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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without significant deviations
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)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Thirty female CBA/J mice (SPF) at 8 weeks old were obtained from Charles River Japan Hino Breeding Center. CBA/J mice are a substrain of CBA/JN mice recommended in OECD TG442B, and the testing facility has a historical control data of CBA/J mice with regard to LLNA: BrdU-ELISA.
Animals were quarantined and acclimatized until 8 days after the acceptance under group housing of 10 or less animals per cage. The animals were acclimatized couture 2 days, and 7 animals of minimum animal number were used for the pre-screen test. The remaining 23 animals were acclimatized until 15 days after the acceptance. In the main study, the animals were allocated to five groups (four animals per group) by body weight-stratified randomization using body weights on 15 days after the acceptance. First sensitization was done at the next day of grouping. Three animals not selected at grouping were excluded from this study. On the first sensitization day, the animals were 9 weeks old in the pre-screen test and 10 weeks old in the main study. After the grouping, animals were housed in one animal per cage in the pre-screen test, and four animals per cage in the main study. In addition, clinical conditions and excretions were observed once or more a day until the first sensitization. The animals were identified by marker on the tail with red ink before the grouping and with other colors after the grouping. Cages were identified by individual cards, and racks were identified by indications of the study number.
Housing and Feeding Conditions
Animals were maintained throughout overall housing period including quarantine and acclimatization in the quarantine room No. 2 and the animal room No. 5 in the main building. These rooms were maintained at a temperature of 21.9 - 23.3 °C (acceptable range: 21 °C to 25 °C), a relative humidity of 50.6 - 63.3% (acceptable range: 40% to 70%), 10 - 15 times/hour of air change and 12 hours/day of light cycle (lighting: 07:00 - 19:00). The animals were housed in a polycarbonate cage (W265 x D426 x H150 mm) before grouping and in a polycarbonate cage (W225 x D338 x H140 mm) after grouping with animal bedding (Sunflake, lot no. 120724, Charles River Japan). Cages with animal bedding were changed once a week, at grouping and termination of breeding. Rack and water bottles were changed at grouping. The animals were free access to MIF pelleted diet (lot no. 121002, Oriental Yeast) and chlorinated water via water bottles. The chlorinated water was prepared at 3 - 5 ppm of chlorine level by adding sodium hypochlorite (Purelox, OYALOX) to Hita City supply. Diets, bedding and housing materials were sterilized by autoclave (121 °C for 30 min.) prior to use. Analyses of the diets and the bedding were performed in Eurofins Analytics, and the analytical data were provided by the manufacturer. The tested parameters met the requirements in CERI Hita according to the “Toxic Substances Control Act of US-EPA” (1979). Contaminants in drinking water were analyzed twice a year according to the water regulations of the “Ordinance on drinking water quality standards” [Ordinance No. 101, 135 and 174 of MHLW]. Contaminants in the water were confirmed to be in the stated ranges at the latest test results obtained before animal receipt.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.5, 1, 2.5, 5, 10, 25 and 50% (w/v%) in the pre-test
0.5, 1, 2.5% (w/v%) in the main-test
25% alpha-hexylcinnamaldehyde (HCA) as positive control
vehicle only for negative control
No. of animals per dose:
1 animal per dose in the pre-test and 4 animals per dose in the main test
Details on study design:
Pre-screen Test
The purpose of pre-screen test was to confirm toxic effect of the test substance such as irritation effect, and to select dose concentration for the main study. Seven dose levels were set ranging from 0.5 to 50% as the maximum concentration which could be prepared. Lower six doses were set serially according to the OECD TG442B. One animal was used for each group.
Preparation of the vehicle and test substance formulations
Vehicle: Acetone was mixed with olive oil (volume ratio: 4:1 v/v) to make AOO.
Test substance: The test substance of 0.500 g was weighed and AOO was added to fill the volume of 1 mL to make 50.0 w/v% formulation. Lower concentrations were prepared by serial dilution with AOO. The vehicle and the test substance formulations were prepared on each sensitization day.
Sensitization
Twenty-five microliters of each formulation were applied to the dorsum of each ear of the animals using a micropipette. The sensitization was at approximately the same time daily each day. This method is a described in OECD TG442B.
Clinical observation
All animals were observed once or more daily from the first sensitization day (Day 1) to the terminal day (Day 6). The erythema was scored according to guideline from 0 (no erythema) to 4 (Severe erythema (beet redness) to eschar formation preventing grading of erythema).
Body weights were measured on Day 1 and Day 6 using an electric balance (SARTORIUS).
