Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Based on the recent LLNA study, di-tert-dodecyl polysulfide is a weak sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21 Sep 2021 to 27 Oct 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Remarks:
Inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approximately 10 weeks old)
- Weight at study initiation: 21.2 to 25.5 g
- Housing: Up to 5 animals of the same dosing group together in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material equipped with water bottles. Animals were separated during designated procedures/activities. For psychological/environmental enrichment, animals were provided with paper and shelters (disposable paper corner home), except when interrupted by study procedures/activities.
- Diet: Pelleted rodent diet (SM R/M-Z) was provided ad libitum throughout the study, except during designated procedures.
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 (actual daily mean: 20)
- Humidity (%): 40-70 (actual daily mean: 44-65)
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 06 Oct 2021 To: 25 Oct 2021
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 10, 25 and 50 %;
Dose concentrations used are in compliance with the OECD test guidelines for LLNA studies.
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
Two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days.
- Compound solubility: Not indicated
- Irritation: Animals were observed for signs of irritation
- Systemic toxicity: Animals were observed for signs of systemic toxicity
- Ear thickness measurements: Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.

MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: If the results indicate a SI = 3, the test item may be regarded as a skin sensitizer. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures, including all amendments. The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation.

TREATMENT PREPARATION AND ADMINISTRATION:
- Preparation of Test Item: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels. Any residual volumes were discarded.
- Induction (Days 1, 2 and 3): The dorsal surface of both ears was topically treated (25 µL/ear) with the test item, at approximately the same time on each day. The formulations were stirred with a magnetic stirrer until dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Excision of the Nodes (Day 6): Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine. After five hours, all animals were euthanized. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

ANALYSIS
- Tissue Processing for Radioactivity (Day 6): Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
- Radioactivity Measurements (Day 7): Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid. Radioactivity measurements were performed using a scintillation counter. Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
- A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The SI values calculated for the positive control concentrations 5, 10 and 25% were 1.2, 2.2 and 5.6, respectively. An EC3 value of 13.5% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 16.3, 12.8, 9.0, 10.9 and 8.0%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at the testing laboratory is an appropriate model for testing for contact hypersensitivity.
Parameter:
SI
Value:
1.6
Test group / Remarks:
Dose of 10%
Parameter:
SI
Value:
2.5
Test group / Remarks:
Dose of 25%
Key result
Parameter:
SI
Value:
4.4
Test group / Remarks:
Dose of 50%
Key result
Parameter:
EC3
Value:
32
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.

DETAILS ON STIMULATION INDEX CALCULATION: Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 841, 1363 and 2331 DPM, respectively. The mean DPM/animal value for the vehicle control group was 535 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.6, 2.5 and 4.4, respectively.

EC3 CALCULATION: The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 32% was calculated.

CLINICAL OBSERVATIONS: No mortality occurred. No clinical signs of systemic toxicity were observed in the animals, with the exception of one animal that showed partly closed eyes on Day 4. This was considered not to have affected the results as this animal did not respond differently compared to the other animals of this group and the clinical sign was only seen on one day.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

SIGNS OF TOXICITY: The very slight irritation of the ears as shown by all animals treated at 50% on Day 4 was considered not to have a toxicologically significant effect on the activity of the nodes.

Pre - Screen Test


At 100%, only very slight irritation of the ears was noted between Days 3 and 5. However, clinical signs of systemic toxicity (hunched posture, partly closed eyes) were also noted between Days 2 and 5 and therefore this concentration did not meet the selection criteria for the main study. At 50%, no signs of systemic toxicity were noted, and only very slight irritation of the ears were observed between Days 3 to 5. Therefore, this concentration was selected as highest concentration for the main study. All animals showed bald spots on the head on Day 4, but this was considered not toxicologically relevant. 


