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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name: Dimethyl Sulphide
- Synonyms: DMS, dimethyl sulfure, sulfure de dimethyle, dimethyl sulfide
- Batch number: C8753
- Description: Colorless liquid
- Storage condition: At room temperature
- Purity: 99.7%
- Purity test date: 09/01/2015
- Expiration date of the lot/batch: 08 April 2017

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 12 weeks old
- Weight at study initiation: 18.2 g to 20.9 g
- Housing:group of two (preliminary test) or four (main test) in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted maintenance diet, ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 2
- Humidity (%):30 to 70
- Air changes (per hr):12
- Photoperiod (hrs dark / hrs light):12h/12h

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100%, 50%, 25%, 10% and 5%
No. of animals per dose:
5
Details on study design:
TREATMENT
Rationale for concentration selection
The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
the vehicle was selected on the basis of producing a homogeneous preparation suitable for application of the test item,
the concentrations were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc,
the maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an increase of the ear thickness = 25%.

Preliminary test
Treatment groups
To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was performed in four animals.

The treatment groups for the preliminary test are detailed in the following table:

Group Number of animals Concentration (%)
Left ear Right ear
1 2 females 25 100
2 2 females 10 50

Application
On Days 1, 2 and 3, a dose-volume of 25 µL of the appropriate dose formulation preparation was applied to the dorsal surface of both ears (one concentration per ear), using an adjustable pipette fitted with a plastic tip.
In order to avoid licking, to ensure an optimized application of the test materials and to facilitate ear thickness measurement, the animals were placed under light isoflurane anesthesia during the administration.
No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.
As the dose formulations were solutions, they were stirred manually before the dosing procedure.

Main test
Treatment groups
The treatment groups for the main test are detailed in the following table:

Group Number of animals
Treatment Concentration (%)
3 4 females Vehicle 0
4 4 females Test item 5
5 4 females Test item 10
6 4 females Test item 25
7 4 females Test item 50
8 4 females Test item 100
9 4 females 25% of HCA

Application
On Days 1, 2 and 3, a dose-volume of 25 µL of the control or dose formulation preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.
As the dose formulations were solutions, they were stirred manually before the dosing procedure.

CLINICAL EXAMINATIONS
Morbidity and mortality
Each animal was checked for mortality and morbidity once a day during the acclimation, treatment and observation periods, including weekends and public holidays.

Data from mortality observations collected during the acclimation phase are not presented in the study report as no mortality and no clinical signs occurred during this period.

Clinical signs
Each animal was observed once a day from Day 1 to Day 6 (at approximately the same time each day) for the recording of clinical signs.

Body weight
The body weight of each animal was recorded once during the acclimation period, and then on the first day of treatment and on the day of sacrifice.

Ear thickness measurements and recording of local reactions
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as potent irritants may yield positive lymphoproliferative responses of equivocal significance in the LLNA.

On Days 1, 2 and 3 (before each cutaneous application) and on Day 6 (immediately after sacrifice), the thickness of both ears of each preliminary test animal and the left ear of each main test animal was measured, using a micrometer.
The increase in ear thickness was expressed as percentages between Day 1 and Day 3, and between Day 1 and Day 6, and was used to determine the irritation level due to the test item application.

No measurement of ear thickness was performed for the main test animals of the positive control group.

The irritation level of the test item was determined according to the following table:

% increase in ear thickness Irritation level Interpretation
< 10% I Non-irritant
= 10 - < 25% II Slightly irritant
= 25% III Irritant

AURICULAR LYMPH NODE CELL PROLIFERATION ASSAY
Intravenous injection of 3H-TdR and sampling of auricular lymph nodes
Lymph node cell proliferative response was measured as described by Kimber and Dearman (1). On Day 6 of the main test, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 to 21 µCi of 3H-TdR (specific activity of 20 Ci/mmol) via the tail vein.
In the following 5 h to 5 h 30 min, the main test animals were sacrificed by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the Auricular Lymph Nodes (ALN) were excised. Two ALN were collected per animal. ALN were then pooled for each experimental group before processing.
A single cell suspension of ALN was prepared, for each group, by mechanical disaggregation in Petri dishes using the plunger of a syringe.

Preparation of auricular lymph node cell suspensions and determination of proliferation
Cell suspensions were washed once with 15 mL of 0.9% NaCl. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic (TCA) at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three milliliters of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using ¿-scintillation counting.
The results are expressed as disintegrations per minute (dpm) per group and as dpm/node.

Stimulation Indices (SI) were calculated according to the following formula:

SI = dpm per node of the treated group / dpm per node of the vehicle control group

Acceptance criteria
The study is considered valid if the SI for the positive control is higher than the threshold positive value of 3.

Evaluation of results
The test item is considered 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 chemical toxicity, radioactivity levels and ear thickness are also taken into account to evaluate the data.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
SI = 8.64

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: 5 % = 0.66 10 % = 1.03 25 % = 1.88 50% = 0.99 100% = 1.68

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Dimethyl Sulphide, tested as a solution after preparation at concentrations of 5, 10, 25, 50 and 100% in Acetone/Olive Oil (4/1; v/v), gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.
Executive summary:

The potential of Dimethyl Sulphide to induce contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). This study was conducted according to the OECD test guideline no. 429. A test to select the vehicle was conducted to ensure solubility of the test item in standard vehicles used in LLNA. Then, to assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item, by topical route to the dorsal surface of both ears (one concentration per ear) on Days 1, 2 and 3 at concentrations of 10, 25, 50 or 100% under a dose-volume of 25 µL. From Day 1 to Day 3 then on Day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and then on Days 1 and 6. On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination. In the main test, five groups of four female mice received the test item by topical route to the dorsal surface of both ears on Days 1, 2 and 3 at concentrations of 5, 10, 25, 50 or 100% under a dose-volume of 25 µL. One negative control group of four females received the vehicle (Acetone/Olive Oil (4/1; v/v)) under the same experimental conditions. Additionally, one positive control group of four females received the positive control, a-hexylcinnamaldehyde (HCA), at 25%in a mixture Acetone/Olive Oil (4/1; v/v) under the same experimental conditions. From Day 1 to Day 3 then on Day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on Days 1 and 6. After 2 days of resting, on Day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-TdR. The results are expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

A solution was obtained at the highest concentration of 50% in Acetone/Olive Oil (4/1; v/v). No unscheduled deaths and no clinical signs were observed during the observation period. Body weight of animals was unaffected by the test item treatment. No local reactions were observed in any animals. In all test item-treated groups, the mean increase in ear thickness was below 10%. The test item was therefore considered as non-irritant. As the acceptance limit of 25% was not reached at any concentrations, no bias in the results of proliferation measurements linked to an excessive irritant potential was considered.The SI of the positive control was > 3; this experiment was therefore considered valid. No significant lymphoproliferation was noted with the test item at any tested concentrations as all SI values were below the cut-off value of 3.

Dimethyl Sulphidegave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.