rel-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Endpoint summary

Description of key information

Skin sensitisation (OECDTG442B): Sensitiser 1B

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2016 - 9 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The test was conducted before the REACH regulation came into force requesting in vitro testing first.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

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

Specific details on test material used for the study:

Hysandol called Timberol in the test report may be a multi of the cis and trans-isomer, while Hysandol is a mono-trans isomer. The cis isomer is expected to have the same results for this endpoint because it is stereo isomer.

Species:

mouse

Strain:

other: CBA/N

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Central Lab. Animal Inc., Republic of Korea
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 9 weeks old
- Weight at study initiation: 16.7-21.2 g (set 1) and 16.5-20.8 g (set 2)
- Housing: 2- 3 animals per cage, housed in polysulfone cages (200W x 320D x 140H (mm))
- Diet (e.g. ad libitum): Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C), ad libitum
- Water (e.g. ad libitum): Public tap water in Cheongju-si (filtered and irradiated by ultraviolet light), ad libitum
- Acclimation period: 4 days (dose range finding study and main study/set 2) and 11 days (main study/set 1)
- Indication of any skin lesions: General health examinations were conducted by a staff veterinarian on the last day of the quarantine-acclimation period: no clinical abnormalities or abnormal body weight gains were observed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3–22.8°C (set 1) and 21.1–22.3°C (set 2)
- Humidity (%): 45.7–59.1% (set 1) and 49.5–55.4% (set 2)
- Air changes (per hr): 10–15 (filtered air)
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 10 May 2016 To: 15 May 2016 (set 1) and From: 10 November 2016 To: 15 November 2016 (set 2)

Vehicle:

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

Concentration:

0, 25, 50 and 100% (set 1); 0, 1, 5 and 10% (set 2)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: the test substance was dissolved in the vehicle in a preliminary solubility test.
- In the dose range finding study, clinical signs, body weights, ear thickness, ear weights and erythema score by the toxicity of the test substance were evaluated.

MAIN STUDY: the main study was conducted in two sets, with lower treatment doses used in the second set.

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. The dose level of the positive substance was selected at 25%. Negative control animals were dosed with the vehicle, acetone/olive oil solution.

OBSERVATIONS:
- Clinical signs: All animals were observed for mortality, general condition and clinical signs for 6 days.
- Body weights: Body weights were recorded prior to dosing (day 1) and on the day of necropsy (day 6).
- Erythema: Both ears of each mouse were observed for erythema and scored for 6 days.
- Ear thickness: Ear thickness measurement was taken using a thickness gauge (543-681B, Mitutoyo Co., Japan) on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6 (the day of necropsy).

On day 5, a volume of 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected (1mL, 26G) interperitoneally. 24 hours after BrdU injection, necorpsy was conducted on all animals. The draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline for each animal.

Evaluation criteria:
SI Result
SI < 1.6 Negative
SI ≥ 1.6 Positive

Determination of cellular proliferation: BrdU was measured by ELISA: 100 μL of the LNC suspension was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC suspension, anti-BrdU antibody was added to each well and allowed to react. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. Absorbance at 370 nm with a reference wavelength of 492 nm was measured.

BrdU labelling index = (ABSem - ABSblank,em) - (ABSref-ABSblank,ref)
with ABS = Absorption; em = emission wavelength; ref= reference substance

SI= mean of BrdU labelling index in the test substance / mean of BrdU labelling index in the negative control

Acceptability criterium: positive control should have a SI ≥ 1.6.

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.

Homogeneity of variance for body weights, ear thickness, ear weight and stimulation index: Bartlett’s test (α=0.05); homogeneous data; Dunnett’s ttest (α=0.05 and 0.01, one-tailed): multiple comparisons between the negative control group and each of the test substance groups. Since not significant, Kruskal-Wallis test was employed on heterogeneous data and Steel’s test was applied for multiple comparisons (α=0.05 and 0.01, onetailed) between the negative control group and each of the test substance groups or positive control group.

Erythema score: Kruskal-Wallis test on heterogenous data; Steel’s test for multiple comparisons (α=0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or the positive control group.