Ear thickness measurements
Ear thickness was measured on Day 1 (before sensitization), Day 3 and Day 6 using a vernier caliper (Digimatic caliper, Mitutoyo). Measurements were conducted twice on each day, and the average values were calculated.
Treatment of the animals after examinations
The animals following all examinations were euthanized by cervical dislocation on Day 6.
Evaluation of the result
Following symptoms may indicate systemic toxic effect. Any concentration which showed the symptom was excluded from the dose concentration in the main study.
- Excessive irritation (erythema score ≥3 or the ear thickness on Day 6 increases above 25% compared with that on Day 1 .)
- Severe systemic toxicity
Main Study
Dose setting: In the pre-screen test, no systemic toxicity was observed at lower doses of 50%. However, erythema and increases of ear thickness were observed at doses of 5% or more. Therefore, maximum dose of no appear the excessive irritation was judged at 2.5%.
Based on the above result, 2.5% was set at the highest dose in the main study, and added at 1.0 and 0.5%.
Group allocation
Vehicle control (AOO) and positive control (25.0 w/v% HCA) groups were set besides the test substance groups. Four animals were used for each group.
Preparations of the vehicle, test substance formulation, positive control substance and BrdU solution
Vehicle
Acetone of 4 mL and olive oil of 1 mL was mixed to make AOO.
Test substance formulations
The test substance of 0.050 g was weighed, and A00 was added to make 2 mL of 2.50 w/v% test substance formulation. The test substance formulation of 2.50 w/v% was collected at volume of 0.4 mL, and A00 of 0.6 mL was added to make 1.00 w/v% test substance formulation. The test substance formulation of 1.00 w/v% was collected at volume of 0.5 tI1L, and AOO of 0.5 mL was added to make 0.50 w/v% test substance formulation.
Positive control substance solution
HCA of 0.250 g was weighed, and AOO was added to make 1 ml. of 25.0 w/v% HCA solution. The solution was subdivided into three airtight-shading glass bottles and stored in the cool place (tolerance temperature: l-10°C). This solution was prepared on the day before the first sensitization under yellow light condition.
BrdU solution
5-Bromo-2’-deoxyuridine (BrdU, lot no. M1B6181, Nacalai Tesque) of 0.200 g was weighed, and physiological saline (lot no. K2H73, Otsuka Pharmaceutical Factory) was added. The BrdU was dissolved by ultrasonic irradiation to make 20 ml of 10 mg/mL solution. The BrdU solution was sterilized by a sterilizing filter ( pore size: 0.20 µm, ADVANTEC) and stored in an airtight plastic tube in the cool place (tolerance temperature: 1 – 10 °C) until administration. Post-operation of the sterilization was performed in a clean bench. This solution was prepared on the two days before the first sensitization day.
Sensitization
The vehicle, test substance formulations and positive control substance solution were applied in the same way as the pre-screen test.
Administration of BrdU solution
A half milliliter of BrdU solution was administrated once intraperitoneally with a syringe (Terumo) and a needle (Terumo) approximately 48 hours after the final sensitization.
Clinical observations
Clinical observations were performed in the same way as for the pre-screen test. However, scoring of the erythema was not executed.
Body weights measurements
Body weights were measured on day 1 and 6. The mean values and the standard deviations were calculated for each group on both days.
Collection and weight measurement of lymph nodes
Approximately 24 hours after the BrdU administration, animals were euthanized by cervical dislocation and the bilateral auricular lymph nodes were taken. The lymph nodes were carefully dissected, trimmed the fascia and fat, and weighed both side together. The mean values and standard deviations of the lymph nodes weights were calculated for each group. The lymph nodes were stored individually in a bio medical freezer (preset temperature: -20 °C).
Measurement of BrdU uptake quantity
The frozen auricular lymph nodes were defrosted to room temperature, and the lymph nodes were homogenized and suspended in physiological saline (lot no. lH98N, Otsuka Pharmaceutical Factory). This suspension was filtered by a cell strainer, and dispensed into a 96 well microplate at three wells for each animal. Thereafter, the incorporated BrdU was measured by ELISA (Cell Proliferation ELISA, BrdU colorimetric, lot no. 13719900, Roche Diagnostics) using microplate reader FLUOstar OPTIMA (BMG LABTECH). Mean values of the absorbance (OD370 nm - OD492 nrn) obtained from three wells per animal were calculated as individual BrdU labelling index.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The Dunnet’s test was used to compare with the vehicle control group, signicance level oftests were 5% by the StatLight of statistical tool.
Positive control results:
Mean lymph nodes weight of the positive control group was 8.3 mg and was 193% compared with the vehicle control group.
Parameter:
SI
Remarks on result:
other: SI values of the BisP-IOTD treatment groups were 1.4, 1.5 and 1.7 at 0.5, 1 and 2.5%, respectively, with an increase of dose-related manner and maximum SI was over 1.6, as limit of no skin sensitizing.

