Didodecyl 3,3'-thiodipropionate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-Mar-2022 to 14-Jun-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012 Part B. Skin Sensitization: Local Lymph Node Assay

Version / remarks:

July 2012

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Didodecyl 3,3'-thiodipropionate

EC Number:

204-614-1

EC Name:

Didodecyl 3,3'-thiodipropionate

Cas Number:

123-28-4

Molecular formula:

C30H58O4S

IUPAC Name:

didodecyl 3,3'-sulfanediyldipropanoate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF-Quality
- Age at study initiation: approximately 11 weeks old
- Weight at study initiation: 20.3 to 26.2 g
- Housing: up to 5 animals of the same sex and same dosing group together; polycarbonate cages containing sterilized wooden fibers as bedding material
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures
- Water (e.g. ad libitum): municipal tap-water was freely available to each animal via water bottles
- Acclimation period: at least 5 days before the commencement of dosing
- Indication of any skin lesions: Before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

- IN-LIFE DATES: From: 01-Mar-2022 To: 13-Jun-2022

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10, 25, 50% (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 50% was maximum technically achievable concentration, 25% was tested in addition
- animals: 2 females per dose
- Treatment: on three consecutive days
- Irritation: Very slight irritation was observed in both animals at 50% and in one animal at 25% on Days 2 and/or 3. White test item remnants were present on the dorsal surface of the ears of all animals on Days 1-3, which did not hamper scoring of the skin reactions.
- Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. No effect on body weight was observed in both animals treated at 50%. At 25%, slight to moderate body weight loss was observed in both animals, as there were no corroborative findings, it was considered that the results for these animals were not affected.
- Ear thickness measurements: At 25 and 50%, ear thickness did not exceed 25% on Day 3 or Day 6. Mean ear punch weight at 25 and 50% was 33.12 and 28.57 mg, respectively.
- Macroscopic examination and weight of lymph node: 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 lymph node weight at 25 and 50% was 3.78 and 4.87 mg, respectively.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Four groups of five animals were treated with one test material concentration or vehicle control per group (Group 1: Vehicle control, Group 2: 10% w/w, Group 3: 25% w/w, Group 4: 50% w/w). The highest test material concentration was selected from the pre-screen test.
Induction - Days 1, 2 and 3: Animals were treated with 25 µL/ear of either test material or vehicle. The formulations were stirred with a magnetic stirrer until dosing.
Excision of the Nodes - Day 6: i.v. injection of 20 μCi of 3H-methyl thymidine per animal in PBS. After five hours, all animals were euthanized and both ears (left and right) were punched in the apical area using a biopsy punch (Stiefel, Ø 8 mm => 0.5 cm²). Pool weights of biopsy punches were immediately determined for each animal. Both auricular draining lymph nodes (left and right) of the mice were 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. Pool weights of the lymph nodes were immediately determined for each animal.
Tissue Processing for Radioactivity - Day 6: A single cell suspension of lymph node cells (LNC) was prepared. 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: DNA precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid. Radioactivity measurements were performed , 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).
Clinical Observations: Post-dose observations: once daily on Days 1-6 (on Days 1-3 at least 3 hours after dosing). Ear thickness measurements: prior to dosing on Days 1 and 3, and on Day 6.
Body Weights: Animals were weighed individually on Day 1 (pre-dose) and 6 (prior to necropsy).
Irritation: observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing)

- Criteria used to consider a positive response:
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. If the results indicate a SI ≥ 3, the test material may be regarded as a skin sensitizer.


TREATMENT PREPARATION AND ADMINISTRATION:
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.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by the testing facility. In this study, performed in June 2022, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Alpha-Hexylcinnamaldehyde, technical grade (HCA). The females were approximately 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. SHBL6908 (Sigma-Aldrich, Steinheim, Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v; AcOO).
The SI values calculated for the material concentrations 5, 10 and 25% were 2.0, 5.2 and 7.8 respectively. An EC3 value of 8.4% 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 9.0, 10.9, 8.0, 13.5 and 12.2%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

control

Results 2

Parameter:

SI

Value:

1.4

Test group / Remarks:

10% test substance

Results 3

Parameter:

SI

Value:

1

Test group / Remarks:

25% test substance

Results 4

Parameter:

SI

Value:

0.8

Test group / Remarks:

50% test substance

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
Mean DPM (number of disintegrations per minute)/animal values for the experimental groups treated with test material concentrations at 10, 25 and 50% were 675, 454 and 376 DPM, respectively. The mean DPM/animal value for the vehicle control group was 475 DPM.

DETAILS ON STIMULATION INDEX CALCULATION:
The SI values calculated for the test material concentrations of 10, 25 and 50% were 1.4, 1.0 and 0.8, respectively.

EC3 CALCULATION:
There was no indication that the test material elicits a SI ≥ 3 when tested up to 50% (technically maximum achievable concentration).

BODY WEIGHTS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Mean body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Slight body weight loss was noted in two animals at 10%, one animal at 25%, and one animal of the control group, this body weight loss was considered not test material-related due to absence of a dose-related response.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness):
Irritation: Very slight irritation in all test item-treated animals between Days 1 - 3, (considered not to have a toxicologically significant effect on the activity of the nodes). White test item remnants were present on the dorsal surface of the ears of all animals at 50% on Days 2 and 3, which did not hamper scoring of the skin reactions.
Ear thickness and ear weight: did not exceed 25% on Day 3 or 6. Mean ear weight at 10, 25 and 50% were 30.19, 30.28, 31.04 mg, respectively. The mean ear weight for the vehicle control group was 29.54 mg. No test item-related effect was observed on ear thickness or ear weight.

