Registration Dossier

Administrative data

Description of key information

- LLNA, Not sensitising (similar to OECD 429, GLP, read-across, WoE, Rel.2);
- GPMT, Not sensitising (OECD 406, GLP, read-across, WoE, Rel.2).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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.
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)
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
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.
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.
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

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

 

 

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.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 22 March 1989 to 27 April 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study performed in compliance with GLP and according to EU Method B.6 (1981 version), therefore positive control data were not required. Based on current requirements, the sensitivity and the reliability of the method cannot be ascertained. This study is therefore used within a weight-of-evidence approach.
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
(adopted in 1981)
Deviations:
yes
Remarks:
positive control data are not included
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time of study completion, the LLNA method was not adopted.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Darley Oaks, Burton-on Trent, Staffordshire - UK (delivered on 17th March 1989)
- Age at study initiation: young
- Weight at study initiation: 364-495g
- Housing: Animals were housed in groups of ten in stainless steel cages fitted with mesh floors and of the approximate dimensions 85 x 57 x 25cm.
- Diet (e.g. ad libitum): ad libitum (pelleted diet with additional vitamin C (FDI, SQC guinea pig diet, Special Diets Services, Witham, Essex, England))
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 5 days (preliminary study) and 17 days for main study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-25°C (3 days at 16°C between 22nd April 1989 and 25th April 1989)
- Humidity (%): 40-67%.
- Air changes (per hr): no data (air-conditioned room)
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal
Vehicle:
other: light liquid paraffin
Concentration / amount:
5 %
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Day(s)/duration:
Day 7 / 48 hrs
Adequacy of induction:
other: skin not pre-treated with SLS but the test material is a skin irritant
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Remarks:
left flank
Concentration / amount:
100 %
Day(s)/duration:
Day 21 / 24 hrs
Adequacy of challenge:
highest non-irritant concentration
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Ethanol
Remarks:
right flank
Concentration / amount:
50 %
Day(s)/duration:
Day 21 / 24 hrs
Adequacy of challenge:
not specified
No. of animals per dose:
20
Details on study design:
RANGE FINDING TESTS:
Intradermal Injection Concentration Ranging Study: Before the start of the main study an injection concentration ranging study was performed to determine a suitable concentration of the test material for the injection stage of the main study. 0.1 mL aliquots of the supplied test material and 50%, 25%, 10%, 5% and 1% v/v dilutions in light liquid paraffin were each injected intradermally into the flanks of one guinea pig. The animal was observed daily for five days and the response at each injection site noted. A 5% dilution was chosen for the main study injection stage, this being the highest concentration that caused an acceptable localised response during the observation period.

Preliminary Topical Irritancy Ranging Study: In an attempt to determine the maximum non-irritating concentration and a minimum irritant concentration of the test material a dose ranging study was performed using eight animals that had been previously treated with Freund's Complete Adjuvant by injection. Four concentrations of the test material were used. These were the test material as supplied and 50%, 25% and 12.5% v/v dilutions in ethanol. An area 8cm x 5cm was clipped free of fur over the back and flanks of the four animals and four 2 x 2cm patches of Whatman No. 3 filter paper, each saturated with a different concentration of the test material, were placed onto the skin, two patches on each flank. Strips of 5cm wide 'Blenderm' surgical tape were placed over the patches to act as occlusive barriers and the patches held in place for twenty four hours by encircling the trunk of
the animal with 5cm wide 'Elastoplast' elastic adhesive bandage.
Twenty four and forty eight hours after removing the patches and dressings the animals were examined under a standard light source designed to comply with the requirements of BS 950 Part 1 (artificial daylight for the assessment of colour) and any reaction at the treated sites assessed.
Results: One animal used for this preliminary study was humanely killed prior to the assessment of the treatment sites. This action was necessary as the animal had sustained a broken leg. None of the remaining animals exhibited dermal responses to the applications of test material, therefore, the undiluted test material was used for both induction and challenge topical applications.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (Day 0: intradermal injection; Day 7: Topical Application)
- Exposure period: 48h for topical application
INTRADERMAL: 3 pairs of intradermal injection (0.1 mL) on Day 0 as follows:
1.) 50% FCA in distilled water
2.) 5% v/v test material in light liquid paraffin.
3.) 10% v/v dilution of the test material in FCA emulsified with an equal volume of distilled water to give a 5% concentration of the test material.
TOPICAL: 7 days after intradermal injections, the test substance (100%) was applied (patches of Whatman No. 3 filter paper, 4cm x 2cm) and covered with a strip of "Blenderm" surgical tape secured in place and wrapped with "Elastoplast" elastic adhesive bandage (occlusive tape).
- Control group: similarly treated with the exception that ethanol was topically applied instead of the test substance.
- Site: dorsal area between the shoulders

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 21
- Exposure period: 24 h
- Test groups: 100% on the left flank, 50% v/v in ethanol on the right flank. Similarly treated than topical indution (patches 2cm x 2cm)
- Control group: similarly treated with the exception that the test substance was omitted
- Site: left flank (undiluted test material) and right flank (diluted test material)
- Concentrations: 100 and 50 %
- Evaluation (hr after challenge): 48 and 72 hours (24 and 48 hours after removing the patches)
Challenge controls:
The 20 guinea pigs used as Control Group (see Field: "Details on study design") represents the challenge (naive control) group in the current assay.
Positive control substance(s):
no
Positive control results:
Not Applicable
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% and 100% Test material in Ethanol
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% and 100% Test Material in Ethanol
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% or 100% Test material in Ethanol
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% and 100% Test Material in Ethanol
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
one dermal reaction observed for 100% test material only
Remarks on result:
no indication of skin sensitisation
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% and 100% Test Material in Ethanol. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: one dermal reaction observed for 100% test material only.

none

Interpretation of results:
GHS criteria not met
Conclusions:
The test material is not classified as skin sensitiser under the test conditions
Executive summary:

In a dermal sensitisation study performed according to the EU test method B.6 and in compliance with GLP, the test material was tested in female Hartley guinea-pigs using the Guinea-Pig Maximisation Test method (20 treated animals + 10 controls).

