[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - February 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

EC Number:

275-156-8

EC Name:

[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Cas Number:

71048-82-3

Molecular formula:

C13H20O

IUPAC Name:

[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN.
- Age at study initiation: 7-8 weeks
- Body weight at study initiation: 16.0 - 22.3 g
- Housing: Individually in plastic shoebox-style cages.
- Diet: Purina Rodent Chow 5002, ad libitum
- Water: City of Raleigh tap water, ad libitum
- Acclimation period: 8-days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.2-27.2
- Humidity (%): 12 -72
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0.25, 0.5, 1.0, 2.5, 5.0%

No. of animals per dose:

5 animals

Details on study design:

Mice were treated daily (24 hrs ± 2 hrs) for three consecutive days. The mice were restrained by hand and a 25 µL volume of test, or control article was applied to the dorsum of each ear using a calibrated Finnpipet. The animals were allowed to rest (no dosing) on Days 4 & 5. The mice were observed daily for signs of toxicity and for mortality. On Day 6, mice were injected in the lateral tail vein with 0.25 mL containing 2 µCi of 125I- labeled Iododeoxyuridine and 10.5 M FuDR 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% tricholoroacetic acid (TCA) and refrigerated at approximately 4°C. Approximately 17 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 at 0.5, 1, and 5%

Statistics:

To test if the compound was a sensitizer, a one-sample t test was performed for testing if the individual untransformed SI values of each dose level of each compound were different than 3.0. 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. 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 test substance data had a significant quadratic term and, therefore, the quadratic model was the better model for determination of the EC-3. 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 and the linear model was therefore the better model. All calculations were performed using Microsoft Excel and SAS, version 6.12. PROCs GLM, FREQ, NPAR1WAY, and MEANS were utilized.

Results and discussion

Positive control results:

In the case of the positive control (isoeugenol), analyses of the natural logarithm of the DPM values had the following results: Bartlett's Chi Square test for homogeneity was found to be non-significant (P = 0.7361), and a one-way analysis of variance was used using dose (significant at p=0.0001). The Dunnett's t test was performed using an alpha of 0.05 and showed that the 5% isoeugenol was significantly different from control. The 5% concentration of isoeugenol resulted in a group SI greater than 3. Statistical analysis (one-sided one-sample Student's t tests) showed that this SI value was statistically significantly greater than 3.0 (using P ≤ 0.05). A quadratic regression model fur isoeugenol resulted in a non-significant quadratic term. 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. The EC-3 was found to be 0.595% using the predicted regression equation SI = -0.2187 + 4.6731I x Dose (in percent). The EC-3 potency value in µg/cm2 for isoeugenol was calculated. In order to calculate the potency value, the conversion from millilitres to grams assumed a conversion of 1mL = 1g and was based on an exposure area of 1 cm2 per mouse ear, and the application of 25 µL. For isoeugenol with an EC-3 of 0.595%, the EC-3 potency value was calculated to be 149 µg/cm2.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

0.866

Remarks on result:

other: see 'Any other information on results incl. tables' field

Results 2

Parameter:

SI

Value:

1.3

Test group / Remarks:

0.25%

Remarks on result:

other: 0.25%

Results 3

Parameter:

SI

Value:

1.8

Test group / Remarks:

0.5%

Remarks on result:

other: 0.5%

Results 4

Parameter:

SI

Value:

3.2

Test group / Remarks:

1.0%

Remarks on result:

other: 1.0%

Results 5

Parameter:

SI

Value:

6

Test group / Remarks:

2.5%

Remarks on result:

other: 2.5%

Results 6

Parameter:

SI

Value:

6.3

Test group / Remarks:

5.0%

Remarks on result:

other: 5.0%

Cellular proliferation data / Observations:

Calculation of EC3:
A quadratic regression model of the data for the test substance had a significant quadratic term and was therefore the preferred model for the determination of the EC-3. The EC-3 was calculated to be 0.866% using the predicted regression equation SI = 0.7410 + (2.9177 x Dose) - (0.3575 x Dose^2). The EC-3 potency value in µg/cm2 for the test substance was calculated. In order to calculate the potency value, the conversion from millilitres to grams assumed a conversion of 1mL = 1g and was based on an exposure area of 1 cm2 per mouse ear, and the application of 25 µL. For the test substance with an EC-3 of 0.866%, the EC-3 potency value was calculated to be 217 µg/cm2.

Other:
Animal #7 was omitted from calculations due to a poor injection and Animal #41 was omitted due to a sample spill during sample processing. Animals # 11, 19, 25, 32, and 34 were DPM outliers (outside of 3 standard deviations from the group mean). All other calculated DPM values were used in data calculations. Analyses of the natural logarithm of DPM values for the test substance had the following results: Bartlett's Chi Square test for homogeneity was found to be non-significant and a one-way analysis of variance was used using dose (significant at p=0.0001). The Dunnett's t test was performed using an alpha of 0.05 and showed that the 1%, 2.5% and 5% dose groups were significantly different from control.

No irritation or other adverse toxic effects were noted in any of the mice used in this study. There was slight weight loss (less than 0.5 g) in 3 mice (Animals # 11 and # 13 dosed at 0.25%, and Animal #14 dosed at 0.5%). Animal # 25 lost 1.3 g (2.5% dose group). The animals appeared healthy otherwise, and the reason for this weight loss is not known.

Applicant's summary and conclusion

Interpretation of results:

other: Skin sensitiser 1A in accordance with EU CLP (EC No. 1272/2008 and its amendments)

Conclusions:

Under the conditions of this study the substance is a skin sensitizer (1A) in the LLNA test (OECD TG 429).

Executive summary:

In a local lymph node assay, performed comparable to OECD Guideline 429 and GLP, nine groups of 5 CBA/J female mice were treated on the dorsal surface of both ears once per day for 3 days with 0.25, 0.5, 1.0, 2.5, and 5.0% of the test substance, with the vehicle alone (acetone/olive oil in a ratio of 4:1) or with the positive control (isoeugenol) at 0.5, 1, and 5%. The mice were observed daily and no irritation at the dosing site or other signs of toxicity were noted. Three days after the final auricular application, the animals were injected intravenously with 125I- labelled iododeoxyuridine to label proliferating cells. 125I-incorporation was quantified using a gamma counter. The test substance at 1, 2.5, and 5.0% had stimulation indices (SI) > 3. Only for the test substance at 5.0% this was statistically significant (Student's t test). The 5% concentration of isoeugenol resulted in a group SI statistically significantly greater than 3. Since the data indicated that the test article was a skin sensitizer, the EC3 was calculated to be 0.866% and the EC-3 potency value was calculated to be 217 µg/cm2.