Cyclopentaneacetic acid, 3-oxo-2-pentyl-, methyl ester, (1R,2S)-rel-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From 8 October to 14 October 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed in compliance with GLP and similarly to the OECD test guideline No. 429. The maximum concentration tested was not justified: a screening study was not performed and ear thickness measurements were not included. Therefore, this study was used within a weight-of-evidence approach to support the absence of skin sensitisation properties of the registered substance. The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Maximal dose tested was 40%, this did not affect the relevance of the test

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3-oxo-2-pentylcyclopentaneacetate

EC Number:

246-495-9

EC Name:

Methyl 3-oxo-2-pentylcyclopentaneacetate

Cas Number:

24851-98-7

IUPAC Name:

methyl (3-oxo-2-pentylcyclopentyl)acetate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): ST 35 C 03
- Substance type: pure active substance

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratory (Bar Harbor, ME, - USA)
- Age at study initiation: 6 to 7 weeks of age
- Weight at study initiation: 17.1-22.3 g
- Housing: individually in plastic shoebox-style cages at the North Carolina State University College of Veterinary Medicine.
- Diet (e.g. ad libitum): Purina Rodent Chow 5002 and City of Raleigh tap water ad libitum.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3-23.4
- Humidity (%): 28-73. Some humidity readings were slightly outside the range recommended in the OECD Guideline 429 (at least 30%) on October 3 and 4 (both 28%). This deviation is not expected to have any effect on data quality or integrity, as per a letter on file from the Director of Laboratory Animal Resources.
- Air changes (per hr): not mentioned in the study report
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

The test article, ST 35 C 03, was tested at 1%,, 5%, 10% , 20% and 40%, 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: 8 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 35 C 03, was tested at 1%, 5%, 10%, 20% and 40%, 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 (labelled by tail markings) were injected in the lateral tail vein with 0.25 ml containing 2 µCi of I-125 labelled 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, VWR) and refrigerated at approximately 4°C. Approximately 18.5 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 of variance was used using dose (concentration). 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 data from the concentration tested 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 concentration of chemical required to elicit an SI of 3 (EC-3) was determined from the appropriate regression equation.

Ear thickness measurements were checked for 10%-or-greater increases between Days 1 and 3. When applicable, the lowest concentration to elicit such an increase (minimal irritating concentration or MIC10) was determined. The MIC10 was then compared to the calculated EC-3. If the MIC10 was greater than the EC-3 (MIC10 > EC-3), confounding irritation is unlikely to have affected the LLNA. If the MIC10 was less than the EC-3 (MIC10 < EC-3), the concentration of the chemical that elicited the increase may have produced an LLNA stimulation index close to 3 because of the physiology surrounding the primary irritation reaction (Anderson et al., 2003).
All calculations were performed using Microsoft® Excel and SAS®, version 8. PROCs GLM, FREQ, NPARIWAY, and MEANS were utilized.

Results and discussion

Positive control results:

An EC-3 of 0.98% was determined for the positive control isoeugenol in the current study using a linear regression method with a 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 245 µg/cm2 was calculated. These data would classify isoeugenol as a moderate sensitizer (potency value between 100 and 1000 µg/cm2) and are consistent 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: SI = 1.7, 1.0, 1.0, 1.2 and 1.7 for 1, 5, 10, 20 and 40%, respectively

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM = 20.3, 34.2, 20.4, 19.6, 24.6 and 33.63, for AOO, 1, , 5, 10, 20 and 40 %, respectively.

Any other information on results incl. tables

No irritation or other adverse toxic effects were visible in any of the mice used in the repeat study. Three of the 48 mice assigned to the study lost minimal amounts of weight between randomization and lymph node harvest (0.2-0.4 grams). The cause of this mild weight loss, distributed across treatment groups, is unknown. Body weights at lymph node harvest ranged from 17.7 to 23.0 grams.

Individual and group DPM values and group SI values appear in Tables 1 and 2 (See Tables of Results in “Attached background material”), respectively. Except for that of animal #48, a DPM outlier, all calculated DPM values were used in data calculations and statistical analyses.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

With all SI values less than 3, an EC-3 potency value could not be calculated. LLNA results showed the test material to be non sensitizing at all concentrations tested in this study (1%-40%).

Executive summary:

In a dermal sensitization study performed similarly to the OECD test guideline No. 429 and in compliance with Good Laboratory Practice, the test material was tested in female CBA/J Hsd mice using the Local Lymph Node Assay

In the main test, mice were allocated to the following groups:

• Five treated groups of five animals receiving the test material at the concentration of 1.0, 5, 10, 20 or 40 % in the vehicle acetone/olive oil (4:1; v/v),

• One negative control group of eight animals receiving the vehicle,

• Three positive control groups of five animals receiving the reference item, Isoeugenol, a moderate sensitizer, at the concentration of 0.5, 1 or 5 % in the vehicle.

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of125I- Iododeoxyuridine (day 6). The obtained values were used to calculate Simulation Indices (SI).

The mice were observed daily and no irritation or other signs of toxicity were noted.

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 (40%) of the test article (ST 35 C 03) had an SI = 1.7. 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 1.6.

For isoeugenol with an EC-3 of 0.98%, the EC-3 potency value was calculated to be 245 µg/cm². Since no concentration tested resulted in an SI> 3, no reliable EC-3 value could be calculated for the test material. 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). This is consistent with previously reported results. The test material was non-sensitizing at the doses tested (1%-40%).

Under the test conditions, the test material is not classified as a dermal sensitizer in the murine Local Lymph Node Assay according to the Directive 67/548/EEC and the Regulation (EC) No. 1272/2008 (CLP).

The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification).