Cyclohexanepropanol, 2,2,3,6-tetramethyl-.alpha.-propyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08-02-2007 to 02-04-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Method equivalent to guideline, well documented and performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

478-330-3

EC Name:

-

Cas Number:

95851-08-4

Molecular formula:

C16H32O

IUPAC Name:

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

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: refrigerated at 2-8 °C, under nitrogen
- Other: liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/J strain

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (documented in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Microbiological status of animals, when known: No issues reported within the study.
- Age at study initiation: approximately 6 to 7 weeks old (prior to acclimatisation) and 7-8 weeks prior to dosing
- Weight at study initiation: 15.7 – 22.2 grams
- Housing: Group housed, in labelled shoebox style cages during acclimatisation then individually housed, in labelled shoebox style cages during the study period
- Diet (e.g. ad libitum): Free access to rodent diet (certified, recognised supplier) d libitum
- Water (e.g. ad libitum): mains tap water ad libitum
- Acclimation period: 6 days
- Indication of any skin lesions: None reported.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (actual: 19.3 to 21.2°C)
- Humidity (%): 40 to 80 (actual: 23 – 54% ; this deviation would not be considered significant to the conclusions of the study)
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark
- IN-LIFE DATES: From: 14-02-2007 To: 20-02-2007

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0% (vehicle control), 1%, 5%, 10%, 20% and 40% in 4:1 A:OO vehicle
ISOEUGENOL (positive control), 5.0% in 4:1 A:OO vehicle
HYDROXY CITRONELLAL (reference item), 15% and 60% in 4:1 A:OO vehicle

No. of animals per dose:

Main test: 8 mice 0% (vehicle control) and 5 mice per test item dose group 1%, 5%, 10%, 20% and 40%

Details on study design:

RANGE FINDING TESTS:
Not applicable.

MAIN STUDY
- Compound solubility: Fully soluble in vehicle. The choice of AOO (4:1) as a vehicle was based on NIH Publication No. 99-449, "The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals / Compounds," in which AOO was the vehicle of choice.
- Irritation: None observed.
- Systemic toxicity: None observed. Thirteen of the treated mice lost weight during that period (0.1 – 0.8 grams). This mild weight loss occurred in treatment and control (vehicle, hydroxycitronellal, and isoeugenol) groups and is therefore considered unrelated to treatment. Body weights at lymph node harvest ranged from 16.2 to 22.4 grams. The cause of this mild weight loss, distributed across treatment groups, is unknown. Applicant assessment indicates: the slight body weight declines would generally not be considered toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent or reported in the study.
- Ear thickness measurements: mean ear thickness values did not increase by 10% or more at any tested concentration of either isoeugenol (5.0%), Hydroxy citronellal (15% or 60%) or test item (1.0% - 40%).
- Erythema scores: No irritation was reported.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Following excision of the nodes. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Five groups of five animals were treated with one test item concentration per group. One group of five animals was treated with vehicle.
- Induction: The dorsal surface of both ears was topically treated (25 μL/ear) with the test item concentration, at approximately the same time on each day with monitoring for local and systemic toxicity during dosing. A rest period was allowed on days 4 and 5. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Node excision: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 2 μCi of 125-I labelled IuDR and 1x10^-5 M FuDR. After approximately five hours, all animals were terminated. Nodes were dissociated using the frosted ends of glass slides. The cell suspension was washed with Hanks' balanced salt solution (HBSS) and then with PBS prior to being resuspended in 5% trichloroacetic acid (TCA) and refrigerated at approximately 4°C. Approximately 20 hours later, the cells were centrifuged and resuspended in fresh 5% TCA. The radioactivity was measured using a gamma counter.
- Tissue processing and radioactivity measurements: See above. Results from each cell suspension counted on the gamma counter were recorded in counts per minute (CPM). The CPM values were converted to disintegrations per minute (DPM) by dividing by the gamma counter efficiency and multiplying by 100. After the DPM values had been calculated, the mean "blank" DPM was subtracted from each individual DPM to obtain corrected DPM values. The mean corrected DPM and standard error of the mean (SE) were determined for each group. The stimulation index (SI) was then calculated by dividing the treatment-group mean DPM by the control (vehicle)-group mean DPM.

Observations:
- Mortality/Viability: Once daily
- Bodyweights: On Day 1 (pre-dose) and Day 6 (post termination).
- Clinical Observations: Once daily
- Irritation: Once daily.
- Ear Thickness: Ear measurements were taken prior to dosing on Days 1 and 3

Positive control substance(s):

other: Isoeugenol (CAS No 97-54-1) and Hydroxy citronellal (CAS 107-75-5)

Statistics:

A one-sample t test was performed to test whether the individual untransformed SI value for each dose level of each test/reference item was greater than or equal to 3. The natural log transformed DPM values for each compound were compared with vehicle using a Bartlett's Chi-Square test for variance homogeneity. If the Bartlett's Chi-Square results were found to be nonsignificant, a one-way analysis of variance (ANOVA) was performed using dose (concentration). If the ANOVA was found to be significant, then a Dunnett' s t test was performed using an alpha of 0.05. If the Barlett's Chi-Square was found to be significant, nonparametric analyses were conducted, specifically a Kruskal-Wallis (KW) test If the non-parametric analyses-were found to be significant, then a Jonckheere's-Terpera test was performed for dose-dependent trends.

Calculations were performed using Microsoft® Excel and SAS®, version 9.1

Results and discussion

Positive control results:

The concurrent positive control (isoeugenol) had an EC3 = 1.7%. This was considered consistent with previously reported results.

