3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)pent-4-en-2-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 Aug 2001 to 10 Sept 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline Study performed according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Humidity readings in the animal room (52-81°C) exceeded the 70% limit value recommended by OECD Guideline. This deviation is not expected to have a major impact on test results

GLP compliance:

yes

Remarks:

GLP compliance statement

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Details on test material:

- Substance type: pure active substance
- Storage condition of test material: ca. 4°C under nitrogen in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN - USA
- Age at study initiation: > 6-8 weeks
- Weight at study initiation: 16.6-24.3 g
- Housing: mice were housed individually in plastic shoebox-style cages.
- Diet (e.g. ad libitum): ad libitum (Purina Rodent Chow 5002)
- Water (e.g. ad libitum): City of Raleigh tap water (ad libitum)
- Acclimation period: yes


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 24.8 °C
- Humidity (%): 52-81 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 5 Sept 2001 To: 10 Sept 2001

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

The test article, ST 23 C C01, was tested at 1%, 2.5%, 5%, 10% and 20%, final concentrations in acetone/olive oil (AOO; 4:1). The control articles included the vehicle (AOO) and a known sensitizer, isoeugenol, at 0.5%, 1.0%, and 5.0%.

No. of animals per dose:

5 animals per dose (excepted for vehicle control: 6 animal per dose)

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: A substance is considered a sensitizer if at least one concentration of the test material results in a
stimulation index (SI) greater than or equal to 3.

TREATMENT PREPARATION AND ADMINISTRATION:
The test article, ST 23 C 01, was tested at 1%, 2.5%, 5%, 10%, and 20%, final concentrations in acetone/olive oil (AOO; 4:1). The control articles included the vehicle (AOO) and a known sensitizer, isoeugenol, at 0.5 %, 1.0%, and 5.0%. The mice were restrained by hand, and 25 µl of test or control article was applied daily for three consecutive days to the dorsum of each ear using a calibrated Finnpipette. The animals were allowed to rest without dosing on Days 4 and 5. The mice were observed daily for signs of toxicity and mortality
On Day 6, individually numbered mice (labeled by tail markings) were injected in tbe lateral tail vein with 0.25 ml containing 2 µCi of I-125 labeled luDR and 10.5 M FuDR (Sigma) in phosphate buffered saline (PBS). Approximately 5 hours later, the mice were euthanized by CO2 asphyxiation, and the auricular lymph nodes were excised. The 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; LabChem) and refrigerated at approximately 4°C. Approximately 19 hours later, the cells were centrifuged and resuspended in fresh 5% TCA. The radioactivity was measured using a gamma counter (Packard Instruments).

Positive control substance(s):

other: isoeugenol (CAS: 97-54-1)

Statistics:

The natural log transformed DPM values for each compound were compared against vehicle by first performing a Bartlett's Chi-Square test for variance homogeneity. If this was found to be non-significant, a one-way analysis ofvariance was used using dose. If this 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, non-parametric analyses were performed. Specifically, a Kruskal-Wallis test was performed. If this was found to be significant, then a Jonckheere's-Terpera test was performed for dose-dependent trends.
A confirmatory analysis was performed against the known standard isoeugenol at three concentrations, 0.5%, 1%, and 5% using the above methods.
The fitted quadratic model of the ST 23 C 01 data had a non-significant quadratic term (p=0.3087) and, therefore, the linear model was the better model for determination ofthe EC-3. A fitted linear equation was used to fit the data from the concentrations tested.
A fitted linear equation was used to determine the concentration of isoeugenol required to elicit a stimulation index of 3 (EC-3). The quadratic model had a non-significant quadratic term (p=0.5024) and the linear model was therefore the better model.

Results and discussion

Positive control results:

A quadratic regression model for isoeugenol resulted in a non-significant quadratic term (p=0.5024). The model was re-fit using the linear term only. This resulted in a better model and the EC-3 concentration for isoeugenol was determined using a fitted linear equation. An EC-3 of 0.64% was determined for the positive control isoeugenol in the current study using a linear regression method with an good fit. This is in agreement with the mean EC-3 value of isoeugenol of 1.2 +/- 0.6 % (Basketter & Cadby, Contact Dermatitis 2004 Jan;50(1):15-7). A potency value of 160 µg/cm2 was calculated. These data would classify isoeugenol as a moderate sensitizer (potency value between 100 and 1000 µg/cm2) and are consistant with previously reported results; this demonstrate the capability of the test laboratory to identify positive dermal sensitizers.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