Key result

Parameter:

other: EC1.6 value (%)

Value:

12.73

Parameter:

SI

Value:

1.33

Test group / Remarks:

1%

Remarks on result:

other: 1% test group

Parameter:

SI

Value:

1.2

Test group / Remarks:

5%

Remarks on result:

other: 5% test group

Parameter:

SI

Value:

1.27

Test group / Remarks:

10%

Remarks on result:

other: 10% test group

Parameter:

SI

Value:

1.91

Test group / Remarks:

25%

Remarks on result:

other: 25% test group

Parameter:

SI

Value:

3.53

Test group / Remarks:

50%

Remarks on result:

other: 50% test group

Parameter:

SI

Value:

2.35

Test group / Remarks:

100%

Remarks on result:

other: 100% test group

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
SI= mean of BrdU labelling index in the test substance/mean of BrdU labelling index in the negative control

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

CLINICAL OBSERVATIONS: There were no abnormal clinical signs or deaths in any dosing group during the observation period in both sets.

BODY WEIGHTS (mean)
- Set 1:
Negative control: 19.1–19.5 g (prior to dosing until day 6 after dosing.)
Positive control: 18.5–19.6 g. There were no significant differences when compared to the negative control group.
The test substance groups at 25, 50 and 100%: 19.3–18.9, 18.9–19.0 and 18.9–19.0 g, respectively. There were no significant differences when compared to the negative control group.
- Set 2:
Negative control: 18.7–18.9 g (prior to dosing until day 6 after dosing).
Positive control: 18.6–19.2 g. There were no significant differences when compared to the negative control group.
The test substance groups at 1, 5 and 10%: 18.6–18.5, 19.4–19.6 and 18.7–19.1 g, respectively. There were no significant differences when compared to the negative control group.

ERYTHEMA SCORE:
- Set 1:
Negative control: 0.0–0.0
Positive control: 0.0–0.7. There was a significant increase when compared to the negative control group (p<0.01: day 4, 5 and 6).
Test substance groups at 25, 50 and 100%: 0.0–0.3, 0.0–0.6 and 0.0–0.5, respectively. There was a significant increase when compared to the negative control group (p<0.05: Days 4, 5 and 6: 50 and 100%).

- Set 2:
Negative control: 0.0–0.0
Positive control: 0.0–1.8. There was a significant increase when compared to the negative control group (p<0.01: day 2, 3, 4, 5 and 6).
Test substance groups at 1, 5 and 10%: 0.0–0.0, 0.0–0.1 and 0.0–1.0, respectively. There was a significant increase when compared to the negative control group (p<0.01: days 4, 5 and 6: 10%).

EAR THICKNESS:
- Set 1:
Negative control: 0.20–0.20 mm
Positive control: 0.19–0.21 mm. There was a significant increase when compared to the negative control group (p<0.01: day 3, p<0.05: day 6).
Test substance group at 25, 50 and 100%: 0.19–0.20, 0.20–0.21 and 0.19–0.21 mm, respectively. There was a significant increase when compared to the negative control group (p<0.01: Day 3: 25, 50 and 100%, Day 6: 50 and 100%).

- Set 2:
Negative control: 0.19–0.19 mm
Positive control: 0.19–0.21 mm. There was a significant increase when compared to the negative control group (p<0.01: day 6).
Test substance group at 1, 5 and 10%: 0.19–0.19, 0.19–0.19 and 0.19–0.20 mm, respectively. There was a significant increase when compared to the negative control group (p<0.05: day 6: 10%, p<0.01: day 3: 10%).

EAR WEIGHTS (mean):
- Set 1:
Negative control: 12.9 mg.
Positive control: 14.9 mg. There was a significant increase when compared to the negative control group (p<0.01).
Test substance groups at 25, 50 and 100%: 15.3, 16.0 and 18.9 mg, respectively. There was a significant increase when compared to the negative control group (p<0.01: 25, 50 and 100%).


