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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
02 October 2012 - 15 February 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions therefore considered to be reliability 1. Read-across is considered to be scientifically valid and reliability 2.
Reference:
Composition 0
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 lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age/Weight at study initiation: on the beginning of the treatment period, the animals of the preliminary test were approximately 12 weeks old and had a mean body weight of 21.4 g (range: 20.3 g to 23.4 g), the animals of the first main test were approximately 8 weeks old and had a mean body weight of 20.2 g (range: 18.2 g to 22.3 g) and the animals of the second main test were approximately 8 weeks old and had a mean body weight of 18.3 g (range: 17.6 g to 19.4 g).
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 6 days before the beginning of the study.

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

IN-LIFE DATES: 10 October 2012 to 11 February 2013
No. of animals per dose:
Number:
- preliminary test: 4 nulliparous and non pregnant females,
- main tests:
- first main test: 28 nulliparous and non pregnant females,
- second main test: 20 nulliparous and non pregnant females.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The maximum concentration tested in the both main tests 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 should be 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% (when test item can be sampled by a pipette), 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 ≥ 25% increase of the ear thickness.

No. of animals per dose:
4 per dose.
Details on study design:
RANGE FINDING TESTS:
The maximum concentration tested in the both main tests 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 should be 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% (when test item can be sampled by a pipette), 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 ≥ 25% increase of the ear thickness.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test item was prepared at the chosen concentrations in the vehicle.
The positive control was dissolved in AOO at the concentration of 25% (v/v).
- Administration:
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form 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 sites. No rinsing was performed between each application.
Positive control substance(s):
other: α-hexyl cinnamaldehyde (HCA)
Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group (see the table below).
Parameter:
SI
Remarks on result:
other: EC3 = 75%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
First assay: DPM per node: Group 3, Vehicle: 377.50 Group 4, 2.5%: 242.25 Group 5, 5%: 188.63 Group 6, 10%: 194.13 Group 7, 25%: 279.25 Group 8, 50%: 230.13 Group 9, HCA: 2665.00 Seconde assay: DPM per node: Group 10, Vehicle: 184.25 Group 11, 50%:: 415.88 Group 12, 75%: 519.13 Group 13, 100%: 2240.13 Group 14, HCA: 1065.50

STIMULATION INDEXES

First main test

 

Treatment

Concentration
(%)

Irritation level

Stimulation Index
(SI)

Test item

2.5

I

0.64

Test item

5

I

0.50

Test item

10

I

0.51

Test item

25

I

0.74

Test item

50

I

0.61

HCA

25

-

7.06

-: not recorded.

I: non-irritant (increase in ear thickness < 10%).

Second main test

 

Treatment

Concentration
(%)

Irritation level

Stimulation Index
(SI)

Test item

50

I

2.26

Test item

75

I

2.82

Test item

100

I

12.16

HCA

25

-

5.78

-: not recorded.

I: non-irritant (increase in ear thickness < 10%).

 

Interpretation of results:
other: Sensitising cat. 1B
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Propane-2-thiol gave a positive result in the murine Local Lymph Node Assay, indicative of skin sensitization properties.
According to the EC3 of 75%, propane-2-thiol should be considered as a weak sensitizer (CiToxLAB, 2013). According to the criteria of CLP Regulation, the test item should be classified as skin sensitizer (category 1 and sub-category 1B) and assigned the signal word “warning” and the hazard statement “H317: May cause an allergic skin reaction”.

According to the criteria of CLP Regulation, the test item should be classified as skin sensitizer (category 1 and sub-category 1B) and assigned the signal word “warning” and the hazard statement “H317: May cause an allergic skin reaction”.

Executive summary:

Local Lymph Node Assay (LLNA) (CiToxLab, 2013). This study was conducted in compliance with the principles of Good Laboratory Practice. To assess the irritant potential of propane-2-thiol (through ear thickness measurement), a preliminary test was first performed in order to define the propane-2-thiol concentrations to be used in the main test. Two groups of two female mice received propane-2-thiol, by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at the concentrations of 5, 10, 25 or 50% 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 at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. On completion of the observation period, the animals were sacrificed then discarded without macroscopicpost-mortemexamination. In the first main test, five groups of four female mice received propane-2-thiol by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 2.5, 5, 10, 25 or 50% under a dose‑volume of 25 µL. In the second main test, three groups of four female mice received propane-2-thiol by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 50, 75 or 100% under a dose‑volume of 25 µL. In both tests, 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,α‑hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions. For each main test, 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 groups, and the local reactions were recorded. Each animal was observed at least 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 of3H-TdR. The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI). No unscheduled deaths and no clinical signs indicative of systemic toxicity were observed during the observation period of both main tests. Body weight of animals was unaffected by propane-2-thiol treatment. No local reactions were observed in any animals of both main tests. No notable increase in ear thickness was observed at any tested concentrations. The SI of the positive control of both main tests was > 3; these main tests were therefore considered valid. No notable lymphoproliferation was noted with propane-2-thiol at any tested concentrations in the first main test. In the second main test, a dose-related increase in the SI was recorded at concentrations ≥ 50%, and the threshold positive value of 3 was exceeded at the concentration of 100%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. Based on the results of the second main test, the EC3value is equal to 75.5%.

