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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 July 2016 to 24 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
other: read-across target
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across from Dioctyltin bis (2-ethylhexylmercaptoacetate) (DOTE) (CAS No. 15571-58-1) can be justified by structural analogy (Dioctyltin and two thiogylocolate ligands).
Reason / purpose for cross-reference:
read-across source
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
10 %
Key result
Parameter:
SI
Value:
0.2
Test group / Remarks:
5 %
Key result
Parameter:
SI
Value:
0.4
Test group / Remarks:
2.5 %

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl 10-ethyl-4,4-dioctyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate
EC Number:
239-622-4
EC Name:
2-ethylhexyl 10-ethyl-4,4-dioctyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate
Cas Number:
15571-58-1
Molecular formula:
C36H72O4S2Sn
IUPAC Name:
8-Oxa-3,5-dithia-4-stannatetradecanoic acid, 10-ethyl-4,4-dioctyl-7-oxo-, 2-ethylhexyl ester
impurity 1
Chemical structure
Reference substance name:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate
EC Number:
248-227-6
EC Name:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate
Cas Number:
27107-89-7
Molecular formula:
C38H74O6S3Sn
IUPAC Name:
2-ethylhexyl 10-ethyl-4-({2-[(2-ethylhexyl)oxy]-2-oxoethyl}sulfanyl)-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecan-1-oate
impurity 2
Chemical structure
Reference substance name:
2-ethylhexyl mercaptoacetate
EC Number:
231-626-4
EC Name:
2-ethylhexyl mercaptoacetate
Cas Number:
7659-86-1
Molecular formula:
C10H20O2S
IUPAC Name:
2-ethylhexyl sulfanylacetate
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Housing: a maximum of four animals were housed in a standard polypropylene cage (Size L 290 x B 220 x H 140 mm) with stainless steel mesh top grill
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 - 22.6°C
- Humidity (%): 48 - 67%
- Air changes (per hr): 12 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours flourescent light and 12 hours dark cycle

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5, 5.0 and 10.0 % (v/v)
No. of animals per dose:
4 per dose
Details on study design:
PRE-SCREEN TESTS:
The pre-screen test was performed under conditions identical to the main study. On day 1, the body weight of each animal was recorded and clinical signs were observed. From days 1 to 3, dorsum of each ear of each animal in each group was applied with 25 µL of respective concentration of test material formulation. Ear thickness measurements were performed using Digital Vernier calipers on Day 1 (pre-dosing), Day 3 (approximately at 48 hours after the first dose) and on Day 6. On Day 6, an ear thickness of each animal was determined and was humanely euthanised by CO2 asphyxiation for ear punch weight determination.
- The concentration of 10 % and 5 % (v/v) in Acetone: Olive oil (4:1 v/v) did not cause any local skin irritation or systemic toxicity in the pre-screen test. Hence, 10, 5 and 2.5 % (v/v) in Acetone: Olive oil (4:1 v/v) were selected as a high, mid and low dose concentrations respectively for the main study.


MAIN STUDY
- Day 1 to 3: On Day 1, the body weight of each animal was recorded and clinical signs were observed and recorded. From days 1 to 3, the dorsum of each ear of each animal in groups was applied with 25 µL of Acetone: Olive oil (4:1, v/v) - negative control; 25% (v/v) a-hexylcinnamaldehyde in Acetone: Olive oil (4:1, v/v) - positive control; or test material at 10%, 5% and 2.5% (v/v) in Acetone: Olive oil (4:1, v/v) respectively.
- Day 4: No treatment was given; only clinical signs and local irritation were observed and recorded.
- Day 5: Each animal was injected with 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution intra-peritoneally.
- Day 6: The body weight, ear thickness and clinical signs of each animal in all the groups were recorded. Approximately 24 hours after BrdU injection, animals were humanely euthanised by CO2 asphyxiation. The draining lymph nodes were carefully dissected and trimmed off fascia and fat and transferred to labelled 1.5 mL centrifuge tube. Ear punch weights were recorded for each individual ear of each animal using punch method.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
10 %
Key result
Parameter:
SI
Value:
0.2
Test group / Remarks:
5 %
Key result
Parameter:
SI
Value:
0.4
Test group / Remarks:
2.5 %

Any other information on results incl. tables

- Clinical Signs and Mortality:

Pre-Screen Test: Pre-screen test animals showed clinical signs of lethargy and abdominal breathing at a dose of 100 % (as such) and 50 % (v/v) in Acetone: Olive oil (4:1 v/v). Lethargy was observed at dose of 25% (v/v) in Acetone: Olive oil (4:1 v/v). One Treatment related animal mortality was observed at the dose of 100 % (as such) on day 4. However, animals treated with doses of 10 % and 5 % (v/v) in Acetone: Olive oil (4:1 v/v) did not show any clinical signs or mortality during the course of the treatment and observation periods.

