number of cases

14

14

14

9

10

10

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In conclusion, under the conditions of the present assay, DI-TERT-DODECYL DISULPHIDE, tested in a suitable vehicle, was shown to have a slight sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value was 46.7 % (w/v).
Executive summary:

The aim of the study was to determine the skin sensitisation potential of DI-TERT-DODECYL DISULPHIDE following dermal exposure. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, corresponding to the regulatory guidelines being followed. Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline no. 429, the test item was tested for formulation compatibility in acetone:olive oil 4:1 (v:v) mixture (abbreviated as AOO). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (undiluted). The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 100 % (undiluted) and 50 % (w/v) in AOO. Based on the observations recorded in the preliminary test, the 100 % (undiluted) was selected as top dose for the main test. In the main assay, twenty-eight female CBA/CaOlaHsd mice were allocated to seven groups of four animals each:

- five groups received DI-TERT-DODECYL DISULPHIDE (formulated in AOO) at 100 % (undiluted), 50, 25, 10 and 2 % (w/v) concentrations,

- the negative control group received the vehicle (AOO),

- the positive control group received 25 % (w/v) HCA (dissolved in AOO).

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. No marked body weight loss (=5%) was observed on the mean body weight of the groups in the study. The stimulation index values were 3.1, 3.2, 1.7, 1.4 and 0.9 at concentrations of 100 % (undiluted), 50, 25, 10 and 2 % (w/v), respectively. The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay. 

In conclusion, under the conditions of the present assay, DI-TERT-DODECYL DISULPHIDE, tested in a suitable vehicle, was shown to have a slight sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value was 46.7 % (w/v).

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
09 May 2011 - 17 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of the treatment period, the animals of the preliminary and main tests were approximately 8 weeks old
- Weight at study initiation: a mean body weight +- standard deviation of 21.0 g +/- 0.9 g
- Housing: the animals were group housed by two (preliminary test) or four (main test) in polycarbonate cages with stainless steel lids
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter)
- Acclimation period: the animals were acclimated to the study conditions from 10 to 14 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: From: 19 May 2011 To: 14 June 2011.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10, 25, 50 or 100% in a mixture acetone/olive oil (4/1; v/v).
No. of animals per dose:
7 groups of 4 animals.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the first choice vehicle was a mixture of Acetone/Olive Oil (4/1; v/v) (AOO). Satisfactory solubility of the test item in AOO
(i.e. solution to the naked eye at the concentration of 50% was obtained).
- Irritation: the test item was not excessively irritant (increase in ear thickness not above 25%) whatever the concentration
- Lymph node proliferation response: incorporation of tritiated methyl thymidine.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item should be regarded as a skin sensitizer when the SI for a dose group is = 3 together with
consideration of a dose-response relationship. Other relevant criteria such as radioactivity levels and ear thickness were also taken into account to evaluate the data.

TREATMENT PREPARATION AND ADMINISTRATION:
On days 1, 2 and 3, a dose-volume of 25 µL of the appropriate dosage form preparation was applied to the dorsal surface of both ears (one concentration per ear), using an adjustable pipette fitted with a plastic tip.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The study was considered valid as the SI for the positive control was higher than 3 (3.15).
Key result
Parameter:
EC3
Remarks:
%
Value:
31
Parameter:
SI
Remarks:
5%
Value:
0.18
Test group / Remarks:
5%
Parameter:
SI
Remarks:
10%
Value:
0.64
Test group / Remarks:
10%
Parameter:
SI
Remarks:
25%
Value:
2.11
Test group / Remarks:
25%
Parameter:
SI
Remarks:
50%
Value:
5.94
Test group / Remarks:
50%
Parameter:
SI
Remarks:
100%
Value:
4.71
Test group / Remarks:
100%
Parameter:
SI
Remarks:
positive control
Value:
3.15
Test group / Remarks:
positive control
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
See attached table

DETAILS ON STIMULATION INDEX CALCULATION
See attached table

EC3 CALCULATION
EC3 = 31%

CLINICAL OBSERVATIONS:
- Clinical signs and mortality
Neither mortality nor clinical signs were observed during the observation period.
- Local irritation
Dryness of the skin of the ear was noted on day 6 in all animals given 50 and 100%. In addition, erythema and scabs on ear were noted in 1/4 females given 100%.
No notable increase in ear thickness was observed at any of the tested concentrations.

