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Description of key information

Skin: Read-across to structurally similar substance: Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE)

Patel (2016)

Under the conditions of this study the SI values for the 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, therefore the test material is not a sensitiser.

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:
21 July 2016 to 08 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In house
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 23.07 to 26.89 g
- 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 having facilities for holding pelleted feed and drinking water in water bottle filled with stain less steel sipper tube. Clean sterilised paddy husk was provided as bedding material.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: Veterinary examination including skin lesions of all the animals was performed on the day of receipt and on the 5'" day of acclimatisation.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.0 to 22.6 °C
- Humidity: 48 to 67 %
- Air changes: 12 to 15 air changes per hour
- Photoperiod: 12 hours fluorescent light und 12 hours dark cycle
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5, 5 and 10 %
No. of animals per dose:
4 animals per dose
Details on study design:
PRE-SCREEN TESTS:
- The objective of the pre-screen test was to determine the maximum dose level of the test material that could be employed in the main study.
- A pre-screen test was conducted as a part of the study. Graded doses from 2.5 % (G5) up to 100 % (G1), were used for the pre-screen test.
- The pre-screen test was performed under conditions identical to the main study, i.e. clinical signs of systemic toxicity and local irritation at the application site, body weight and ear thickness and ear punch weight were recorded. In the pre-screen the assessment of lymph node proliferation was not done. Two animals per group were used. On day 1 body weight of each animal was recorded and clinical signs were observed. From Days 1 to 3, 25 μL of respective concentration of test material formulation, was applied to the dorsum of each ear of each animal in each group. Ear thickness measurements were performed using Digital Vernier callipers on Day 1 (pre-closing), 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.
- Concentrations above 10 % caused local skin irritation and clinical sign of systemic toxicity. The concentration of 10 and 5 % (v/v) test material 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 concentration, respectively for the main study as recommended in "OECD Test Guideline No. 442B - "Skin Sensitization: Local Lymph Node Assay: Breda-ELISA" guideline.


TREATMENT PREPARATION AND ADMINISTRATION:
- Each day prior to dosing, the required quantity of test material, and positive control 25 % (v/v) a-Hexylcinnamaldehyde in Acetone: Olive oil (4:1 v/v) was prepared.

MAIN STUDY
- Day 1 to 3: On Day 1, body weight of each animal was recorded and clinical signs were observed and recorded. From days 1 to 3, 25 μL of Acetone: Olive oil (4:1, v/v), 25 % (v/v) n-Hexylcinnamaldehyde in Acetone: Olive oil (4:1, v/v) and 2.5, 5 and 10 % (v/v) test material in Acetone: Olive oil (4:1, v/v) respectively, was applied to the dorsum of each ear of each animal in groups G6, G7, G8, G9 and G10.
- Day4: 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 auricular lymph nodes from each test animal were carefully dissected, trimmed of fascia and fat, and then transferred to labelled 2 mL centrifuge tube. Ear punch weights were recorded for each individual ear using punch method.
- Preparation of Cell Suspensions: A 0.3 mL of phosphate buffered saline (PBS) was added to the centrifuge tube that contained the collected lymph nodes. The lymph nodes were crushed with a plunger of the syringe followed by passage through a 70 µm cell strainer fitted to a 50 mL graduated centrifuge tube. The single cell suspension was finally made up to 15 mL with phosphate buffered saline (PBS).