Pre-screen Test

Clinical observations: No abnormalities were observed at doses of 2.5% or less. However, erythema was observed at doses of 5% or more, and increases of ear thickness were observed at doses of 25% or more.

Body weights: Body weights on Day 6 were decreased at doses of 1% or more compared with those on Day 1. However, a decrease of body weights has not shown dose relationship.

Thickness of ear: There were no increases of 25% or more in ear thickness on Day 6 compared with Day 1 at doses of 2.5% or less. However, increases of 25% or more in ear thickness were seen at doses of 5% or more.

Main Study

Clinical observations: No abnormalities were noted in any treatment or control groups. Neither dead nor moribund animal were found.

Body weights: Body weights on Day 6 were increased at all groups compared with those on Day 1. In the positive control group, 0.4g of decrease of body weights was observed.

Lymph node weights: Mean lymph nodes weight of the vehicle control was 4.3 mg, those of the 0.5%, 1% and 2.5% test substance groups were 4.3 mg, 4.6 mg and 5.3 mg, respectively. As stated above, the mean lymph nodes weights of the 0.5%, 1% and 2.5% groups were 100%, 107% and 123 %, respectively, compared with the vehicle control group. Mean lymph nodes weight of the positive control group was 8.3 mg and was 193% compared with the vehicle control group.

Stimulation Index (SI): Mean SI of the positive control group was 3.0 and this value met the forming condition of this study (≥1.6).

Mean Sls of the test substance groups were 1.4, 1.5 and 1.7 in the 0.5%, 1% and 2.5% groups, respectively. SI of 1% and 2.5% groups were shown a significant increase compared with the vehicle control and the increase was dose dependence.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Mean SI of the 2.5% goup of the test substance was 1.7 and thus more than 1.6 (threshold for classification). SI of 1% and 2.5% groups showed a signicant dose-depnedent increase compare with the vehicle control. Consequently, it was concluded that BisP-IOTD had a possibility to be a skin sensitizing material under the conditions of this study.
Executive summary:

Skin sensitization potential of BisP-IOTD was examined by local lymph node assay: BrdU-ELISA in female CBA/J mice.

In the pre-screen test, 0.5, 1, 2.5, 5, 10, 25 and 50% groups were set using one mouse per group, neither irritate appearance nor other toxic symptoms were observed at 2.5 % or less. At 5% or more, erythema and increases of ear thickness were observed. Therefore, 2.5% was set at the highest dose in the main study, and 1 and 0.5% were added. AOO was applied as a vehicle control and 25.0 w/v% alpha-Hexylcinnamaldehyde (HCA) was applied as a positive control in the main study. 4 animals were used in each group.

In the main study, all animal data were used for the assessment as neither excessive irritation nor systemic toxicity was observed in any group. SI of the positive control was 3.0 and it exceeded 1.6, which was an approval condition of the study. Therefore, the main study was judged to be valid. SI values of the BisP-IOTD treatment groups were 1.4, 1.5 and 1.7 at 0.5, 1 and 2.5%, respectively, with an increase in a dose-related manner and maximum SI was over 1.6, as limit of no skin sensitizing. SI values of 1 and 2.5% showed a significant increase compared to the vehicle control group. Consequently, it was concluded that BisP-IOTD had a possibility to be a skin sensitizing material under the conditions of this study.

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

The skin sensitization potential of BisP-IOTD was examined by local lymph node assay.

In the pre-screen test 2.5% was defined as the highest dose in the main study, and 1 and 0.5% doses were added. AOO was applied as a vehicle control and 25.0 w/v% alpha-Hexylcinnamaldehyde (HCA) was applied as a positive control in the main study.

In the main study, all animal data were used for the assessment as neither excessive irritation nor systemic toxicity was observed in any group. SI of the positive control was 3.0 and exceeded 1.6, which was an approval condition of the study. Therefore, the main study was judged to be valid. SI values of the BisP-IOTD treatment groups were 1.4, 1.5 and 1.7 at 0.5, 1 and 2.5%, respectively, with an increase in a dose-related manner and maximum SI was over 1.6, as limit of not skin sensitising. SI values of 1 and 2.5% showed a significant increase compared to the vehicle control group. Consequently, it was concluded that BisP-IOTD had a possibility to be a skin sensitizing material under the conditions of this study.


Migrated from Short description of key information:
The substance showed dose dependent increase in SI index resulting in 1.7 and thus above the threshold for classification as skin sensitiser. Therefore BisP-IOTD is considered a skin sensitising substance.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the outcome of a LLNA test according to OECD method 442B the substance is considered as skin sensitising and thus is classified accordingly as skin sensitiser category 1 according to CLP (Regulation EC No 1272/2008) and as Xi, R43 according to DSD (Directive 67/548/EEC).