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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 28 August 2001 to 10 September 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study performed in compliance with GLP and similarly to the OECD test guideline No. 429 although conducted one year before its adoption. The maximum concentration tested was not justified: a screening study was not performed and ear thickness measurements were not included. However, the substance being a skin irritant, it probably could not be tested at higher concentrations. Nevertheless, this study was used within a weight-of-evidence approach to support the absence of skin sensitisation properties of the substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
dose selection was not justified, no preliminary study, no ear thickness measurements
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): ST 23 C 01
- Substance type: pure active substance
- Physical state: liquid
- Storage condition of test material: in its original container at room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN - USA
- Age at study initiation: > 6-8 weeks
- Weight at study initiation: 16.6-24.3 g
- Housing: mice were housed individually in plastic shoebox-style cages.
- Diet (e.g. ad libitum): ad libitum (Purina Rodent Chow 5002)
- Water (e.g. ad libitum): City of Raleigh tap water (ad libitum)
- Acclimation period: yes


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 24.8 °C
- Humidity (%): 52-81 %. Humidity readings in the animal room (52-81°C) exceeded the 70% limit value recommended by OECD Guideline. This deviation is not expected to have a major impact on test results.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 5 Sept 2001 To: 10 Sept 2001

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1, 2.5, 5, 10 and 20 %
No. of animals per dose:
5 animals per dose (excepted for vehicle control: 6 animal per dose)
Details on study design:
PRE-SCREEN TESTS: not performed
- Compound solubility: soluble at the dose prepared (i.e. up to 20 % in AOO)

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: individual 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 23 C 01, was tested at 1%, 2.5%, 5%, 10%, and 20%, 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 tbe 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; LabChem) and refrigerated at approximately 4°C. Approximately 19 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) at 0.5, 1.0 or 5.0 %
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 ofvariance 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 ST 23 C 01 data had a non-significant quadratic term (p=0.3087) and, therefore, the linear model was the better model for determination ofthe EC-3. A fitted linear equation was used to fit the data from the concentrations tested.
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 (p=0.5024) and the linear model was therefore the better model.

Results and discussion

Positive control results:
A quadratic regression model for isoeugenol resulted in a non-significant quadratic term (p=0.5024). 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. An EC-3 of 0.64% was determined for the positive control isoeugenol in the current study using a linear regression method with an 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 160 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
Key result
Parameter:
SI
Value:
1
Variability:
0.2
Test group / Remarks:
1.0%
Key result
Parameter:
SI
Value:
1.9
Variability:
0.4
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
1.2
Variability:
0.3
Test group / Remarks:
5.0%
Key result
Parameter:
SI
Value:
1.4
Variability:
0.1
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
2.7
Variability:
0.5
Test group / Remarks:
20%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
DPM = 31.1, 31.5, 57.8, 38.9, 42.5 and 85.4, for AOO, 1, 2.5, 5, 10 and 20%, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
Calculated by dividing the treatment group mean DPM by the control (vehicle) group mean DPM.

EC3 CALCULATION
Not applicable, all SI < 3

CLINICAL OBSERVATIONS:
No irritation or other adverse effects were noted in any of the mice used in this study.

BODY WEIGHTS
Not reported

Any other information on results incl. tables

Irritation:

All animal appeared healthy and showed no sign of irritation at the dosing site

Table 7.4.1/1: DPM measurements

Treatment (% of test material)

N° of animal

Mean DPM

Standard Deviation

Standard error of the mean

1

5

31.5

10.8

5.4

2.5

5

57.8

27.8

12.4

5

5

38.9

23.5

10.5

10

5

42.5

6.2

2.8

20

5

85.4

32.3

14.5

AOO

6

31.1

17.7

7.2

Isoeugenol

0.5 %

5

49.5

29.3

13.1

Isoeugenol

1.0 %

5

132.2

122.7

54.9

Isoeugenol

5.0 %

5

757.8

244.9

109.5

 

AOO= Acetone- Olive Oil (4:1) = vehicle

 

 

Table 7.4.1/2: Stimulation Index Following Exposure to Test & Control Material

Treatment (% of test material)

Mean Stimulation Index (SI)

Standard error of the mean

1

1.0

0.2

2.5

1.9

0.4

5

1.2

0.3

10

1.4

0.1

20

2.7

0.5

Isoeugenol 0.5 %

1.6

0.4

Isoeugenol 1.0 %

4.3

1.8

Isoeugenol 5.0 %

24.4

3.5

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test material is not classified as skin sensitizer according to the Regulation (EC) No. 1272/2008.
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/J Hsb strain mouse following topical application to the dorsal surface of the ear. The method was performed in compliance with GLP and similarly to the OECD test guideline No. 429 although conducted one year before its adoption.

 

Five groups, each of five animals, were treated for three consecutive days with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 20, 10, 5, 2.5 or 1.0%. A further group of six animals was treated with acetone/olive oil 4:1 alone. Three concurrent positive control groups, using five animals each, were also performed with the known sensitizer, Isoeugenol at concentration of 0.5, 1 or 5% w/w acetone/olive oil 4:1. 25%

The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 25 I- Iododeoxyuridine.

Stimulation index for 20, 10, 5, 2.5 or 1.0%. in acetone/olive oil 4:1 were 1.0, 1.9, 1.2, 1.4 and 2.7, respectively. No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations of 10%, 5% or 2.5 w/w.

The 5% concentration of isoeugenol resulted in a group SI greater than 3. Statistical analysis (one-sample t tests) showed this SI value was statistically significantly greater than 3.0 (p ≤ 0.05). The 1% concentration had an SI of 4.3. This concentration is normally considered non-sensitizing. Statistical analysis of the 1% concentration showed that this SI = 4.3 was not statistically significantly greater than 3. For isoeugenol with an EC-3 of 0.64%, the EC-3 potency value was calculated to be 160 µg/cm², thus, demonstrating the sensitivity and reliability of the test system.

 

Under the test conditions, the test material is not classified as skin sensitizer according to the Regulation (EC) No. 1272/2008.

In this study, the maximum concentration tested was not justified: a screening study was not performed and ear thickness measurements were not included. However, the substance being a skin irritant, it probably could not be tested at higher concentrations. Nevertheless, this study was used within a weight-of-evidence approach to support the absence of skin sensitisation properties of the substance.