OBSERVATIONS
- Clinical Observations: Each mouse was carefully observed once daily for clinical signs and for local irritation at the site of application and for systemic toxicity. Animals were observed for mortality and morbidity twice daily. All observations were recorded and maintained for each mouse. Erythema scoring of each ear was scored using the following:
No erythema = 0
Very slight erythema (barely perce1itible) = 1
Well defined erythema = 2
Moderate to severe erythema = 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema = 4
- Body Weight: Individual animal body weight was recorded at receipt and on experimental Day 1 and on Day 6.
- Ear Thickness: Ear thickness measurement was performed and recorded using a thickness gauge (e.g. Digital Vernier Callipers) on Day 1 (pre-dose), Day 3 (approximately at 48 hours after the first dose) and on Day 6.
- Necropsy: At the end of observation period, all animals were euthanised by carbon dioxide anaesthesia. After euthanising the animals were subjected to ear punch weight and lymph node collection procedure.
- Ear Punch Weights: Both the ears from each mouse in all groups were collected and ear punch weights were measured and recorded at necropsy.
- Determination of Cellular Proliferation: The BrdU content in DNA of lymphocytes was measured by using a commercially available ELISA kit. Node cell suspensions were mixed thoroughly with vortex then 100 μL of lymph node cell suspension was added to the wells of a flat-bottom microplate in triplicate. Simultaneously, three blank wells were prepared by adding 100 μL of PBS. After filling all sample wells and blank wells, the plate was centrifuged at 300 X g for 10 minutes and the supernatant volume was removed by flicking off. The assay plate was dried completely in a hot-air oven at 60°C for 1 hour. A volume of 200 μL Fix-Denat solution was added to each well and plate was allowed to stand for 30 minutes at room temperature (15 to 25°C). The Fix-Denat solution was removed completely by flicking off and tapping and 100 μL of anti-BrdU-POD antibody working solution was added to each well and allowed it to react for 1 hour at room temperature (15 to 25°C). After one hour incubation period, the anti-BrdU-POD antibody solution was removed completely and 200 μL of wash solution was added to each well, the wash solution was discarded completely by tapping. The wash step was repeated twice (three times total). A volume 100 μL of substrate solution was added to each well and was allowed to stand for 15 minutes at room temperature in a dark place. Absorbance (ABS) was measured at 370 nm with a reference wavelength of 492 nm.

CALCULATION OF RESULTS
- Results for each treatment group are expressed as the mean Stimulation Index (SI). BrdU labelling index and SI was calculated using:
SI = (Mean BrdU labelling index for each Test group/ positive control group) / Mean BrdU labelling index for concurrent vehicle control group
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
One-way ANOVA with an appropriate post hoc test was performed for the body weight, ear thickness and ear punch weight data by using SPSS statistical software, version 22.
Positive control results:
- 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).
- 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).
- The Sl values of 25 % a-Hexylcinnamaldehyde in Acetone:Olive oil (4:1 v/v) treated group (Positive control) was found to be 0.9 (SI ≥1.6, considered as a sensitiser).
Key result
Parameter:
SI
Value:
0.6
Test group / Remarks:
2.5 % Test material
Key result
Parameter:
SI
Value:
0.6
Test group / Remarks:
5 % Test material
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
10 % Test material
Cellular proliferation data / Observations:
CLINICAL SIGNS AND MORTALITY
- Pre-Screen Test: Pre-screen test animals showed clinical signs of lethargy and abdominal breathing at dose of 100 % (as such) and 50 % (v/v). Lethargy was observed at a dose of 25 % (v/v). Among the animals treated with any dose of test material from 100 to 5 % no mortalities were observed. Animals treated with doses of 10 and 5 % test material did not show any clinical signs during the course of the treatment and observation periods.
- Main Test: In the main test among animals receiving the test material there were no mortalities and no animals showed any clinical signs during the course of treatment and observation periods.

BODY WEIGHTS
- Pre-Screen Test: Normal increases in body weight were observed in animals treated with doses of 100, 50, 25, 10 and 5 %.
- 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 moderate to severe erythema [Grade 3] was observed beginning on Day 2 and at each assessment interval thereafter; scores did not decline from Day 3 lo Day 6. In the 50 % dose group well defined erythema [Grade 2] was observed beginning on Day 2. Severity increased to 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 at doses of 10 or 5 %.
- Main Test: No erythema was observed at any dose concentration of test material. Among animals in the Positive Control [Group G7] very slight erythema (barely perceptible) was observed in two animals on day 1 and in all animals 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. At doses of 100, 50 and 25 % test material, there was a progressive increase in ear thickness within each group which did not decline by Day 6.
- Main Test: There was no significant change in mean ear thickness 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 any dose of test material from 5 up to 100 %.
- Main Test: No significant change in mean ear punch weights were noted in groups treated with any dose of test material when compared with vehicle control group. There was significant increase noted in the mean ear punch weights in G7: 25 % a-Hexylcinnamaldehycle 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 Sl values of 25 % a-Hexylcinnamaldehyde in Acetone: Olive oil (4:1, v/v) treated group (Positive control) was found to be 1.9 (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 0.9, 0.6 and 0.6 (SI ≤ 1.6), respectively.