Table 1. Pre-Screen Test: Body Weights and Skin Reactions








































































































































































TI 1   (%)



Animal



 



Day 1



 



Day 2



 



Day 3



 



Day 4



 



Day 5



 



Day 6



 



 



 



 



bw


(g) 2



Erythema3



 



Erythema



 



Erythema



 



Erythema



 



Erythema



 



Erythema



 



bw


(g)



 



 



 



left



right



left



right



left



right



left



right



left



right



left



right



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



50



81



24.7



0



0



0



0



1



1



1



1 k



1



1



0



0



25.6



 



82



25.6



0



0



0



0



1



1



1



1 k



1



1



0



0



25.7



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



100



83



27.0



0



0



0



0 og



1



1 og



1



1 k



1



1 og



1



1



27.3



 



84



24.1



0



0



0



0 og



1



1 og



1



1 k



1



1



0



0



23.6




  1. partly closed eyes, g. hunched posture, k. bald spots on the head


1   TI = test item (% w/w).


2   Body weight (grams).


3   Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear):


    0 = No erythema


    1 = Very slight erythema (barely perceptible)


 


Table 2. Pre-Screen Test: Ear Thickness Measurements




















































































































































TI 1  (%)



Animal



Day 1



 



Day 3



 



 



 



Day 6



 



 



 


  

Left



Right



Left



 



Right



 



Left



 



Right



 



 



 



(mm)



(mm)



(mm)



% 2



(mm)



% 2



(mm)



% 2



(mm)



% 2



 



 



 



 



 



 



 



 



 



 



 



 



50



81



0.225



0.230



0.230



2



0.240



4



0.225



0



0.230



0



 



82



0.220



0.230



0.230



5



0.235



2



0.220



0



0.225



-2



 



 



 



 



 



 



 



 



 



 



 



 



100



83



0.225



0.225



0.230



2



0.225



0



0.225



0



0.230



2



 



84



0.220



0.225



0.225



2



0.230



2



0.230



5



0.225



0


              

Left (mm) = thickness of left ear in millimeters; right (mm) = thickness of right ear in millimeters.


1   TI = test item (% w/w).


2   Percent increase compared to Day 1 pre-dose value. A 25% value is used as the threshold for selection for  use in the main study.


Table 3. Main Study: Body Weights and Skin Reactions


























































































































































































































































































































































































































































































































































Group



TI (%)



Animal



 



Day 1



 



Day 2



 



Day 3



 



Day 4



 



Day 5



 



Day 6



 



 



 



 



 



bw


(g) 2



Erythema3



 



Erythema



 



Erythema



 



Erythema



 



Erythema



 



Erythema



 



bw


(g)



 



 



 



 



left



right



left



right



left



right



left



right



left



right



left



right



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



1



0



1



25.5



0



0



0



0



0



0



0



0



0



0



0



0



26.7



 



 



2



22.9



0



0



0



0



0



0



0



0



0



0



0



0



22.3



 



 



3



22.7



0



0



0



0



0



0



0



0



0



0



0



0



22.7



 



 



4



23.7



0



0



0



0



0



0



0



0



0



0



0



0



23.7



 



 



5



22.4



0



0



0



0



0



0



0



0



0



0



0



0



24.5



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



2



10



6



22.9



0



0



0



0



0



0



0



0



0



0



0



0



23.1



 



 



7



23.6



0



0



0



0



0



0



0



0



0



0



0



0



24.6



 



 



8



24.3



0



0



0



0



0



0



0



0



0



0



0



0



26.0



 



 



9



22.9



0



0



0



0



0



0



0



0



0



0



0



0



23.9



 



 



10



23.8



0



0



0



0



0



0



0



0



0



0



0



0



23.6



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



3



25



11



22.7



0



0



0



0



0



0



0



0



0



0



0



0



23.8



 



 



12



24.3



0



0



0



0



0



0



0



0



0



0



0



0



26.3



 



 



13



23.9



0



0



0



0



0



0



0



0



0



0



0



0



26.0



 



 



14



21.4



0



0



0



0



0



0



0



0



0



0



0



0



22.1



 



 



15



23.2



0



0



0



0



0



0



0



0



0



0



0



0



24.4



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



4



50



16



21.4



0



0



0



0



0



0



1



1



0



0



0



0



21.9



 



 



17



24.0



0



0



0



0



0



0



1



1



0



0



0



0



26.0



 



 



18



22.5



0



0



0



0



0



0



1



1



0



0



0



0



23.4



 



 



19



23.4



0



0



0



0



0



0



1a



1a



0



0



0



0



24.0



 