BODY WEIGHTS
The body weight change of test item treated animals was similar to that of vehicle treated animals.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
TERTIODODECYLMERCAPTAN induced contact hypersensitivity in the murine Local Lymph Node Assay.
According to the EC3 value of 31%, it was a weak sensitizer.
Executive summary:

The potential of the test item, TERTIODODECYLMERCAPTAN, to induce contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). This study was conducted in compliance with the principles of Good Laboratory Practice.

 

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

In the main test, 28 female CBA/J mice were allocated to 7 groups of 4 animals:

.            five treated groups receiving the test item at the concentration of 5, 10, 25, 50 or 100% in a mixture acetone/olive oil (4/1; v/v) (vehicle),

.            one negative control group receiving the vehicle,

.            one positive control group receiving the positive control, a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in a mixture acetone/olive oil (4/1; v/v).

 

During the induction phase, the test item, vehicle or positive control item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6. Following the solubility assay, a mixture acetone/olive oil (4/1; v/v) was chosen as vehicle. The concentrations selected for the preliminary test were 10, 25, 50 and 100%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (100%).

 

Neither mortality nor clinical signs were observed during the observation period. Dryness of the skin of the ear was noted on day 6 in all animals given 50 and 100%. In addition, erythema and scabs on ear were noted in 1/4 females given 100%. No notable increase in ear thickness was observed at any of the tested concentrations. As acceptance criteria were met, this experiment was therefore considered valid.

 

The results are presented in the following table:

 

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

5

non-irritant

0.18

Test item

10

non-irritant

0.64

Test item

25

non-irritant

2.11

Test item

50

non-irritant

5.94

Test item

100

slightly irritant

4.71

HCA

25

-

3.15

A significant lymphoproliferation (SI > 3) was noted at concentrations=50% with a clear evidence of a dose-response relationship between all concentrations. In the absence of excessive local irritation, the significant lymphoproliferative responses were attributed to delayed contact hypersensibility. The EC3value is equal to 31%.

The test item, TERTIODODECYLMERCAPTAN, induced contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3value obtained, the test item should be considered as a weak sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
A positive control was not tested but the test substance was a skin sensitizer in this test.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed before the publication of the REACH regulation
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lebeau breeding centre (78950 Gambais, France)
- Age at study initiation: no data
- Weight at study initiation: 328 +/-18 g for the males and 314 +/- 10 g for the females
- Housing: individually in polycarbonate cages
- Diet (e.g. ad libitum): Guinea-pigs sustenance ref. 106 (UAR, 91360 Villemoisson/Orge, France)
- Water (e.g. ad libitum): filtered tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
25%
Day(s)/duration:
D1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
D8
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
D22
No. of animals per dose:
Thirty guinea-pigs (15 males and 15 females) were allocated to 2 groups: a control group (5 males and 5 females) and a treated group (10 males and 10 females)
Details on study design:
The sensitization potential of TPS 44 was evaluated after a 10-day induction period during which the animals were treated with the vehicle (control group) or the test substance (treated group). On day 1, in the presence of Freund's adjuvant, 0.1 ml of the test substance was administered by intradermal route at a concentration of 25% in an injectable isotonic solution of 0.9% NaCl. On day 8, 0.5 ml of the test substance in its original form was applied by cutaneous route. After a period of 12 days without treatment, a challenge cutaneous application of 0.5 ml of the vehicle (left flank) and 0.5 ml of the test substance in its original form (right flank) were then performed on all animals. The substances were prepared on a dry compress, then applied to the skin and held in place for 24 hours by means of an occlusive dressing. The cutaneous reactions were then evaluated at the challenge application site, 24 and 48 hours after removal of the dressing. No histological examination was performed on the cutaneous samples.
Positive control substance(s):
no
Positive control results:
Not tested but the test substance was a skin sensitizer in this test.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
14
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
9
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
other: Not tested but the test substance was a skin sensitizer in this test.
During the study, no clinical signs were observed and no
death occurred. The body weight of the animals of the
treated group was unaffected when compared to that of the
control group.