INTERPRETATION OF RESULTS
- The SI values for the 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.
Interpretation of results:
other: Not classified in accordance with EU Criteria
Conclusions:
Under the conditions of this study the SI values for the 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, therefore the test material is not a sensitiser.
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442B, under GLP conditions using the Local Lymph Node Assay: BrdU-ELISA.

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.

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 - G6), 25 % a-Hexylcinnamaldehyde in Acetone: Olive oil (4:1, v/v) (Positive Control Group - G7) and 2.5, 5 and 10 % (v/v) test material in Acetone: Olive oil (4:1 v/v) (G8 - Low Dose, G9 - Mid Dose and G 10 - High 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 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-dosing), Day 3 (approximately at 48 hours after the first dose) and on 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 and 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 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 of the test material groups (G6, G8, G9 and G10]. 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 the 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 lndex (SI) for the 25 % a-Hexylcinnamaldehyde in Acetone: Olive oil (4:1 v/v) group (positive control) was found to be 1.9 (SI1.6, 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 0.9, 0.6 and 0.6 (SI1.6) respectively.

 Under the conditions of this study the SI values for the 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 SI1.6, therefore the test material is not a sensitiser.

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 to Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (EC Number 248-227-6 and CAS No 27107-89-7) based on structural similarity and hydrolytical behaviour, see attached justification.
Reason / purpose for cross-reference:
read-across source
Key result
Parameter:
SI
Value:
0.6
Test group / Remarks:
2.5 % Test material
Key result
Parameter:
SI
Value:
0.6
Test group / Remarks:
5 % Test material
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
10 % Test material
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin: Read-across to structurally similar substance: Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE)

Patel (2016)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442B, under GLP conditions using the Local Lymph Node Assay: BrdU-ELISA. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

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 - G6), 25 % a-Hexylcinnamaldehyde in Acetone: Olive oil (4:1, v/v) (Positive Control Group - G7) and 2.5, 5 and 10 % (v/v) test material in Acetone: Olive oil (4:1 v/v) (G8 - Low Dose, G9 - Mid Dose and G 10 - High 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 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-dosing), Day 3 (approximately at 48 hours after the first dose) and on 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 and 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 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 of the test material groups (G6, G8, G9 and G10]. 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 the 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 lndex (SI) for the 25 % a-Hexylcinnamaldehyde in Acetone: Olive oil (4:1 v/v) group (positive control) was found to be 1.9 (SI1.6, 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 0.9, 0.6 and 0.6 (SI1.6) respectively.

 Under the conditions of this study the SI values for the 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 SI1.6, therefore the test material is not a sensitiser.

Skin: Read-across to structurally similar substance (mixture MOTE: DOTE. 70:30) (CAS No 27107-89-7 and CAS 15571-58-1)

Hagemann (1993)

A sensitisation test in albino guinea pigs was performed to determine the contact allergenic potency of the test material in albino guinea pigs. The test was conducted according to the standardised OECD 406 guideline under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

After the first challenge application 90% of the animals of the test group and 60 % of the animals of the control group showed skin reactions 24 and 48 hours after removing the dressings. After a second challenge application, using a lower concentration of the test article, 20 and 0 % of the animals of the control group and 0 and 10 % of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings, respectively.

Since 2/10 animals of the control group also showed skin reactions observations made in the test group are considered to be of no toxicological relevance. Frequency of observed skin reactions in control and test group are considered to indicate the absence of skin sensitisation.

According to the maximisation grading the test material showed a weak grade of skin-sensitising (contact allergenic) potential in albino guinea pigs.

Under the conditions of this study the test material is not sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the Regulation (EC) No. 1272/2008, the substance does not require classification as a skin sensitiser.