Main Test: In the main test, animals receiving the test material did not show any clinical signs or mortality during the course of the treatment and observation periods.

 

- Body Weight:

Pre-Screen Test: Normal increases in body weight were observed in animals treated with doses of 100, 50, 25, 10 and 5 % (v/v) in Acetone: Olive oil (4:1 v/v).

Main Test: No treatment related changes in body weight were observed in any treated groups.

 

- Skin Erythema:

Pre-Screen Test: In the 100 % dose group well defined erythema (Grade 2) was observed beginning on Day 2. Moderate to severe erythema (Grade 3) was observed beginning on Day 3 and at each assessment interval thereafter; scores for Days 4 through 6 could not be read due to skin damage and did not decline from Day 3 to Day 6. In the 50 % dose group well defined erythema (Grade 2) was observed beginning on Day 3. Severity increased to a maximum of Grade 3 on Day 4 and declined to Grade 2 by Day 6. In the 25 % dose group well defined erythema (Grade 2) was observed beginning only on Day 4. Severity decreased to slight (Grade 1) by Day 6. No erythema was observed in mice at doses of 10 or 5 % (v/v) in Acetone: Olive oil (4:1 v/v).

Main Test: No erythema was observed at any of the tested doses in Acetone: Olive oil (4:1 v/v). However, very slight erythema (barely perceptible) was observed in two animals of group 07 (Positive Control) on day 1 and in all animals of this group from Day 2 to Day 5.

 

- Ear Thickness:

Pre-Screen Test: There was no within-group change in mean ear thickness noted in groups treated with 10 or 5 % test material in Acetone: Olive oil (4:1 v/v). At doses of 100, 50 and 25 % in Acetone: Olive oil (4:1 v/v), there was a progressive increase in ear thickness within each group which did not decline by Day 6.

Main Test: No significant change in mean ear thickness was noted in groups treated with the test material when compared with vehicle control group. There was significant increase observed in the mean ear thickness in G7: 25 % a -Hexylcinnamaldehyde in Acetone: Olive oil (4:1, v/v) treated group (Positive control) when compared to vehicle control group (G6).

 

- Ear Punch Weights:

Pre-Screen Test: No change in mean ear punch weights were noted in groups treated with 10 and 5 % (v/v) in Acetone: Olive oil (4:1 v/v), whereas 100, 50 and 25 % (v/v) in Acetone: Olive oil (4:1 v/v) showed increase in ear punch weight.

Main Test: No significant change in mean ear punch weights were noted in groups treated with the test material when compared with vehicle control group. There was significant increase noted in the mean ear punch weights in G7: 25 % a -Hexylcinnamaldehyde in Acetone: Olive oil (4:1, v/v) treated group (Positive control) when compared to vehicle control group (G6).

 

- BrdU Labelling Index and Stimulation Index:

BrdU labelling index and Stimulation Index (SI) results for each treatment group were expressed and calculated. The SI values of 25 % a-Hexylcinnamaldehyde in Acetone: Olive oil (4:1, v/v) treated group (Positive control) was found to be 2.0 (SI 1.6, considered as a sensitiser) and the SI values of the test material in Acetone: Olive oil (4:1, v/v) at 10, 5 and 2.5 % (v/v) were found to be 0.7, 0.2 and 0.4 (SI 1.6), respectively.

 

INTERPRETATION OF RESULTS.

The SI values for test material treated mice in the main test at all concentrations from 2.5 up to 10.0% (v/v) in Acetone: Olive oil (4:1 v/v) were SI 1.6.These data strongly support the conclusion that the test material is not a sensitiser and should be classified as a non-sensitising chemical.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study the test material was not a sensitiser to skin.
Executive summary:

The skin sensitisation potential of the test material was determined in accordance with the standardised guideline OECD 442B, under GLP conditions.

The study was conducted in two phases, a pre-screen test and the main test. The pre-screen test was conducted under conditions identical to the main study. Clinical signs of systemic toxicity and local irritation at the application site, body weight (Days 1 and 6), and ear thickness (Days 1, 3 and 6) and ear punch weight (Day 6) were recorded. In the pre-screen the assessment of lymph node proliferation was not done.

