Cyclohexanecarboxylic acid, 1-(2-ethylbutyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 24, 2010 - December 13, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Temporary deviations from the minimum level of temperature and relative humidity occurred.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Temporary deviations from the minimum level of temperature and relative humidity occurred.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

Temporary deviations from the minimum level of temperature and relative humidity occurred.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1-(2-ethylbutyl)cyclohexane-1-carboxylic acid

EC Number:

619-508-4

Cas Number:

381209-09-2

Molecular formula:

C13 H24 O2

IUPAC Name:

1-(2-ethylbutyl)cyclohexane-1-carboxylic acid

Details on test material:

- Name of test material (as cited in study report): CAT-Acid
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark
- Density: 0.98 g/mL
- pH (1% in water, indicative range): 5.4-5.2

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France.
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. On Day 6, the animals were group housed in cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: at least 5 days.
- Health inspection: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.9 – 21.5
- Humidity (%): 21 - 60
1. Temporary deviations from the minimum level of temperature and relative humidity occurred.
Evaluation: The minimum relative humidity of 21% occurred on a single day of the acclimatization period of main study animals. Given that on other study days of the main study the relative humidity was 39% at the lowest (i.e. only just outside protocol specifications), and that the temperature deviation was marginal, no adverse effect on the study outcome was expected. The minimum level of relative humidity and the temperature deviation was considered to have no adverse effect on the study outcome based on laboratory historical data.
The study integrity was not adversely affected by the deviation.
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: November 24, 2010 to December 13, 2010

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 25, 50 and 100%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
A Pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give moderate irritation at the most (maximum grade 2) and is the highest possible concentration that can technically be applied.
Two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids).
The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old; source: Janvier, Le Genest-Saint-Isle, France). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 12.5 cm). Animals were sacrificed after the final observation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI = 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.
The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
In order to obtain homogeneity, the test substance was heated in a water bath to a maximum of 51.5 ºC for approximately 1 hour followed by shaking prior to weighing the required amount of test substance. The formulations were heated in a water bath to a maximum temperature of 50.7ºC for approximately 10 minutes. The test substance (formulations) were allowed to cool down to a temperature below 40ºC prior to dosing.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that the vehicle was administered instead of the test substance.

Excision of the nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 µCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6:
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) stored in the refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the OECD scoring system. Furthermore, a description of all other (local) effects was recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
0: No erythema
1: Very slight erythema (barely perceptible)
2: Well-defined erythema
3: Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth)
4: Severe erythema (beet redness) to eschar formation preventing grading of erythema

Oedema formation:
0: No oedema
1: Slight oedema (barely perceptible)
2: Moderate oedema
3: Severe oedema

Necropsy: All animals were subjected to necropsy for gross macroscopic examination.

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.7

Test group / Remarks:

25% Substance concentration

Results 2

Parameter:

SI

Value:

2

Test group / Remarks:

50% Substance concentration

Results 3

Parameter:

SI

Value:

3.8

Test group / Remarks:

100% Substance concentration

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 774, 900 and 1678 DPM respectively. The mean DPM/animal value for the vehicle control group was 443 DPM.

Any other information on results incl. tables

Tables and figures of the Pre-screen test and Main study have been included in the attached document "LLNA tables and figures".

Results Pre-screen test:

Slight irritation of both ears was observed among all animals at 50% and 100%, between Days 2 and 4 (50%) or between Days 2 and 6 (100%). No signs of systemic toxicity were observed in any of the animals examined. The ears and fur of all animals felt greasy on Days 2 and 3 of treatment, which was considered to be due to presence of the test substance. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

Other results - main study:

Slight irritation of both ears was observed among all animals at 50% and 100%, between Days 2 and 6, which was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined. The ears and fur of all animals treated with the test substance felt greasy on Days 2 and 3 of treatment, which was considered to be due to presence of the test substance.

 

The auricular lymph nodes of all animals at 100% and two animals at 50% appeared larger in size when compared to the other treated groups. The auricular lymph nodes of the animals at 25% and those of the control group were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

    

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Assessment of Contact Hypersensitivity to CAT-Acid in the Mouse (Local Lymph Node Assay, 5 females/dose) was conducted according to OECD 429 guidelines and GLP principles.

The results indicate that the test substance could elicit an SI = 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 77.8% was calculated.

Based on these results, according to the recommendations made in the test guidelines, CAT-Acid would be regarded as skin sensitizer.
According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, CAT-Acid should be classified as skin sensitizer (Sub-category 1B as the EC3 value >2%) and labeled as H317: May cause an allergic skin reaction.