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Description of key information

LLNA assay

The potential of tert-dodecanethiol, to induce contact hypersensitivity, was evaluated in the murine LLNA guideline study [OECD TG 429] (Gerbeix, 2011). A preliminary test was first performed in order to define the concentrations of test item to be used in the main test. 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%). 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. 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. The SI for the5, 10, 25, 50 and 100%groups were 0.18, 0.64, 2.11, 5.94 and 4.71, respectively. The threshold positive value of 3 for the SI was exceeded in the positive control group (SI = 3.15). This experiment was therefore considered valid. A dose-related increase in the SI was recorded 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 hypersensitivity. The calculated EC3 value was 31%. Under the experimental conditions of this study, tert-dodecanethiol induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. Based on the EC3value obtained, tert-dodecanethiol should be considered a weak sensitizer.

 

Buehler assay

In a guideline study [OECD TG 406], Hartley guinea pigs (5/sex/group) were treated with a 0.5 mL induction dose (15% occlusive epicutaneous) of tert-dodecanethiol in acetone, or a known sensitiser applied to the back (Pence, 1983). The animals were exposed for 6 hours, once a week, for 3 weeks. Two control groups (5/sex/group) were not exposed during this induction period. Two weeks following the application of the last induction patch, a 0.5 mL challenge dose oft-dodecyl mercaptan in acetone (5% occlusive epicutaneous) or a known sensitizer were applied to the test and control animals for a 4 to 6 hour exposure period. A depilatory agent was applied to this area the following day and washed off. The challenge was scored for erythema and edema at 24, 48, and 72 hours following the challenge dose. Based on the response of one test animal, tert-dodecanethiol was considered to be a weak dermal sensitiser in guinea pigs (Hazleton Laboratories, Inc., 1983).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 May 2011 - 17 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study (report not available for review)
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
not specified
Type of study:
Buehler test
Species:
guinea pig
Strain:
Hartley
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
15%
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
5%
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10
Details on study design:
Adult guinea pigs of the Hartley strain weighing 300-500 g were used as  the experimental  animals. Preliminary tests established the induction  dose level (producing very slight irritation) and the challenge dose  level (highest non irritating concentration) of the test material to be  15% and 5% respectively. The carrier vehicle used for tertiary dodecyl  mercaptan was acetone. Four groups each containing five male and five  female guinea pigs were used in this study. One group received induction  and challenge doses of a known sensitizer, dinitrochlorobenzene (DNCB). A  second group served as a control, and received only the challenge dose of  the known sensitizer. The remaining two groups were set up similarly,  however, the test material replaced the known sensitizer. Two groups of  guinea pigs were clipped free of hair in the upper left quadrant of their  backs. A 0.5 ml induction dose of test material or known sensitizer was  applied to the back and a patch was taped in place. Rubber damming was  then wrapped around the animal to secure the patch and the guinea pig was  restrained for 6 hours. This procedure was carried out once a week for  three weeks. The two control groups received no treatment during this  induction phase. Two weeks following the application of the last  induction patch, the lower left quadrant of both test control animals was  shaved. A 0.5 ml challenge dose was applied to the shaved back for a 4 to  6 hour exposure period. A depilatory agent was applied to this area the  following day and washed off. The challenge site was scored for erythema  and edema 24, 48 and 72 hours following the challenge dose according to  the system of Draize. The animals were sacrificed following the 72 hours  observation. A skin section from the challenge site was fixed in 10%  neutral buffered formalin for histopathologic evaluation.
Positive control substance(s):
yes
Remarks:
DNCB
Positive control results:
All ten DNCB-induced animals had very slight to well-defined erythema at  24 hours after DNCB challenge, while two of these animals had very slight  edema as well.  Edema was no longer evident at the 48-hour scoring  interval. Erythema, however, persisted in 80% of these animals, thus  satisfying the validation criteria regarding the reliability and sensitivity of the test system.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10

In this study the following results were noted. No deaths occurred and all animals appeared normal throughout the study. Guinea pigs receiving the test material, 5.0% tertiary dodecyl mercaptan (Sulfole 120) in acetone, during the challenge phase only exhibited no dermal irritation at 24, 48 or 72 hours and those animals exposed to the same challenge dose following the induction with 15% tertiary dodecyl mercaptan (Sulfole 120) in acetone and rest period exhibited well-defined erythema in one animal at 24 hours that subsided to very slight at 48 hours and cleared by 72 hours. Based on the response in this one animal, tertiary dodecyl mercaptan is considered a slight dermal sensitizer in guinea pigs.

Interpretation of results:
GHS criteria not met
Executive summary:

Adult guinea pigs of the Hartley strain weighing 300-500 g were used as the experimental animals. Preliminary tests established the induction dose level (producing very slight irritation) and the challenge dose level (highest non irritating concentration) of the test material to be 15% and 5% respectively. The carrier vehicle used for tertiary dodecyl mercaptan was acetone. Four groups each containing five male and five female guinea pigs were used in this study. One group received induction and challenge doses of a known sensitizer, dinitrochlorobenzene (DNCB). A second group served as a control, and received only the challenge dose of the known sensitizer. The remaining two groups were set up similarly, however, the test material replaced the known sensitizer.

Two groups of guinea pigs were clipped free of hair in the upper left quadrant of their backs. A 0.5 ml induction dose of test material or known sensitizer was applied to the back and a patch was taped in.place. Rubber damming was then wrapped around the animal to secure the patch and the guinea pig was restrained for 6 hours. This procedure was carried out once a week for three weeks. The two control groups received no treatment during this induction phase. Two weeks following the application of the last induction patch, the lower left quadrant of both test control animals was shaved. A 0.5 ml challenge dose was applied to the shaved back for a 4 to 6 hour exposure period. A depilatory agent was applied to this area the following day and washed off. The challenge site was scored for erythema and edema 24, 48 and 72 hours following the challenge dose according to the system of Draize*. The animals were sacrificed following the 72 hours observation. A skin section from the challenge site was fixed in 10% neutral buffered formalin for histopathologic evaluation.

In this study the following results were noted. No deaths occurred and all animals appeared normal throughout the study. Guinea pigs receiving the test material, 5.0% tertiary dodecyl mercaptan (Sulfole 120) in acetone, during the challenge phase only exhibited no dermal irritation at 24, 48 or 72 hours and those animals exposed to the same challenge dose following the induction with 15% tertiary dodecyl mercaptan (Sulfole 120) in acetone and rest period exhibited well-defined erythema in one animal at 24 hours that subsided to very slight at 48 hours and cleared by 72 hours. Based on the response in this one animal, tertiary dodecyl mercaptan is considered a slight dermal sensitizer in guinea pigs.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the available data, and CLP criteria: Skin sensitization cat. 1B.