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Description of key information

Read-across from analogue: Key study. Method according to OECD 429 / EU B.42 (LLNA), GLP study. The substance was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. The stimulation index values were 7.0, 6.0, 5.4 and 1.3 at concentrations of 100 % (undiluted), 50, 25 and 10 % (w/v), respectively. The calculated EC3 value was 16.2% (w/v).

Based on the available information for the read-across approach, the target substance is a sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 November 2014 - 02 December 2014
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
Qualifier:
according to guideline
Guideline:
EU Method B.42 (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:
TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE (France)
- Age at study initiation: 10 weeks old.
- Weight at study initiation: 21.2-23.2 g
- Housing: Group caging / mice were provided with glass tunnel-tubes. Cage type: Type II. polypropylene / polycarbonate.
- Diet (e.g. ad libitum): ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”, ad libitum.
- Water (e.g. ad libitum): tap water, ad libitum.
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 h dark / 12 h light (from 6 a.m. to 6 p.m.)
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25, 50 and 100%.
No. of animals per dose:
4 animals / group
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test. Acetone:olive oil 4:1 (v:v) mixture (AOO) was tested and was considered to be suitable for the test. The highest achievable concentration of the test item was 100 % (undiluted) in AOO.
- Preliminary Irritation/Toxicity Test:
2 animals per dose were exposed to 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 with a body weight measurement and the radioactive proliferation assay was not performed. 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. 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. Mortality and signs of systemic toxicity were observed. One animal in the 100 % (undiluted) was found dead on Day 3, however the second animal at 100 % had no signs of toxicity at all so the single mortality is considered of equivocal relationship with test item treatment. Intermittent tremors were observed for both animals in the 50 % (w/v) dose group on Day 3. Erythema (erythema score: 1) was detected on the ears of the animals on Day 3. The surviving animals were symptom-free in the rest of the experiment. No marked body weight loss was detected on the mean body weight values of the groups. The revealing ear punch weights were within the historical control range. The draining auricular lymph nodes of the animals were visually examined: they were slightly enlarged in both dose groups (subjective judgement by analogy with observations of former experiments). Based on these results, the 50 % (w/v) dose was considered to be acceptable for the main test. The 100 % (undiluted) dose group range finding part was equivocal, so a 100 % (undiluted) group was included in the main study (4 animals) as an additional dose group. Therefore, 100 % (undiluted), 50, 25 and 10 % (w/v) doses were examined in the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
1) That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
2) The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
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. 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). Five hours later, the mice were euthanized and the auricular lymph nodes were extracted from the animals. A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and the samples were examined in a scintillation counter.

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 body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection).

EVALUATION OF RESULTS:
Radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes. 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 and data permitted, the EC3 value of the test item was calculated (EC3 means the effective chemical concentration required for SI=3).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Key result
Parameter:
SI
Value:
7
Test group / Remarks:
100 % (undiluted)
Key result
Parameter:
SI
Value:
6
Test group / Remarks:
50% (w/v)
Key result
Parameter:
SI
Value:
5.4
Test group / Remarks:
25% (w/v)
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
10% (w/v)
Key result
Parameter:
EC3
Value:
16.2
Variability:
%
Test group / Remarks:
mean
Cellular proliferation data / Observations:
- Disintegrations per minute (DPM): Test item 100 % (w/v) in AOO: 20140.5 DPM Test item 50 % (w/v) in AOO: 17214.5 DPM Test item 25 % (w/v) in AOO: 15522.5 DPM Test item 10 % (w/v) in AOO: 3844.5 DPM

Clinical observations:

No mortality or signs of systemic toxicity were observed during the study. Eythema (erythema score: 1) was detected on the ears of the animals in the 100 % (undiluted) and 50 % (w/v) dose groups on Days 2 -3.

Body weight measurement:

No treatment related effects were observed on the mean body weight changes, however marked body weight loss (>5%) was observed for one animal in the 50% (w/v) dose group and for one animal in the positive control group.

Proliferation assay:

Test Group Name

Measured DPM / group

DPM

Number
of lymph nodes

DPN

Stimulation Index

Background

(5 % (w/v) TCA)

30
31

-

-

-

-

Negative (vehicle) control (AOO)

2897

2866.5

8

358.3

1.0

Test item 100 % (undiluted)

20171

20140.5

8

2517.6

7.0

Test item 50 % (w/v) in AOO

17245

17214.5

8

2151.8

6.0

Test item 25 % (w/v) in AOO

15553

15522.5

8

1940.3

5.4

Test item 10 % (w/v) in AOO

3875

3844.5

8

480.6

1.3

Positive control(25 % (w/v) HCA in AOO)

24832

24801.5

8

3100.2

8.7

The stimulation index values were 7.0, 6.0, 5.4 and 1.3 at concentrations of 100 % (undiluted), 50, 25 and 10 % (w/v), respectively.

