Reaction mass of butyl palmitate and butyl oleate and butyl (9Z,12Z)-octadeca-9,12-dienoate and 482-680-2

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-23 to 2009-12-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Deviations:
- The humidity of the animal room was lower than the SOP stated minimum value of 30% on 7, 8, and 10-13 December 2009 (19%-25%). The temperature of the animal room exceeded the SOP stated range of 18°C – 26°C on 8 December 2009 (range = 17.4°C – 29.0°C). The magnitude and duration of these changes did not have an effect on the study outcome.
- As per protocol, the mice were supposed to be 8-12 weeks of age at start of dosing. The mice were approximately 7 weeks of age on the first day of dosing. This deviation did not have an impact on the study evaluation.

Deviations:

yes

Remarks:

For the deviations explanation, please check the textbox above "Version/remarks"

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of butyl palmitate and butyl oleate and butyl (9Z,12Z)-octadeca-9,12-dienoate and 482-680-2

EC Number:

944-892-8

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of butyl palmitate and butyl oleate and butyl (9Z,12Z)-octadeca-9,12-dienoate and 482-680-2

Test material form:

liquid: viscous

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Weight Range: 16.6 to 21.9 grams on 09 Dec 2009, the day before dosing.
Age: Approximately 7-8 weeks at start of dosing.
Indeitification: Animal numbers marked on tails in permanent ink.
Animal housing: Group housed with up to 5 mice per cage during acclimation and then housed individually for remainder of study.
Cage identification: Labeled with study number, animal number, treatment, and dose.
Room temperature: 17.4°C - 29.0°C.
Cage Bedding: Bed-o' cobs.
Relative Humidity: 19% - 48%.
Lighting: 12-hour light/12-hour dark cylce.
Acclimation: 7 days.
Food: PMI(R) LabDiet(R) (5002 Certified Rodent Diet)
Water: Town of Cary tap water ad libitum.
Clinical Observations: Mice were monitored daily.

The were no contaminants in the food or water that were known to influence the outcome of the study. The animal supplier's Animal Health Report (performed quarterly) does not indicate levels of bacteria or other pethogens that would have changed the outcome of the study.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10%, 25%, and 50% v/v in AOO and 100% (undiluted)

No. of animals per dose:

8 animals/vehicle control group (AOO).
5 animals/test-article treatment group.
5 animals/positive control group (isoeugenol)

Details on study design:

The mice were restrained by hand, and 25 µL of test or control article was supplied daily for 3 consecutive days to the dorsum of each ear using a calibrated Finnpipette(R). The animals were allowed to rest without dosing on Days 4 and 5. The mice were observed daily for signs of toxicity or irritation.

Ear measurements were taken prior to dosing on Days 1 and 3 using an Oditest micrometer (Kroeplin; The dyer Company, Lancaster, PA). Percentage increase in ear thickness from Day 1 to Day 3 was calculated.

Assay procedures were performed as per SOP BRT 601-04. In brief, on Day 6, individually nulbered mice wre injected in the lateral tail vein with 0.25 mL containing 2 µCi of 125I-labelled 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 and then with PBS prior to being resuspended in 5% trichloroacetic acid (TCA) and refrigerated at approximately 4°C. The following day, the cells were centrifuged and resuspended in fresh 5% TCA. The radioactivity was measured using a gamma counter (Packard Instrument).

Positive control substance(s):

other: Isoeugenol

Statistics:

The natural log-transformed DPM values for the test article and positive control were compared against vehicle for variance homogeneity using Bartlett’s chi-square test. Compounds with nonsignificant Bartlett’s chi-square values were evaluated with one-way analysis of variance (ANOVA) using dose (concentration). If the ANOVA values fit (p < 0.05), Dunnett’s t test was applied to determine which concentrations were different from vehicle by statistically significant margins. For compounds with significant Bartlett’s chi-square terms, the nonparametric Kruskal-Wallis (KW) test was applied. Significant (p < 0.05) KW test results were further analyzed with the Jonckheere–Terpstra test for dose-dependent trends.
The concentration of chemical required to elicit an SI of 3 (EC-3) was determined from the appropriate regression equation. Stimulation index values from the test article concentrations were fit using a quadratic equation. If the quadratic term did not fit, linear regression was used. Because the positive control had only two data points, a linear equation was used to fit the positive control SI data.
To determine EC-3 potency, the derived EC-3 value is expressed in terms of dose per unit area of skin, assuming a conversion of 1 milliliter to 1 gram. With 25 L of test article applied to an exposure area of 1 cm2 per mouse ear, potency values were calculated by multiplying the EC-3 by 250.
Ear thickness measurements were checked for 10%-or-greater increases between Day 1 and Day 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.
All calculations were performed using Microsoft® Excel and SAS®, version 9.1. The following SAS statistical procedures were utilized: GLM, FREQ, NPAR1WAY, and MEANS.