In the pre-screen test, two animals for each dose were tested. The vehicle diluent was Acetone: Olive oil (4: 1, v/v). The dose groups G1-G5 received concentrations as follows: G 1 - 100% (undiluted; as such), G2 – 50 %, G3 – 25 %, G4 - 10 % and G5 5 % (v/v) test material in the vehicle diluent. For the animals in each test group a volume of 25 μL of the respective test concentration was applied bilaterally on the dorsum of the ears for three consecutive days (Days 1, 2 and 3). On Day 6, animal ear thickness and ear punch weight were measured. Clinical signs of lethargy and abdominal breathing were observed in Groups G1 (100 %) and G2 (50 %). Lethargy was observed in group G3 (25 %). In Group G1 100 % (v/v), one treatment related animal mortality was observed on Day 4. In the 100 % dose group well defined erythema (Grade 2) was observed beginning on Day 2. Moderate to severe erythema (Grade 3) was observed beginning on Day 3 and at each assessment interval thereafter; scores for Days 4 through 6 could not be read due to skin damage and did not decline from Day 3 to Day 6. In the 50 % dose group well defined erythema (Grade 2) was observed beginning on Day 3. Severity increased to a maximum of Grade 3 on Day 4 and declined to Grade 2 by Day 6. In the 25 % dose group well defined erythema (Grade 2) was observed beginning only on Day 4. Severity decreased to slight (Grade 1) by Day 6. No erythema was observed in mice at doses of 10 or 5 % (v/v) test material in Acetone: Olive oil (4:1 v/v). There was no within-group change in mean ear thickness noted in groups treated with 10 or 5 % test material in Acetone: Olive oil (4:1 v/v). At doses of 100, 50 and 25 % in Acetone: Olive oil (4:1 v/v), there was a progressive increase in ear thickness within each group which did not decline by Day 6. The pre-screen test results were sufficient to support dose selection for the Main Test as recommended in the OECD guideline. The dose of 10 % was the highest non-irritating dose and was selected as the high dose for the Main Test. Doses of 5 % - mid dose and 2.5 % - low dose (v/v) were also selected for the main test

In the main test, each group consisted of 4 animals per group. The animals were allocated into the following groups: Acetone: Olive oil (4:1, v/v) (Vehicle Control Group), 25 % a-Hexylcinnamaldehyde in Acetone: Olive oil (4: 1, v/v) (Positive Control Group) and 10, 5 and 2.5 % (v/v) test material in Acetone: Olive oil (4:1, v/v) (High Dose, Mid Dose and Low Dose, respectively). A volume of 25 μL of the respective concentration of the test material was applied bilaterally on the dorsum of the ears of each animal for three consecutive days (days 1, 2 and 3). On Day 4, the animals were left undisturbed. On Day 5, 0.5 mL of BrdU solution (10 mg/mL) was injected intraperitoneally to each animal in all the groups. Individual animal body weights were recorded on Day 1 and Day 6. Ear thickness of each animal was recorded on Day 1 (pre-dose), Day 3 and Day 6. Each mouse was carefully observed once daily for clinical signs of systemic toxicity and for local irritation at the application site. Animals were observed for mortality and morbidity twice daily. Erythema on each ear was scored using the standard scoring scale. On Day 6, after body weight and ear thickness measurement, animals were humanely euthanised. Both ears of each animal were isolated and ear punch weights were measured and recorded. After isolation of bilateral ears, bilateral draining auricular lymph nodes were collected and processed separately in phosphate buffered saline (PBS) to produce single cell suspensions of lymph nodes. BrdU content in DNA of lymphocytes was measured by using a commercially available ELISA kit.

There was no mortality or clinical signs observed during the course of the test material treatment and the subsequent observation period. No treatment related change in body weights was observed from Day 1 to 6. There was no erythema found at the site of application on Day 1 to Day 6 in animals in the controls or any of the test material groups. In the positive control group (25 % a-Hexylcinnamaldehyde in Acetone: Olive oil (4:1, v/v); group G7) there was a very slight erythema (barely perceptible) observed at the site of application on Day 1 (two animals) and from Day 2 to Day 5 in all animals.

No statistically significant change in mean ear thickness or ear punch weights was found in groups treated with the test material when compared with vehicle control group. Statistically significant increases in mean ear thickness and ear punch weight were observed for animals of the positive control group (G7) when compared with vehicle control group.

The Stimulation Index (SI) for the 25 % a-Hexylcinnamaldehyde in Acetone: Olive oil ( 4:1, v/v) group (positive control) was found to be 2.0 (SI 1.6, considered as a sensitiser) which supports the conclusion the study is valid. The SI values for the test material groups at 10, 5 and 2.5 % (v/v) were 0.7, 0.2 and 0.4 (SI 1.6) respectively.

Under the conditions of this study the test material was not a sensitiser to skin.