All the calculated SI values for the test material were smaller than 3, for that reason no reliable EC-3 could be calculated (EC-3 > 20 %). (See Table 2 in "Remarks on results including tables and figures"). Calculation of the EC-3 potency value assumed a conversion of 1 ml = 1 g and was based on an exposure area of 1 cm2 per mouse ear and the application of 25 µL. The EC-3 potency value for isoeugenol was determined to be 320 µg/cm2. With an EC-3 greater than 20% for ST 23 C 01, the potency value of the test article can only be estimated to be greater than 5'000 µg/cm2.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

DPM measurments for the four first different contrations of test material (1%, 2.5 %, 5%, 10%) were similar to DPM measurement of vehicle. DPM measurements for the 5th and last concentration tested (20%) was roughly 2.5 times higher than vehicle. (See Table 1 in "Remarks on results including tables and figures". )

Any other information on results incl. tables

Irritation: All animal appeared healthy and showed no sign of irritation at the dosing site

Table 1: DPM measurements

Treatment (% of test material)	N° of animal	Mean DPM	Standard Deviation	Standard error of the mean
1	5	31.5	10.8	5.4
2.5	5	57.8	27.8	12.4
5	5	38.9	23.5	10.5
10	5	42.5	6.2	2.8
20	5	85.4	32.3	14.5
AOO	6	31.1	17.7	7.2
Isoeugenol 0.5 %	5	49.5	29.3	13.1
Isoeugenol 1.0 %	5	132.2	122.7	54.9
Isoeugenol 5.0 %	5	757.8	244.9	109.5
AOO= Acetone- Olive Oil (4:1) = vehicle

 

 

Table 2: Stimulation Index Following Exposure to Test & Control Material

Treatment (% of test material)	Mean Stimulation Index (SI)	Standard error of the mean
1	1.0	0.2
2.5	1.9	0.4
5	1.2	0.3
10	1.4	0.1
20	2.7	0.5
Isoeugenol 0.5 %	1.6	0.4
Isoeugenol 1.0 %	4.3	1.8
Isoeugenol 5.0 %	24.4	3.5

 

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Based on the results of this study, the positive control (isoeugenol), with an EC-3 of 0.68% and a potency value of 160 µg/cm2 , would be classified as a moderate sensitizer (potency value between 100 and 1000 µg/cm2) (Gerberick, et al, 2001 American Journal of Contact Dermatitis 42:344-348). This is consistent with previously reported results.
With all SI values less than 3 for ST 23 C 01, a reliable EC-3 potency value could not be calculated. LLNA results showed ST 23 C 01 to be nonsensitizing at all concentrations tested in this study (1%-20%).

Executive summary:

Purpose of the study. The purpose of this study is to evaluate the sensitization potential of ST 23 COl by

measuring its ability to stimulate proliferation of lymphocytes within the auricular lymph nodes of the mouse.

Methods. Mice were treated daily for three consecutive days by direct epicutaneous application of 25 µl of test or

control article to the dorsum of each ear. The test article (ST 23 C 01) was tested at 1.0%, 2.5%, 5.0%,

10% and 20% final concentration in acetone/olive oil (4: 1). The control articles included the vehicle

acetone/olive oil (AOO) 4:1, and a known sensitizer, isoeugenol, that was tested at concentrations of 0.5%,

1.0% and 5.0% in acetone/olive oil (4:1). The mice were observed daily and no irritation or other signs of

toxicity were noted. Three days after the final auricular application, the animals were injected

intravenously (iv) with 125-I labeled IuDR to label proliferating cells. 125-I incorporation was quantified

using a gamma counter.

Results. A substance is considered a sensitizer if at least one concentration of the test material results in a

statistically significant 3-fold or greater stimulation index (SI). The highest dose (20%) of the test article

(ST 23 CO 1) had an SI = 2.7. None of the doses tested had an SI greater than, or equal to 3.

The 5% concentration of isoeugenol resulted in a group SI greater than 3. Statistical analysis (one-sample t

tests) showed this SI value was statistically significantly greater than 3.0 (p ¿ 0.05). The 1% concentration

had an SI of 4.3. This concentration is normally considered non-sensitizing. Statistical analysis ofthe 1%

concentration showed that this SI = 4.3 was not statistically significantly greater than 3.

For isoeugenol with an EC-3 of 0.64%, the EC-3 potency value was calculated to be 160 µg/cm2. Since no

concentration tested resulted in an SI> 3, no reliable EC-3 value could be calculated for ST 23 C0 1

Based on the results from this study, the positive control isoeugenol would be classified as a moderate

sensitizer (potency value between 100-1000 µg/cm2) (Gerberick et al., 2001). This is consistent with

previously reported results. The test compound, ST 23 C0l, was non-sensitizing at the doses tested (1%-20%)