STIMULATION INDEX/EC1.6:
- Set 1:
Negative control: 1.00.
Positive control: 2.41. There was a significant increase when compared to the negative control group.
Test substance groups at 25, 50 and 100%: 1.91, 3.53 and 2.35, respectively. There was a significant increase when compared to the negative control group (p<0.05: 25, 50 and 100%).
- Set 2:
Negative control group: 1.00.
Positive control: 3.16. There was a significant increase when compared to the negative control group (p<0.01).
Test substance groups at 1, 5 and 10%: 1.33, 1.20 and 1.27, respectively. There were no significant differences when compared to the negative control group.

Three concentration showed SI of >1.6, and EC1.6 was calculated to be 12.73%

Estimation of the Proliferative Response of Lymph Node Cells

Treatment Group	Concentration	Stimulation Index	Result
Test Item	25%v/vin acetone/olive oil4:1	1.91	Positive
	50%v/vin acetone/olive oil4:1	3.53	Positive
	100%	2.35	Positive
Positive Control Item	25%v/vin acetone/olive oil4:1	2.41	Positive

Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
0	2101	19.0	21.0	0	0	0	0	0	0	0	0	0
0	2102	16.9	19.1	0	0	0	0	0	0	0	0	0
5	2201	16.5	20.4	0	0	0	0	0	0	0	0	0
5	2202	16.4	19.3	0	0	0	0	0	0	0	0	0
10	2301	16.8	20.0	0	0	0	0	0	0	0	0	0
10	2302	17.5	19.5	0	0	0	0	0	0	0	0	0
25	2401	16.4	19.8	0	0	0	0	0	0	0	0	0
25	2402	16.1	19.5	0	0	0	0	0	0	0	0	0
50	2501	16.8	19.8	0	0	0	0	0	0	0	0	0
50	2502	15.7	19.7	0	0	0	0	0	0	0	0	0
100	2601	19.1	18.6	0	0	0	0	0	0	0	0	0
100	2601	19.2	20.4	0	0	0	0	0	0	0	0	0

0=     No abnormalities detected

Local Skin Irritation

Concentration	Animal Number	Local Skin Irritation
		Day 1	Day 2	Day 3		Day 4	Day 5	Day 6
25%	mean	0	0	0.2		0.3	0.3	0.3
50%	mean	0	0	0.2		0.6	0.6	0.6
100%	mean	0	0.2	0.3		0.5	0.5	0.5
Positive control (SET 1)	mean	0	0	0.1		0.7	0.7	0.7
Negative control (SET 1)	mean	0	0	0		0	0	0
1%	mean	0	0	0		0	0	0
5%	mean	0	0	0.1		0.1	0.1	0.1
10%	mean	0	0.3	0.4		0.8	0.9	1.0
Positive control (SET 2)	mean	0	0.6	1.1		1.7	1.7	1.8
Negative control (SET 2)	mean	0	0	0		0	0	0

Measurement of Ear Thicknessand Mean Ear Thickness Changes – SET 1

Concentration: 100%		Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
overall mean (mm)		0.19		0.21		0.21
overall mean ear thickness change (%)		na		10.5		10.5

Measurement of Ear Thicknessand Mean Ear Thickness Changes – SET 2

Concentration: 25%		Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
overall mean (mm)		0.19		0.21		0.21
overall mean ear thickness change (%)		na		10.5		10.5

na=     Not applicable

Individual Clinical Observations and Mortality Data

Treatment Group	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
Vehicle acetone/olive oil 4:1	2101	0	0	0	0	0	0	0	0	0
	2102	0	0	0	0	0	0	0	0	0
	2103	0	0	0	0	0	0	0	0	0
	2104	0	0	0	0	0	0	0	0	0
	2105	0	0	0	0	0	0	0	0	0
Test Item 25% v/vin acetone/olive oil 4:1	2201	0	0	0	0	0	0	0	0	0
	2202	0	0	0	0	0	0	0	0	0
	2203	0	0	0	0	0	0	0	0	0
	2204	0	0	0	0	0	0	0	0	0
	2205	0	0	0	0	0	0	0	0	0
Test Item 50% v/vin acetone/olive oil 4:1	2301	0	0	0	0	0	0	0	0	0
	2302	0	0	0	0	0	0	0	0	0
	2303	0	0	0	0	0	0	0	0	0
	2304	0	0	0	0	0	0	0	0	0
	2305	0	0	0	0	0	0	0	0	0
Test Item 100%	2401	0	0	0	0·	0	0·	0	0	0
	2402	0	0	0	0·	0	0·	0	0	0
	2403	0	0	0	0·	0	0·	0	0	0
	2404	0	0	0	0·	0	0·	0	0	0
	2405	0	0	0	0·	0	0·	0	0	0
Positive Control Item 25% v/v in acetone/olive oil4:1	2501	0	0	0	0	0	0	0	0	0
	2502	0	0	0	0	0	0	0	0	0
	2503	0	0	0	0	0	0	0	0	0
	2504	0	0	0	0	0	0	0	0	0
	2505	0	0	0	0	0	0	0	0	0