Any other information on results incl. tables

Tabular Results of Main Study

Table 1: Body Weights and Skin Reactions

Group	TI ^1(%)	Animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
			bw	Erythema^3		Erythema^3		Erythema^3		Erythema^3		Erythema^3		Erythema^3		bw
			(g) ^2	left	right	left	right	left	right	left	right	left	right	left	right	(g) ^2
1	0	1	26.2	0	0	0	0	0	0	0	0	0	0	0	0	24.4
		2	23.6	0	0	0	0	0	0	0	0	0	0	0	0	23.7
		3	22.7	0	0	0	0	0	0	0	0	0	0	0	0	21.9
		4	22.8	0	0	0	0	0	0	0	0	0	0	0	0	22.2
		5	22.4	0	0	0	0	0	0	0	0	0	0	0	0	21.2
2	10	6	21.3	0	0	1	1	1	1	0	0	0	0	0	0	21.2
		7	24.4	0	0	1	1	1	1	0	0	0	0	0	0	23
		8	20.7	0	0	1	1	1	1	0	0	0	0	0	0	20.5
		9	24.4	0	0	1	1	1	1	0	0	0	0	0	0	22.8
		10	20.3	0	0	1	1	1	1	0	0	0	0	0	0	20.6
3	25	11	22.2	1	1	1	1	1	1	0	0	0	0	0	0	21.7
		12	22.4	1	1	1	1	1	1	0	0	0	0	0	0	22.1
		13	22.9	1	1	1	1	1	1	0	0	0	0	0	0	23.4
		14	25.6	1	1	1	1	1	1	0	0	0	0	0	0	24
		15	24.1	1	1	1	1	1	1	0	0	0	0	0	0	22.8
4	50	16	23	1	1	1f	1f	1f	1f	0	0	0	0	0	0	23.2
		17	24.4	1	1	1f	1f	1f	1f	0	0	0	0	0	0	24.6
		18	22.7	1	1	1f	1f	1f	1f	0	0	0	0	0	0	23.5
		19	25	1	1	1f	1f	1f	1f	0	0	0	0	0	0	23.8
		20	21.6	1	1	1f	1f	1f	1f	0	0	0	0	0	0	21.2

¹   TI = test material (% 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)

f   White staining of test item remnants on the dorsal surface of the ears did not hamper scoring of erythema.

 

Table 2: 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
0	1	0.22	0.22	0.22	0	0.22	0	0.22	0	0.22	0
	2	0.215	0.22	0.23	7	0.23	5	0.225	5	0.23	5
	3	0.23	0.23	0.22	-4	0.24	4	0.23	0	0.225	-2
	4	0.22	0.22	0.23	5	0.225	2	0.23	5	0.22	0
	5	0.22	0.215	0.215	-2	0.22	2	0.22	0	0.215	0
10	6	0.225	0.225	0.235	4	0.24	7	0.23	2	0.235	4
	7	0.22	0.22	0.23	5	0.235	7	0.23	5	0.225	2
	8	0.22	0.22	0.225	2	0.23	5	0.225	2	0.225	2
	9	0.215	0.215	0.24	12	0.24	12	0.22	2	0.23	7
	10	0.22	0.225	0.22	0	0.21	-7	0.22	0	0.215	-4
25	11	0.215	0.22	0.22	2	0.22	0	0.225	5	0.22	0
	12	0.23	0.225	0.235	2	0.23	2	0.23	0	0.225	0
	13	0.225	0.22	0.235	4	0.23	5	0.23	2	0.225	2
	14	0.22	0.22	0.23	5	0.225	2	0.23	5	0.23	5
	15	0.22	0.22	0.22	0	0.22	0	0.22	0	0.225	2
50	16	0.23	0.23	0.23	0	0.24	4	0.23	0	0.235	2
	17	0.225	0.225	0.235	4	0.23	2	0.23	2	0.23	2
	18	0.23	0.23	0.24	4	0.235	2	0.24	4	0.23	0
	19	0.22	0.22	0.23	5	0.225	2	0.225	2	0.225	2
	20	0.22	0.225	0.225	2	0.235	4	0.225	2	0.23	2

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: Ear Punch Weights, Lymph Node Weights, disintegration per minute and Stimulating Index

Test item (%)	Ear Punch Weights [mg]		Lymph Node Weights [mg]		DPM (disintegration per minute)		SI (Stimulating Index)
	Mean	SD	Mean	SD	Mean	SEM	Mean	SEM
0	29.54	1.16	4.42	0.61	475	132	1.0	0.3
10	30.19	1.46	4.59	0.85	675	185	1.4	0.4
25	30.28	1.13	4.57	1.04	454	257	1.0	0.5
50	31.04	1.48	3.89	0.57	376	19	0.8	0.0

 

Tabular Result of Positive Control

Group	% HCA	Mean
		DPM ± SEM	SI ± SEM
1	0%	344 ± 81	1.0 ± 0.2
2	5%	696 ± 307	2.0 ± 0.9
3	10%	1804 ± 515	5.2 ± 1.5
4	25%	2695 ± 554	7.8 ± 1.6

HCA = Alpha-Hexylcinnamaldehyde

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results and according to the recommendations made in the test guidelines, the test item is not be regarded as a skin sensitizer under the experimental conditions chosen.