β,2,2,3-tetramethylcyclopent-3-ene-1-butanol

Endpoint summary

Description of key information

Skin sensitisation (OECD 442B): skin sensitising

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

Study period:

18 Apr - 21 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

(adopted 22 Jul 2010)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Species:

mouse

Strain:

CBA

Remarks:

CBA/N

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 8 weeks
- Weight at study initiation: 16.6 to 20.1 g for dose range finding study; 16.2 - 21.1 g for Set 1; 16.0 - 20.6 g for Set 2
- Housing: 2 - 3 animals per cage in polysulfone cages (200W x 320D x 140H mm)
- Diet: pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C), ad libitum
- Water: tap water (filtered and irradiated by ultraviolet light), ad libitum
- Acclimation period: 4 to 10 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 22.4 in dose range finding study; 21.1 - 22.8 in Set 1; 21.3 - 22.2 in Set 2
- Humidity (%): 47.0 - 55.8 in dose range finding study; 45.8 - 54.6 in Set 1; 48.7 - 57.1 in Set 2
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Dose range finding study: 5, 10, 25, 50% (w/v) and 100%
Set 1: 10, 25, 50% (w/v) and 100%
Set 2: 0.5, 1 and 5% (w/v)

No. of animals per dose:

2 (range-finding study), 5 (main study)

Details on study design:

RANGE-FINDING STUDY: Dose selection was based on the consecutive doses and dose levels are selected from a series of appropriate concentrations such as 5, 10, 25, 50 and 100%.
- Compound solubility: The test substance was dissolved in aceton/olive oil in a preliminary solubility test. Therefore, aceton/olive oil was utilized as vehicle for this study.
Two animals were observed per dose group. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to dosing (Day 1) and on the day of necropsy, Day 6. Both ears of each mouse were observed for erythema and scored using erythema score. Ear thickness measurement was taken using a thickness gauge on Day 1 (pre-dose), Day 3 and Day 6. Additionally, on Day 6, ear weight was determined by ear punch weight measurement.
- Irritation: not specified
- Systemic toxicity: toxicity was observed in the high dose group (100%)
- Ear thickness measurements: not specified
- Erythema scores: not specified

MAIN STUDY: Based on the result of the dose range finding study, the high dose for the main study was selected at 50%. Two additional low levels (25 and 10%) were produced by applying a geometric ratio of 2 (Set 1). In addition, the positive and negative control groups were included in the main study. Test substance concentrations of 0.5, 1 and 5% were added (Set 2) after discussion with the sponsor.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ELISA BrdU
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the BrdU labelling index of each test group divided by the BrdU labelling index of the vehicle control group. SI < 1.6 is considered as negative result. SI ≥ 1.6 is considered as positive result.

The EC1.6 value was used to classify the test substance according to ECETOC Potency classification as follows:
EC1.6 Value(%) ≥ 10 to ≤ 100 -> Weak
EC1.6 Value(%) ≥ 1 to ≤ 10 -> Moderate
EC1.6 Value(%) ≥ 0.1 to ≤ 1 -> Strong
EC1.6 Value(%) < 0.1 -> Extreme
Cindy A et al. Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency. Cutaneous and Ocular Toxicology, 2007, 26: 135-145.

TREATMENT PREPARATION AND ADMINISTRATION: A volume of 25 μL was applied to the dorsum of both ears of all animals daily for three consecutive days. Negative control animals were dosed with the vehicle, acetone/olive oil solution. Two days after the third application on Day 5, an intraperitoneal injection of 0.5 mL (5 mg/mouse) of BrdU solution (10 mg/mL) was made. Approximately 24 h after BrdU injection, the mice were sacrificed and draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline for each animal. A single cell suspension was prepared by separation through a nylon mesh. In each case, the target volume of the cell suspension was adjusted to the determined optimized volume. The optimized volume was based on the mean absorbance within 0.1 - 0.2 in the negative control group. BrdU was measured by ELISA using a commercial kit. Briefly, 100 µL of the lymph node cell suspension was added to the wells of a microplate in triplicate. After fixation and denaturation of the cell suspension, anti-BrdU antibody was added to each well. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was added. Absorbance was measured at 370 nm with a reference wavelength of 492 nm.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index. Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data. One-way analysis of variance (ANOVA) was employed on homogeneous data. Dunnett’s t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group. Since it was not significant, Kruskal-Wallis test was employed on heterogeneous data and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group. Kruskal-Wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group.

Positive control results:

Body weights: Set 1: The mean body weights were 19.5 - 20.0 g. There were no significant differences when compared to the negative control group. Set 2: The mean values of body weight ranged within 20.0 - 20.7 g. There were no significant differences when compared to the negative control group.
Irritation: Set 1: The mean erythema score was 0.0 - 1.9. There were significant increases when compared to the negative control group (p < 0.01: Days 4, 5 and 6). Set 2: The mean value of erythema score range within 0.0 - 1.0. There was a significant increase when compared to the negative control group (p < 0.01: Days 3, 4, 5 and 6).
Ear thickness: Set 1: The mean ear thickness was 0.19 -0.22 mm. There were significant increases when compared to the negative control group (p < 0.01: Days 1, 3 and 6). Set 2: The mean value of ear thickness ranged within 0.19 -0.20 mm. There was a significant increase when compared to the negative control group (p < 0.01: Day 6).
Ear weights: Set 1: The mean ear weight was 13.9 mg. There was a significant increase when compared to the negative control group (p < 0.05). Set 2: The mean value of ear tissue was 14.0 mg. There were no significant differences when compared to the negative control group.
Stimulation Index: Set 1: The mean stimulation index was 3.18. There was a significant increase when compared to the negative control group (p < 0.05). Set 2: The mean value of stimulation index was 3.46. There was a significant increase when compared to the negative control group (p < 0.01).