0=     No signs of systemic toxicity

 Individual Bodyweights and Bodyweight Changes

Treatment Group	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle acetone/olive oil 4:1	2101	19	21	2
	2102	16.9	19.1	2.2
	2103	15.4	18.4	3
	2104	16.5	18.3	1.8
	2105	17.4	18.3	0.9
Test Item 25% v/vin acetone/olive oil 4:1	2201	16.5	20.4	3.9
	2202	16.4	19.3	2.9
	2203	17.0	18.5	1.5
	2204	16.9	18.3	1.4
	2205	16.5	18.3	1.8
Test Item 50% v/vin acetone/olive oil 4:1	2301	16.8	20.0	3.2
	2302	17.5	19.5	2.0
	2303	16.7	18.8	2.1
	2304	16.6	18.4	1.8
	2305	15.1	18.2	3.1
Test Item 100%	2401	16.4	19.8	3.4
	2402	16.1	19.5	3.4
	2403	16.8	19.1	2.3
	2404	15.5	18.7	3.2
	2405	14.9	17.8	2.9
Positive Control Item 25% v/v in acetone/olive oil 4:1	2501	16.8	19.8	3.0
	2502	15.7	19.7	4.0
	2503	16.7	19.0	2.3
	2504	16.5	18.6	2.1
	2505	16.2	17.6	1.4

Interpretation of results:

other: Skin sensitizing Category 1B

Remarks:

In accordance with EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

The substance produced a SI ≥ 1.6 and an EC1.6 value of 12.73% was calculated. Based on these results, the substance is considered to be a skin sensitiser 1B.

Executive summary:

In a Local Lymph Node Assay (BrdU-ELISA), the skin sensitisation potential of the substance was tested according to OECD TG 442B test guideline and GLP principles. Per concentration, 5 female mice were used. Skin sensitization potential was evaluated by ear thickness measurements. Cellular proliferations and SI were determined. In the first set, at 25, 50 and 100%, SI values were 1.91, 3.53 and 2.35, respectively. In the second set, at 1, 5 and 10%, SI values were 1.33, 1.20 and 1.27, respectively. Negative and positive controls were included and all acceptability criteria were met (SI values positive controls: 2.41 and 3.16). These results show that the substance could elicit a SI ≥ 1.6. An EC1.6 value of 12.73% was calculated. Based on the results, the substance is considered a skin sensitiser 1B.

Reference 2

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:

In a Local Lymph Node Assay (BrdU-ELISA), the skin sensitisation potential of the substance was tested according to OECD TG 442B test guideline and GLP principles. Per concentration, 5 female mice were used. Skin sensitization potential was evaluated by ear thickness measurements. Cellular proliferations and SI were determined. In the first set, at 25, 50 and 100%, SI values were 1.91, 3.53 and 2.35, respectively. In the second set, at 1, 5 and 10%, SI values were 1.33, 1.20 and 1.27, respectively. Negative and positive controls were included and all acceptability criteria were met (SI values positive controls: 2.41 and 3.16). These results show that the substance could elicit a SI ≥ 1.6. An EC1.6 value of 12.73% was calculated.

Based on the results, the substance is considered a skin sensitiser 1B.

Justification for classification or non-classification

Based on the results, the substance is considered a skin sensitiser and needs to be classified for skin sensitisation category 1B and shall be labelled with H317: May cause an allergic skin reaction according to EU CLP (EC No. 1272/2008 and its amendments).