After the challenge application of the test substance, no
cutaneous reactions were observed in the animals of the
control group. A positive response characterised by a
well-defined erythema (score of 2) was observed on the right
flank of 9 treated animals after 24 and 48 hours. No oedema
was noted. The reactions noted in 10 animals (very slight or
well-defined erythema after 24 hours and very slight
erythema after 48 hours) were considered to be inconclusive
evidence of well-defined sensitization and were considered
to be due to a slight sensitization effect.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
TPS 44 induced a positive sensitization reaction in 9 out of 20 guinea-pigs (45%).
Executive summary:

The sensitization potential of di-tert-butyl polysulfides (TPS 44) was evaluated in guinea-pigs by intradermal injection and cutaneous application, according to the maximization method of Magnusson and Kligman, the O.E.C.D. Guideline No. 406 and GLP. Thirty guinea-pigs (15 males and 15 females) were allocated to 2 groups: a control group (5 males and 5 females) and a treated group (10 males and 10 females). The sensitization potential of TPS 44 was evaluated after a 10-day induction period during which the animals were treated with the vehicle (control group) or the test substance (treated group). On day 1, in the presence of Freund's adjuvant, 0.1 ml of the test substance was administered by intradermal route at a concentration of 25% in an injectable isotonic solution of 0.9% NaCl. On day 8, 0.5 ml of the test substance in its original form was applied by cutaneous route. After a period of 12 days without treatment, a challenge cutaneous application of 0.5 ml of the vehicle (left flank) and 0.5 ml of the test substance in its original form (right flank) were then performed on all animals. The substances were prepared on a dry compress, then applied to the skin and held in place for 24 hours by means of an occlusive dressing. The cutaneous reactions were then evaluated at the challenge application site, 24 and 48 hours after removal of the dressing. No histological examination was performed on the cutaneous samples. During the study, no clinical signs were observed and no death occurred. The body weight of the animals of the treated group was unaffected when compared to that of the control group. After the challenge application of TPS 44, no cutaneous reactions were observed in the animals of the control group. A positive response characterised by a well-defined erythema (score of 2) was observed on the right flank of 9 treated animals after 24 and 48 hours. No oedema was noted. The reactions noted in 10 animals (very slight or well-defined erythema after 24 hours and very slight erythema after 48 hours) were considered to be inconclusive evidence of well-defined sensitization and were considered to be due to a slight sensitization effect. TPS 44 induced a positive sensitization reaction in 9 out of 20 guinea-pigs (45%).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In an OECD TG 406 study, Dunkin-Hartley guinea pigs (5/sex/control group; 10/sex/treated group) were tested for skin sensitization to polysulfides, di-tert-dodecyl using the guinea pig maximization test (Guillot, 1986). Induction was accomplished by intracutaneous exposure to 1% of polysulfides, di-tert-dodecyl followed by an epicutaneous occlusive application of undiluted polysulfides, di-tert-dodecyl. During the challenge exposure at 24 hours, a 0.5 ml topical occlusive application of polysulfides, di-tert-dodecyl was carried out with the test article as supplied. From the macroscopic and histological results, it was concluded that polysulfides, di-tert-dodecyl was a weak irritant in four of 20 treated guinea pigs, which could mask possible weak reactions of cutaneous sensitization. No characteristic cutaneous abnormalities were noted in the 10 control guinea pigs.


Considering that this study was insufficient to definitively conclude about the skin sensitization potential of the registered substance, a wider evaluation based on a weight of evidence was performed using studies carried out on similar products, di-tert-dodecyl disulfide and di-tert-butyl polysulfide and its raw material and potential metabolite, tert-dodecyl mercaptan.


 


- Di-tert-dodecyl disulfide


Di-tert-dodecyl disulfide have the same alkyl chains than the registered substance linked by a disulfide bridge intead a polysulfide bridge. Di-tert-dodecyl disulfide is also a component of the registered substance. The registered substance and di-tert-dodecyl disulfide generate a common metabolite, tert-dodecyl mercaptan, after a step of desulphuration.


The skin sensitisation potential of di-tert-dodecyl disulfide was evaluated in an OECD Guideline no. 429 study (Váliczkó, 2016). The test item was tested for formulation compatibility in acetone:olive oil 4:1 (v:v) mixture (abbreviated as AOO). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (undiluted). The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 100 % (undiluted) and 50 % (w/v) in AOO. Based on the observations recorded in the preliminary test, the 100 % (undiluted) was selected as top dose for the main test. In the main assay, twenty-eight female CBA/CaOlaHsd mice were allocated to seven groups of four animals each: five groups received di-tert-dodecyl disulphide (formulated in AOO) at 100 % (undiluted), 50, 25, 10 and 2 % (w/v) concentrations, the negative control group received the vehicle (AOO), the positive control group received 25 % (w/v) HCA (dissolved in AOO).


The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).


No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. No marked body weight loss (=5%) was observed on the mean body weight of the groups in the study. The stimulation index values were 3.1, 3.2, 1.7, 1.4 and 0.9 at concentrations of 100 % (undiluted), 50, 25, 10 and 2 % (w/v), respectively. The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay. 