The obtained data are compatible with a conventional dose response and allow the calculation of the EC3 value according to an adequate scientific method. EC3 means the effective chemical concentration required for SI=3. The calculated EC3 value of the test substance is 16.2 % (w/v).

The appearance of the lymph nodes was normal in the negative (vehicle) control group and in the 10 % (w/v) group. Larger than normal lymph nodes were observed in the 100 % (undiluted) 50 % (w/v) dose groups and in the positive control group. Slightly enlarged lymph nodes were detected in the 25 % (w/v) group.

The DPN values observed for the vehicle and positive control substance were within the historical control change. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 8.7) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Interpretation of results:
other: Skin Sensitizer Cat.1B (CLP Regulation EC no. 1272/2008)
Conclusions:
The substance was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. The stimulation index values were 7.0, 6.0, 5.4 and 1.3 at concentrations of 100 % (undiluted), 50, 25 and 10 % (w/v), respectively. The calculated EC3 value was 16.2% (w/v).
Executive summary:

The skin sensitisation test following dermal exposure was performed according to OECD Guideline 429 and EU method B.42, following the Principles of GLP. Based on the results of the Preliminary Compatibility Test, the test item was formulated in acetone:olive oil 4:1 (v:v) at a highest achievable concentration of 100 % (w/v). The Preliminary Irritation / Toxicity Test was performed in CBA/J Rj mice using two doses: 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-four female CBA/J Rj mice were allocated to six groups of four animals each: four groups received the test substance (formulated in AOO) at 100% (undiluted), 50, 25 and 10 % (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. Erythema (erythema score: 1) was detected on the ears of the animals in the 100 % (undiluted) and 50 % (w/v) dose groups on Days 2-3. No treatment related effects were observed on the mean body weight changes, however marked body weight loss (>5%) was observed for one animal in the 50 % (w/v) dose group. The stimulation index values were 7.0, 6.0, 5.4 and 1.3 at concentrations of 100 % (undiluted), 50, 25 and 10 % (w/v), respectively. The calculated EC3 value was 16.2% (w/v). All validity criteria were fulfilled. In conclusion, under the conditions of the present assay the test substance was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance 7-chloro-3-methyl-1-benzothiophene (SER-4) which shares the aryl functional group chlorophenyl with the substance 1-[(4-chlorophenyl)sulfanyl]propan-2-one (SER-3) also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Parameter:
SI
Value:
7
Test group / Remarks:
100% (undiluted analogue substance)
Key result
Parameter:
SI
Value:
6
Test group / Remarks:
50% analogue substance
Key result
Parameter:
SI
Value:
5.4
Test group / Remarks:
25% analogue substance
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
10% analogue substance
Key result
Parameter:
EC3
Value:
17.8
Interpretation of results:
other: Skin Sensitizer Cat.1B (CLP Regulation EC no. 1272/2008)
Conclusions:
Based on the available information for the read-across approach, the target substance is a sensitizer.
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Read-across from analogue: Key study: The skin sensitisation test following dermal exposure was performed according to OECD Guideline 429 and EU method B.42, following the Principles of GLP. Based on preliminary results, four groups (four animals each) received the test substance (formulated in AOO) at 100% (undiluted), 50, 25 and 10 % (w/v) concentrations. No mortality or signs of systemic toxicity were observed during the study. Erythema (erythema score: 1) was detected on the ears of the animals in the 100 % (undiluted) and 50 % (w/v) dose groups on Days 2-3. No treatment related effects were observed on the mean body weight changes, however marked body weight loss (>5%) was observed for one animal in the 50 % (w/v) dose group. The stimulation index values were 7.0, 6.0, 5.4 and 1.3 at concentrations of 100 % (undiluted), 50, 25 and 10 % (w/v), respectively. The calculated EC3 value was 16.2% (w/v). All validity criteria were fulfilled. In conclusion, the test item was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. Based on the available information for the read-across approach, the target substance is a sensitizer.

Skin sensitisation in vitro: Data-waiving (study scientifically not necessary). Adequate data from skin sensitisation in vivo is available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available results, the substance is classified as Skin Sensitizer Category 1B, H317 according to CLP Regulation (EC) no. 1272/2008.