Propane-2-thiol gave a positive result in the murine Local Lymph Node Assay, indicative of skin sensitization properties. According to the EC3value obtained, propane-2-thiol should be considered as a weak sensitizer.

 

First main test

Treatment

Concentration (%)

Irritation level

Stimulation Index (SI)

Test item

2.5

I

0.64

Test item

5

I

0.50

Test item

10

I

0.51

Test item

25

I

0.74

Test item

50

I

0.61

HCA

25

-

7.06

Second main test

Test item

50

I

2.26

Test item

75

I

2.82

Test item

100

I

12.16

HCA

25

-

5.78

-: not recorded.

I: non-irritant (increase in ear thickness < 10%).

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

Sensitisation data from three structurally-related substances were used to address the skin sensitisation potential of ethanethiol.

The key study represents the worst-case result. Propane-2-thiol was found to be sensitising in a mouse local lymph node assay (CiToxLAB, 2013) resulting in a Stimulation Index of greater than 3 (12.16) at a concentration of 100%. The EC3 concentration was calculated to be 75.5%.

2 -Methylpropane-2-thiol was found to be sensitising in a mouse local lymph node assay (CiToxLAB, 2011a) resulting in a Stimulation Index of greater than 3 at concentrations of 25% and above (3.62, 4.26 and 30.43 at substance concentrations of 25, 50% and 100% respectively). The EC3 value was calculated to be 20%.

Butane-1-thiol was found to be sensitising in a mouse local lymph node assay (CiToxLAB, 2011b) resulting in a Stimulation Index of greater than 3 at concentrations of 50% and above (5.38 and 14.40 at substance concentrations of 50% and 100% respectively). The EC3 value was calculated to be 30%.

Based on the results from these three structurally-related substances, it is considered that ethanethiol would also result in a Stimulation Index of greater than 3 in a mouse local lymph node assay and therefore is should be considered to be a sensitiser.

Read-across justification

To reduce animal testing REACH recommends to make use of a read-across approach where appropriate based on the high accordance in properties relevant for the specific endpoint.

Read-Across Justification

To reduce animal testing REACH recommends to make use of a read-across approach where appropriate based on the high accordance in properties relevant for the specific endpoint.

There are no data on skin sensitisation for ethanethiol, therefore good quality studies on three other Category members, propane-2-thiol, butane-1-thiol and 2-methylpropane-2-thiol, have been read-across to assess the potential for ethanethiol to cause skin sensitisation.

The low molecular weight aliphatic alkylthiols are volatile organic liquids of moderate aqueous solubility and low n-octanol-water partition coefficient. The substances all contain a single thiol (-SH) functional group. The acid dissociation constant (pKa) of the thiol group is approximately 10 therefore the substances can be considered as weak acids which will not be significantly ionised at physiologically relevant pH. The three read-across skin sensitisation studies all three related substances were clearly positive and therefore ethanethiol is also expected to be a skin sensitiser.



Migrated from Short description of key information:
Sensitisation data from three structurally related substances are available for the assessment of the sensitisation potential of ethylmercaptan. Mouse local lymph node assays on 2-methylpropane-2-thiol (CiToxLAB, 2011a), butane-1-thiol (CiToxLAB, 2011b) and propane-2-thiol (CiToxLAB, 2013) found they were all sensitisers with calculated EC3 values of 20%, 30% and 75.5% respectively.

Justification for selection of skin sensitisation endpoint:
The key study was conducted to OECD test guideline 429 and in compliance with GLP. The key study represents the worst-case result.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available weight of evidence for three structurally-related read-across substances, ethylmercaptan requires classification for skin sensitisation as Skin Sensitiser Category 1B: (H317: May cause allergic skin reaction), according to Regulation (EC) No 1272/2008, as amended.