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Remarks:

%

Value:

19.2

Variability:

C.I. -%

Remarks on result:

other: See table below

Results 2

Parameter:

SI

Value:

1.4

Variability:

± 0.3

Test group / Remarks:

1% in acetone:olive oil (4:1)

Results 3

Parameter:

SI

Value:

1

Variability:

± 0.3

Test group / Remarks:

5% in acetone:olive oil (4:1)

Results 4

Parameter:

SI

Value:

1

Variability:

± 0.2

Test group / Remarks:

10% in acetone:olive oil (4:1)

Results 5

Parameter:

SI

Value:

3.6

Variability:

± 0.6

Test group / Remarks:

20% in acetone:olive oil (4:1)

Results 6

Parameter:

SI

Value:

10.2

Variability:

± 2.2

Test group / Remarks:

40% in acetone:olive oil (4:1)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: See tables.

DETAILS ON STIMULATION INDEX CALCULATION: See tables.

EC3 CALCULATION: The data per concentration was fit using a quadratic equation (a linear term and a square term of the concentration). If the quadratic term did not fit, a simple regression model was used. The EC3 was determined from the appropriate regression equation.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed.

BODY WEIGHTS: Thirteen (13) of the 48 mice assigned to the study lost minimal amounts of weight between randomization and lymph node harvest (0.1 - 0.7 grams). The cause of this mild weight loss, distributed across treatment groups, without dose-response, is unknown. Applicant assessment indicates: the slight body weight declines would generally not be considered toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent or reported in the study. Along with no reported correlating clinical signs. Eight (8) of the thirteen (13) were in test item treatment groups and without dose-response in terms of bodyweight declines. Three (3) of thirteen (13) declines were in the vehicle control group.

Any other information on results incl. tables

Table 1. Results from the definitive test

Group	Number	CPM	DPM	Mean DPM	SD	SE	Mean SI	SE	Ears mean % increase	SE
Vehicle Control	1	15.0	13.6
	2	22.2	22.7
	3	59.2	69.7
	4	22.4	23.0
	5	60.6	71.4
	6	19.0	18.7
	7	45.8	52.7
	8	24.2	25.3	37.1	23.7	8.4	-	-	0.7	0.27
Test Item 1%	9	79.6	95.6
	10	30.2	32.9
	11	29.6	32.1
	12	39.8	45.1
	13	50.8	59.0	52.9	26.2	11.7	1.4	0.3	0.7	0.23
Test Item 5%	14	10.6	8.0
	15	51.0	59.3
	16	25.4	26.8
	17	25.2	26.5
	18	52.0	60.5	36.2	22.9	10.3	1.0	0.3	0.6	0.26
Test Item 10%	19	26.0	27.5
	20	40.4	45.8
	21	24.4	25.5
	22	49.2	57.0
	23	34.8	38.7	38.9	13.1	5.8	1.0	0.2	0.7	0.32
Test Item 20%	24	69.6	82.9
	25	158.8	196.1
	26	78.4	94.0
	27	90.6	109.5
	28	147.6	181.9	132.9	52.3	23.4	3.6	0.6	1.1	0.17
Test Item 40%	29	243.0	302.9
	30	414.0	519.9
	31	458.2	577.3
	32	105.0	127.8
	33	299.0	374.0	380.4	178.9	80.0	10.2	2.2	0.7	0.22
Hydroxy citronellal 15%	34	129.4	158.8
	35	110.8	135.2
	36	31.0	33.9
	37	44.6	51.1
	38	47.0	54.2	86.6	56.2	25.2	2.3	0.7	0.4	0.17
Hydroxy citronellal 60%	39	82.4	99.1
	40	44.2	50.6
	41	209.2	260.0
	42	166.6	206.0
	43	336.2	421.2	207.4	145.6	65.1	5.6	1.8	1.0	0.49
Isoeugenol 5.0%	44	203.2	252.4
	45	127.4	156.2
	46	127.6	156.5
	47	341.2	427.5
	48	236.2	294.3	257.4	112.6	50.4	6.9	1.4	1.0	0.48

Where:

SE = standard error of the mean

SD = standard deviation

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is considered to be sensitising to skin with EC3 of 19.2%.

Executive summary:

The study was performed to using the local lymph node assay method under GLP to assess the skin sensitisation potential of the test item in the CBA/J strain mouse following topical application to the dorsal surface of the ear. The test item was tested at concentrations of: 1%, 5%, 10%, 20% and 40% in acetone/olive oil (4:1) and with a 0% vehicle control group. A concurrent positive control consisting of Isoeugenol at 5% and a reference item of hydroxy citronellal at 15% and 60% concentrations was additionally conducted. Observations were made daily and ear thickness measurements conducted daily on days 1 to 3. No irritation or signs of systemic toxicity were observed. Three days after the final auricular application, the animals were injected intravenously with 125-I radiolabelled Iododeoxyuridine to label proliferating cells. 125-I incorporation was quantified using a gamma counter. The SI values calculated for the test item concentrations 1%, 5%, 10%, 20% and 40% were 1.4, 1.0, 1.0, 3.6 and 10.2 respectively. Although only 40% achieved statistical significance (p < 0.05). The results show that the test item elicited an SI ≥ 3. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be 19.2%. The concurrent positive control Isoeugenol had an EC3 value of 1.7% and hydroxy citronellal had an EC3 value of 25.5%. Under the conditions of this study, the test item would be considered to be classified under Regulation (EC) No 1272/2008 as skin sensitizer category 1B.