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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 8 October to 14 October 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to OECD Guideline study but deviating at the level of the max concentration used

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
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
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ST 35 C 03
- Substance type: pure active substance
- Isomers composition: ca. 75 % cis- and 25 % trans- isomers
- Lot/batch No.: 2F12682
- Expiration date of the lot/batch: January 2004
- Analytical purity: 97.1 %

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 ofage
- 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 mentionned 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 (labeled by tail markings) were injected in the lateral tail vein with 0.25 ml containing 2 µCi of I-125 labeled 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. Ifthe 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 consistant with previously reported results; this demonstrate the capability of the test laboratory to identify positive dermal sensitizers.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The calculated EC-3 for ST 48 C 03 is > 40%. For more details, see Remarks on results including tables and figures.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM measured for the first concentration of test material tested (1%) was ~1.5 higher than vehicle DPM. Then, DPM measurement dropped to values identical to vehicle DPM values (for 5% and 10 % of test material) to increase slightly in a linear manner until a DPM value of ~ 1.5 higher than vehicle DPM. For more details, see Remarks on results including tables and figures.

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.

A substance is considered a sensitizer if at least one concentration of the test material results in an SI value greater than or equal to 3. ST 35 C 03 had SI values less than 3 at all tested concentrations (1-40%).

Isoeugenol at 5.0% yielded the only SI greater than or equal to 3, a value shown to be statistically significant by a one-sample, one-sided Student's t test (SI = 14.7).

For ANOVA and nonparametric results, the isoeugenol model had excellent fit. Isoeugenol had anonsignificant Bartlett's Chi-Square term; therefore, parametric results are reliable. Dunnett'stest found only the 5% concentration of isoeugenol to be different from vehicle. The results of the KW and Jonckheere's tests were both significant, which confirms the parametric Dunnett's test results.

The parametric ANOVA did not fit for ST 35 C 03; therefore, parametric results are invalid. The results of the KW and Jonckheere'stests were also nonsignificant, suggesting no dose response or relationship.

The quadratic and linear models had excellent fit for isoeugenol, although the quadratic term wasnonsignificant. The EC-3 (linear model) was calculated to be 0.98%. Neither model fit for ST 35 C 03, thus a valid EC-3 for the test article could not be calculated.

The EC-3 potency value for isoeugenol was determined to be 245 µg/cm2. Calculation of the potency value assumed a conversion of 1 mm = 1 g and was based on an exposure area of 1 cm2 per mouse ear and the application of 25µl.

None of the tested mice experienced 10%-or-greater increases in ear thickness between Day 1 and Day 3, thus there was no irritation reaction to potentially affect the LLNA stimulation indices.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Based on the results of this study, the positive control (isoeugenol), with an EC-3 of 0.98% and a potency value of 245 µg/cm2, would be classified as a moderate sensitizer (potency value between 100 and 1000 µg/cm2) (Gerberick et al., 2001). This is consistent with previously reported results.
LLNA results showed ST 35 C 03 to be non sensitizing at all concentrations tested in this study (1-40%). With an EC-3 greater than 40% (highest dose tested), the EC-3 potency value can be estimated to be greater than 10'000 µg/cm2.
Mean ear thickness values, which were taken on Days 1 and 3, did not increase by 10% or more at any tested concentration of either isoeugenol (0.5% -5.0%) or ST 35 C 03 (1%- 40%), thus there was no irritation reaction to potentially affect the LLNA stimulation indices.
Executive summary:

Mice were treated daily for three consecutive days by directepicutaneousapplication of 25 µl of test or control article to the dorsum of each ear. The test article, ST 35 C 03, was tested at 1%, 5%, 10%, 20%, and 40% final concentrations in acetone/olive oil. The control articles included the vehicle, acetone/olive oil (AOO; 4:1), and a known sensitizer, isoeugenol. Isoeugenol was tested at 0.5%, 1.0%, and 5.0% concentrations in AOO. The mice were observed daily.

Three days after the final auricular application, the animals were injected intravenously with I-125 labeledlododeoxyuridine to label proliferating cells. I-125 incorporation was quantified using a gamma counter.

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. Isoeugenol had an SI value greater than 3 at the 5.0% concentration only (SI = 14.7). ST 35 C 03 had no SI value greater than or equal to 3 at any tested concentration (1% -40%).

Mean ear thickness values, which were taken on Days 1 and 3, did not increase by 10% or more at any tested concentration of either isoeugenol (0.50- 5.0%) or ST 35 C 03 (1.0-40%).

Isoeugenol, with an EC-3 of 0.98% and a potency valueof 245 µg/cm2,would be classified as a moderate sensitizer (potency value between 100 and 1000 µg/cm2) (Gerbericket al., 2001). This is consistent with previously reported results.

LLNA results showed ST 35 C 03 to be nonsensitizing at all concentrations tested in this study (0-40%). With an EC-3 value greater than 40% (highest dose tested), the EC-3 potency value can be estimated to be greater than 10000 µg/cm2.