 



20



21.2



0



0



0



0



0



0



1



1



0



0



0



0



23.0



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



a = partly closed eyes


1   TI = test item (% w/w).


2   Body weight (grams).


3   Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear):


    0 = No erythema


    1 = Very slight erythema (barely perceptible)


Table 4. Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)




































































































































































































































































































































































Group



TI 1 (%)



Animal



Size Nodes 2



 



DPM 3 / Animal



Mean


DPM ± SEM 4



 



 



Mean


SI ± SEM



 



 



 



 



 



left



right



 



 



 



 



 



 



 



1



0



1



n



n



508



 



 



 



 



 



 



 



 



2



n



n



470



 



 



 



 



 



 



 



 



3



n



n



677



 



 



 



 



 



 



 



 



4



n



n



494



 



 



 



 



 



 



 



 



5



n



n



526



535



±



37



1.0



±



0.1



 



 



 



 



 



 



 



 



 



 



 



 



2



10



6



n



n



992



 



 



 



 



 



 



 



 



7



n



n



572



 



 



 



 



 



 



 



 



8



n



n



440



 



 



 



 



 



 



 



 



9



n



n



1094



 



 



 



 



 



 



 



 



10



n



n



1105



841



±



140



1.6



±



0.3



 



 



 



 



 



 



 



 



 



 



 



 



3



25



11



n



n



1563



 



 



 



 



 



 



 



 



12



n



n



1438



 



 



 



 



 



 



 



 



13



n



n



1183



 



 



 



 



 



 



 



 



14



n



n



1696



 



 



 



 



 



 



 



 



15



n



n



936



1363



±



136



2.5



±



0.3



 



 



 



 



 



 



 



 



 



 



 



 



4



50



16



n



n



3388



 



 



 



 



 



 



 



 



17



n



n



1950



 



 



 



 



 



 



 



 



18



n



n



1232



 



 



 



 



 



 



 



 



19



n



n



2364



 



 



 



 



 



 



 



 



20



n



n



2723



2331



±



362



4.4



±



0.7



1   TI = test item (% w/w).


2   Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal).


3     DPM = Disintegrations per minute.


4     SEM = Standard Error of the Mean.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The results indicate that the test item could elicit a SI = 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 32% was calculated. The substance should be classified as skin sensitizer.
Executive summary:

In a GLP compliant Local Lymph Node Assay, performed in accordance with OECD guideline 429, the potential to induce skin sensitization in mice after three epidermal exposures of the animals was evaluated. The six-month reliability check with Alpha-Hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the testing laboratory is an appropriate model for testing for contact hypersensitivity. Test item concentrations selected for the main study were based on the results of a pre-screen test. At 100%, partly closed eyes and hunched posture were noted and therefore this concentration did not meet the selection criteria for the main study. At 50%, no clinical signs were noted and only very slight irritation of the ears was observed and therefore this concentration was selected as highest concentration for the main study.


In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v) (AcOO)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.


All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.


Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 841, 1363 and 2331 DPM, respectively. The mean DPM/animal value for the vehicle control group was 535 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.6, 2.5 and 4.4, respectively.


These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 32% was calculated. According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), The substance should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

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

In a GLP compliant Local Lymph Node Assay (CRL 2021), performed in accordance with OECD guideline 429, the potential to induce skin sensitization in mice after three epidermal exposures of the animals was evaluated. The six-month reliability check with Alpha-Hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the testing laboratory is an appropriate model for testing for contact hypersensitivity. Test item concentrations selected for the main study were based on the results of a pre-screen test. At 100%, partly closed eyes and hunched posture were noted and therefore this concentration did not meet the selection criteria for the main study. At 50%, no clinical signs were noted and only very slight irritation of the ears was observed and therefore this concentration was selected as highest concentration for the main study.


In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v) (AcOO)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.


All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.


Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 841, 1363 and 2331 DPM, respectively. The mean DPM/animal value for the vehicle control group was 535 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.6, 2.5 and 4.4, respectively.


These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 32% was calculated. The substance should be classified as skin sensitizer.


 


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.

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.


Justification : the EC3 in the LLNA is of 32%.

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