Parameter:

SI

Value:

1.44

Test group / Remarks:

0.5% (w/v)

Parameter:

SI

Value:

1.74

Test group / Remarks:

1% (w/v)

Parameter:

SI

Value:

1.35

Test group / Remarks:

5% (w/v)

Parameter:

SI

Value:

2.17

Test group / Remarks:

10% (w/v)

Parameter:

SI

Value:

2.04

Test group / Remarks:

25% (w/v)

Parameter:

SI

Value:

3.08

Test group / Remarks:

50% (w/v)

Parameter:

other: EC1.6

Value:

11.5

IRRITATION, EAR THICKNESS and EAR WEIGHTS:

Erythema Score: Set 1: In the negative control group, the mean erythema score was 0.0 - 0.0 from Day 1 to Day 6 after dosing. In the test substance groups at 10, 25 and 50%, the mean erythema score was 0.0 - 1.6, 0.0 - 1.6 and 0.0 - 2.0, respectively. There were significant increases when compared to the negative control group (p < 0.01: Days 4, 5 and 6 (10, 25 and 50%)).

Set 2: In the negative control group, the mean value of erythema score was 0.0 prior to dosing until Day 6 after dosing. In the test substance groups at 0.5, 1 and 5%, the mean values of erythema score ranged within 0.0 - 0.1, respectively. There were no significant differences when compared to the negative control group.

Ear thickness: Set 1: In the negative control group, the mean value of ear thickness was 0.19 mm prior to dosing until Day 6 after dosing. In the test substance groups at 10, 25 and 50%, the mean ear thickness was 0.19 - 0.21, 0.19 - 0.22 and 0.19 - 0.23 mm, respectively. There were significant increases when compared to the negative control group (p < 0.05: Day 1 (10%), p < 0.01: Day 1 (25%), Day 3 (10, 25 and 50%), Day 6 (10, 25 and 50%).

Set 2: In the negative control group, the mean value of ear thickness ranged within 0.18 - 0.19 mm prior to dosing until Day 6 after dosing. In the test substance groups at 0.5, 1 and 5%, the mean values of ear thickness ranged within 0.18 - 0.19 mm, respectively. There was a significant increase when compared to the negative control group (p < 0.05: Day 6: 5%).

Ear weights: Set 1: In the negative control group, the mean ear weight was 12.3 mg. In the test substance groups at 10, 25 and 50%, the mean ear weight was 14.5, 14.8 and 16.4 mg, respectively. There were significant increases when compared to the negative control group (p < 0.01).

Set 2: In the negative control group, the mean value of ear tissue was 13.6 mg. In the test substance groups at 0.5, 1 and 5%, the mean values of ear tissue were 13.0, 12.9 and 13.1 mg, respectively. There were no significant differences when compared to the negative control group.

DETAILS ON STIMULATION INDEX CALCULATION: Set 1: In the negative control group, the mean stimulation index was 1.00. In the test substance groups at 10, 25 and 50%, the mean stimulation index was 2.17, 2.04 and 3.08, respectively. There were significant increases when compared to the negative control group (p < 0.05: 10 and 50%).

Set 2: In the negative control group, the mean value of stimulation index was 1.00. In the test substance groups at 0.5, 1 and 5%, the mean values of stimulation index were 1.44, 1.74 and 1.35, respectively. There were significant increases when compared to the negative control group (p < 0.05: 1%).

EC1.6 CALCULATION: EC1.6 =c+[(1.6-d)/(b-d)] x (a-c)

a = The dose concentration with higher SI; b = The higher SI value, c = The dose concentration with lower SI; d = the lower SI value

EC1.6 = 11.5%

CLINICAL OBSERVATIONS: Set 1 and Set 2: There were no abnormal clinical sighs or deaths in any dosing group during the observation period.

BODY WEIGHTS: Set 1: In the negative control group, the mean body weights were 19.6 - 20.1 g from Day 1 to Day 6 after dosing. In the test substance groups at 10, 25 and 50%, the mean body weights were 19.7 - 19.8, 19.8 - 20.5 and 19.6 - 19.8 g, respectively. There were no significant differences when compared to the negative control group.

Set 2: In the negative control group, the mean value of body weight ranged within 20.6 - 21.0 g prior to dosing until Day 6 after dosing. In the test substance groups at 0.5, 1 and 5%, the mean values of body weights ranged within 20.2, 20.0 - 20.4 and 19.7 - 19.9 g, respectively. There were no significant differences when compared to the negative control group.

Interpretation of results:

other: Skin Sens Cat 1B according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the local lymph node assay, the test substance revealed a SI ≥ 1.6 at concentrations of 1, 10, 25 and 50%. The calculated EC1.6 value was 11.5%. Therefore, the test substance is considered as weak sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation potential of the test substance was assessed by a LLNA test using the BrdU ELISA method according to OECD Guideline 442B in compliance with GLP (2017). Treatment of CBA/N mice with the test substance revealed stimulation indices of 1.44, 1.74, 1.35, 2.17, 2.04 and 3.08 at test substance concentrations of 0.5, 1, 5, 10, 25 and 50% (w/v) in acetone/olive oil (4:1 v/v), respectively. The EC1.6 value was calculated as 11.5%.

In conclusion, based on the available data the test substace is considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The available data on skin sensitisation meets the criteria for classification as Skin Sens 1B (H317) according to Regulation (EC) 1272/2008.