In conclusion, di-tert-dodecyl disulfide tested in a suitable vehicle, was shown to have a slight sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value was 46.7 % (w/v). Di-tert-dodecyl disulfide was classified as a skin sensitizer 1B.


 


- Di-tert-butyl polysulfide


Di-tert-butyl disulfide have shorter alkyl chains than the registered substance but the same polysulfide bridge.


The sensitization potential of di-tert-butyl polysulfides (TPS 44) was evaluated in guinea-pigs by intradermal injection and cutaneous application, according to the maximization method of Magnusson and Kligman, the O.E.C.D. Guideline No. 406 and GLP (Clouzeau, 1992). Thirty guinea-pigs (15 males and 15 females) were allocated to 2 groups: a control group (5 males and 5 females) and a treated group (10 males and 10 females). The sensitization potential of TPS 44 was evaluated after a 10-day induction period during which the animals were treated with the vehicle (control group) or the test substance (treated group). On day 1, in the presence of Freund's adjuvant, 0.1 ml of the test substance was administered by intradermal route at a concentration of 25% in an injectable isotonic solution of 0.9% NaCl. On day 8, 0.5 ml of the test substance in its original form was applied by cutaneous route. After a period of 12 days without treatment, a challenge cutaneous application of 0.5 ml of the vehicle (left flank) and 0.5 ml of the test substance in its original form (right flank) were then performed on all animals. The substances were prepared on a dry compress, then applied to the skin and held in place for 24 hours by means of an occlusive dressing. The cutaneous reactions were then evaluated at the challenge application site, 24 and 48 hours after removal of the dressing. No histological examination was performed on the cutaneous samples. During the study, no clinical signs were observed and no death occurred. The body weight of the animals of the treated group was unaffected when compared to that of the control group. After the challenge application of TPS 44, no cutaneous reactions were observed in the animals of the control group. A positive response characterised by a well-defined erythema (score of 2) was observed on the right flank of 9 treated animals after 24 and 48 hours. No oedema was noted. The reactions noted in 10 animals (very slight or well-defined erythema after 24 hours and very slight erythema after 48 hours) were considered to be inconclusive evidence of well-defined sensitization and were considered to be due to a slight sensitization effect. TPS 44 induced a positive sensitization reaction in 9 out of 20 guinea-pigs (45%). Di-tert-butyl polysulphide was classified as a skin sensitizer 1B.


 


- Tert dodecyl mercaptan


Tert dodecyl mercaptan is the raw material used to synthetize the registered substance and also a potential degradation product during metabolisation due to clivage of the di- and polysulfide bridge.


The potential of tert dodecyl mercaptan to induce contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). This study was conducted in compliance with the principles of Good Laboratory Practice.


A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.


In the main test, 28 female CBA/J mice were allocated to 7 groups of 4 animals:  five treated groups receiving the test item at the concentration of 5, 10, 25, 50 or 100% in a mixture acetone/olive oil (4/1; v/v) (vehicle), one negative control group receiving the vehicle, one positive control group receiving the positive control, a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%in a mixture acetone/olive oil (4/1; v/v).


During the induction phase, the test item, vehicle or positive control item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6. Following the solubility assay, a mixture acetone/olive oil (4/1; v/v) was chosen as vehicle. The concentrations selected for the preliminary test were 10, 25, 50 and 100%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (100%).


Neither mortality nor clinical signs were observed during the observation period. Dryness of the skin of the ear was noted on day 6 in all animals given 50 and 100%. In addition, erythema and scabs on ear were noted in 1/4 females given 100%. No notable increase in ear thickness was observed at any of the tested concentrations. As acceptance criteria were met, this experiment was therefore considered valid.


A significant lymphoproliferation (SI > 3) was noted at concentrations=50% with a clear evidence of a dose-response relationship between all concentrations. The stimulation index values were 4.71, 5.94, 2.11, 0.64 and 0.18 at concentrations of 100 % (undiluted), 50, 25, 10 and 5 % (w/v), respectively. The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. In the absence of excessive local irritation, the significant lymphoproliferative responses were attributed to delayed contact hypersensibility. The EC3value is equal to 31%. Tert-dodecyl mercaptan was classified as a skin sensitizer 1B.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the available data and criteria of Regulation no. 1272/2008, polysulfides, di-tert-dodecyl must be classified as skin sensitizer cat. 1B.