Dimethylcyclohex-3-ene-1-carbaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-05-2008 to 21-05-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26th November, 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethylcyclohex-3-ene-1-carbaldehyde

EC Number:

248-742-6

EC Name:

Dimethylcyclohex-3-ene-1-carbaldehyde

Cas Number:

27939-60-2

Molecular formula:

C9H14O

IUPAC Name:

(1R,6R)-3,6-dimethylcyclohex-3-ene-1-carbaldehyde; (1R,6R)-4,6-dimethylcyclohex-3-ene-1-carbaldehyde

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME
- Age at study initiation: 9-10 weeks at start of dosing
- Weight at study initiation: 18-23 grams at the outset (Day 1) of the study.
- Housing: Animals were group housed (5 per cage) upon receipt in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals".
- Diet: All animals had access to Harlan Teklad Rodent Chow 7012C ad libitum.
- Water: Tap water was available ad libitum, via water bottles.
- Acclimation period: a minimum of 6 days prior to their first day of dosing.
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 27
- Humidity (%): 22 - 60
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: Diethyl Phathalate Special Odorless (1:3 ETOH/DEP)

Concentration:

1, 2.5, 5, 10 or 25 % (v/v)

No. of animals per dose:

5

Details on study design:

JUSTIFICATION DOSE LEVELS
In general, the doses were selected so that the highest concentration maximizes exposure while avoiding systemic toxicity and excessive local irritation. Doses were selected based on known reported uses of the material.

ANIMAL ASSIGNMENT AND TREATMENT
- Animals were assigned to study groups based on body weight and apparent good health. Mice were given identification numbers and identified by tail mark with an indelible marker. Treatment of animals was in accordance with the study protocol and also in accordance with SOP's which adhere to the regulations outlined in the USDA Animal Welfare Act (9 CFR Parts 1, 2 and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press).

IN-LIFE OBSERVATIONS AND MEASUREMENTS
- Mortality/ morbidity: Daily on Days 1 to 6
- Clinical Observations: Immediately prior to dose administration and immediately post-dose on Days 1 to 3. Clinical observations were performed once daily on Days 4 to 6. Any build up of test article was recorded.
- Dermal Irritation: Daily for signs of erythema and edema. Irritation was scored and recorded using the Draize scoring system. Scoring was performed prior to dosing on Days 1-3.
- Body weight: Animals were weighed on Days 1 and 6.

TREATMENT PREPARATION AND ADMINISTRATION
- Route: Topically on the dorsal surface of both ears
- Frequency: Once daily for 3 consecutive days (Days 1-3). The timing of dose administration remained consistent (± 2 hours) during the dosing phase.
- Procedure: A volume of 25 µL/ear was applied using a micro pipette.
- On Day 6 the mice were injected i.v. with 20 µCi of ³H-thymidine in 250 µL of sterile saline. Five hours later the mice were euthanized by CO2 asphyxiation and the draining auricular lymph nodes were removed.
- At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded.
- The lymph nodes from each group were pooled and a single cell suspension was prepared, Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8°C. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing scintillation fluid. An additional a mL of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid.
- Incorporation of ³H-thymidine was measured in a ß-scintillation counter.

EVALUATION CRITERIA
Increases in 3H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response was that one or more concentration of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.

Positive control substance(s):

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

Statistics:

The evaluation of the equality of means for body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, a Dunnett’s test was used to determine the degree of significance from the control means.

Results and discussion

Positive control results:

The positive control substance, hexyl cinnamic aldehyde, gave a Stimulation Index of 1.75, 3.97 and 7.48 when tested at a concentration of 5, 15 and 35 % v/v, respectively, in diethyl phthalate/ethanol 3:1.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.1

Remarks on result:

other: 1% (v/v)

Results 2

Parameter:

SI

Value:

2

Remarks on result:

other: 2.5% (v/v)

Results 3

Parameter:

SI

Value:

2.9

Remarks on result:

other: 5% (v/v)

Results 4

Key result

Parameter:

SI

Value:

4.2

Remarks on result:

other: 10% (v/v)

Remarks:

Although the data indicated a positive response, the EC3 could not be calculated since a normal dose range curve was not achieved.

Results 5

Key result

Parameter:

SI

Value:

2.7

Remarks on result:

other: 25% (v/v)

Remarks:

While treatment with the test substance did not yield an SI of 3, this may be inaccurate given the other results at lower concentration. It is, however, impossible to make a definitive assessment since individual DPMs were not obtained.

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
- At termination, the lymph nodes from the mice in the vehicle group and the test article treated animals were normal in size and appearance.

EC3 CALCULATION
- Although the data indicated a positive response (10 % (v/v)), the EC3 could not be calcuIated since a normal dose range curve was not achieved.

CLINICAL OBSERVATIONS
- No erythema or edema was noted in any of the mice in the vehicle group or in those dosed with the test article.
- There were no other findings.

MORTALITY
- There was no mortality and all animals appeared normal throughout the study.

BODY WEIGHTS
- There were no statistically significant differences observed between any of the treatment groups.

Applicant's summary and conclusion

Interpretation of results:

other: skin sensitizer 1B

Remarks:

in accordance with EU CLP (EC No. 1272/2008 and its updates)

Conclusions:

The application of the test substance at 10% (v/v) in ETOH/ DEP (1:3) resulted in an increase in isotope incorporation which was greater than 3-fold. Although the EC3 could not be calcuIated because a normal dose range curve was not achieved, based on the positive results of this study, the test substance is considered to be sensitising to the skin.

Executive summary:

The skin sensitisation potential of the test substance has been tested using the Local Lymph Node Assay (LLNA) according to OECD TG 429 and GLP. The application of the test substance at concentrations of 1, 2.5, 5, 10 and 25% (v/v) in ETOH/DEP (1:3) resulted in an increase in isotope incorporation which was greater than 3-fold only at the 10% v/v concentration. Based on this result, no EC3 value could be calculated since a normal dose range curve was not achieved, but the NOEC can be derived and it is 5%. Still, based on the results of this study, the test substance is considered to be sensitising to the skin.