Results and discussion

Positive control results:

The positive control, isoeugenol at 5%, has a statistically significant SI value of 7.9. Isoeugenol has an EC-3 of 1.5 % and an EC-3 potency value of 375 g/cm2, making it a moderate sensitizer (potency value between 100 and 1,000 g/cm2) (Gerberick et al., 2001).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Variability:

SE (standard error of the mean) : 0.2

Test group / Remarks:

5 alpha Avocuta in AOO - 10%

Remarks on result:

other: no indication of skin sensitisation

Results 2

Key result

Parameter:

SI

Value:

1.1

Variability:

SE (standard error of the mean): 0.2

Test group / Remarks:

5 alpha Avocuta in AOO - 25%

Remarks on result:

other: no indication of skin sensitisation

Results 3

Key result

Parameter:

SI

Value:

1.3

Variability:

SE (standard error of the mean): 0.3

Test group / Remarks:

5 alpha Avocuta in AOO - 50%

Remarks on result:

other: no indication of skin sensitisation

Results 4

Key result

Parameter:

SI

Value:

2.7

Variability:

SE (standard error of the mean): 0.5

Test group / Remarks:

5 alpha Avocuta in AOO - 100%

Remarks on result:

other: no indication of skin sensitisation

Results 5

Key result

Parameter:

SI

Value:

7.9

Variability:

SE (standard error of the mean): 0.8

Test group / Remarks:

Isoeugenol in AOO - 5%

Remarks on result:

other: validation of positive control

Results 6

Key result

Parameter:

EC3

Remarks:

%

Value:

> 100

Test group / Remarks:

5 alpha Avocuta

Results 7

Key result

Parameter:

EC3

Remarks:

%

Value:

1.5

Test group / Remarks:

Isoeugenol

Cellular proliferation data / Observations:

The natural log DPM values are not increased in a dose-related manner.

Any other information on results incl. tables

Ear measurement Summary Table

Treatment	Mean % increase	SE (Standard error of the mean)
AOO Vehicle Control	0.2	0.22
5 alpha Avocuta in AOO - 10%	0.6	0.50
5 alpha Avocuta in AOO - 25%	0.8	0.51
5 alpha Avocuta in AOO - 50%	2.2	0.50
5 alpha Avocuta - 100%	2.5	0.56
Isoeugenol in AOO - 5%	1.6	0.63

Applicant's summary and conclusion

Interpretation of results:

other: not considered a sensitizer

Conclusions:

A substance is considered a sensitizer if at least one concentration of the test material results in an SI of 3 or more. None of the SI values for 5 alpha Avocuta(R) are greater than 3 (SI value are 1.0, 1.1, 1.3, and 2.7 for the 10%, 25%, 50%, and 100% concentrations, respectively). The natural log DPM values are not increased in a dose-related manner and the calculated EC-3 is greater than 100%. Therefore, 5 alpha Avocuta(R) is not considered a sensitizer under the conditions of this study.

Executive summary:

This study evaluated the test article 5 α Avocuta® in one local lymph node assay in mice (one study number/protocol for the study). The report contained herein is for test article 5 α Avocuta®.

Mice were treated daily for 3 consecutive days by direct epicutaneous application of 25 µL of test or control article to the dorsum of each ear. Test article 5 α Avocuta® was tested at 10%, 25%, and 50% final concentrations in acetone/olive oil 4:1 (AOO) and at 100% (undiluted). The control articles included the vehicle control (AOO) and a known sensitizer, isoeugenol, which was tested at 5% in acetone/olive oil 4:1 (AOO). The mice were observed daily. Three days after the final auricular application, the animals were injected intravenously with 125I- labeled Iododeoxyuridine to label proliferating cells. 125I-incorporation was quantified using a gamma counter.

There were no compound-related changes in body weights. All animals in the 100% 5 α Avocuta® group had clinical observations of slight ear swelling. But the measured increase in mean ear thickness was less than 10% for this treatment group and all other 5 α Avocuta®, AOO control, and isoeugenol groups. Thus primary irritation is unlikely to have affected the LLNA stimulation indices for this study. There were no other compound-related clinical observations.

A substance is considered a sensitizer if at least one concentration of the test material results in an SI of 3 or more. None of the SI values for 5 α Avocuta® are greater than 3 (SI values were 1.0, 1.1, 1.3, and 2.7 for the 10%, 25%, 50%, and 100% concentrations, respectively). The natural log DPM values are not increased in a dose-related manner and the calculated EC-3 is greater than 100%. Therefore, 5 α Avocuta® is not considered a sensitizer under the conditions of this study.

The positive control, isoeugenol at 5%, has a statistically significant SI value of 7.9. Isoeugenol has an EC-3 of 1.5 % and an EC-3 potency value of 375 µg/cm2, making it a moderate sensitizer (potency value between 100 and 1,000 µg/cm2) (Gerberick et al., 2001). This is consistent with previously reported results.