Di-tert-dodecyl disulphide

Endpoint summary

Description of key information

The skin sensitisation potential of DI-TERT-DODECYL DISULPHIDE was evaluated following dermal exposure (Váliczkó, 2016). 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).

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:

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 conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: Category 1 (sub-category 1B).