The preliminary study determined the concentration to be used for the induction and challenge phases of the main study.

 

The test material diluted in liquid paraffin at 5% (v/v) was administered by injection for intradermal induction. Topical induction was performed with the test material as supplied, 7 days after intradermal injections. For the challenge, the test material was tested at 100% and 50% v/v in ethanol.

 

Following the challenge application, one animal in the test group exhibited responses at the site challenged with the undiluted test material. There were no responses apparent in the control group.

Under the test conditions, the test material is not classified as skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP).

Study performed in compliance with GLP and according to EU Method B.6 (1981 version), therefore positive control data were not required. Based on current requirements, the sensitivity and the reliability of the method cannot be ascertained. This study is therefore used within a weight-of-evidence approach.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to the Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are structurally related, in that both are trans-3,3-dimethyl-5-(2,2,3-trimethyl-cyclopent-3-en-1-yl)pent-4-en-2-ol. As two asymmetric (chiral) carbon atoms are present in this chemical structure (marked with a * in Table 1), the chemical exists as 2x2=4 enantiomers: RR, RS, SR and SS forms. The target substance is a reaction-mass between two of these stereoisomers (RS and SS) whereas the target substance is a mixture of the two others (SR and RR).

3. ANALOGUE APPROACH JUSTIFICATION
Based on structural similarity and comparable physicochemical and toxicological properties, the source and the target substances are expected similar skin sensitisation profile.
The study designs (OECD 429 and 406, GLP) are adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the skin sensitisation studies conducted with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.7.2.

4. DATA MATRIX
Please refer to Iuclid section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
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.
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

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

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the available data on the source substance, the target substance 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 the source substance 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 source substance 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.

 

Based on the available data on the source substance, the target substance 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 source 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.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to the Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are structurally related, in that both are trans-3,3-dimethyl-5-(2,2,3-trimethyl-cyclopent-3-en-1-yl)pent-4-en-2-ol. As two asymmetric (chiral) carbon atoms are present in this chemical structure (marked with a * in Table 1), the chemical exists as 2x2=4 enantiomers: RR, RS, SR and SS forms. The target substance is a reaction-mass between two of these stereoisomers (RS and SS) whereas the target substance is a mixture of the two others (SR and RR).

3. ANALOGUE APPROACH JUSTIFICATION
Based on structural similarity and comparable physicochemical and toxicological properties, the source and the target substances are expected similar skin sensitisation profile.
The study designs (OECD 429 and 406, GLP) are adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the skin sensitisation studies conducted with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.7.2.

4. DATA MATRIX
Please refer to Iuclid section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Positive control results:
Not Applicable
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% and 100% Test material in Ethanol
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% and 100% Test Material in Ethanol
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% or 100% Test material in Ethanol
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% and 100% Test Material in Ethanol
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
one dermal reaction observed for 100% test material only
Remarks on result:
no indication of skin sensitisation
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% and 100% Test Material in Ethanol. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: one dermal reaction observed for 100% test material only.

none

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the available data on the source substance, the target substance is not classified as skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP).
Executive summary:

In a dermal sensitisation study performed according to the EU test method B.6 and in compliance with GLP, the source substance was tested in female Hartley guinea-pigs using the Guinea-Pig Maximisation Test method (20 treated animals + 10 controls).

The preliminary study determined the concentration to be used for the induction and challenge phases of the main study.

 

The source substance diluted in liquid paraffin at 5% (v/v) was administered by injection for intradermal induction. Topical induction was performed with the source substance as supplied, 7 days after intradermal injections. For the challenge, the source substance was tested at 100% and 50% v/v in ethanol.

 

Following the challenge application, one animal in the test group exhibited responses at the site challenged with the undiluted source substance. There were no responses apparent in the control group.

Based on the available data on the source substance, the target substance is not classified as skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP).

Study performed in compliance with GLP and according to EU Method B.6 (1981 version), therefore positive control data were not required. Based on current requirements, the sensitivity and the reliability of the method cannot be ascertained. This study is therefore used within a weight-of-evidence approach.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No study was available on the target substance itself, therefore a read-across approach was used. The source substance is considered adequate for read-across purposes (see Iuclid section 13 for additional justification).

Two skin sensitisation studies were identified on the source substance. Although these studies provided information to assess the skin sensitisation potential of the target substance, none of them was considered as sufficient to be the key study on its own. Therefore, a weight-of-evidence approach was built:

- A Local Lymph Node Assay (LLNA, BRT, 2001, Rel.2)) was performed in compliance with GLP and similarly to the OECD test guideline No. 429 although conducted one year before its adoption. A significant lymphoproliferation was noted in the positive control group, therefore the study was considered valid. The source substance (1%-20%) did not induce delayed contact hypersensitivity in this assay. None of the mice assigned to this study experienced visible irritation or other adverse toxic effects. 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 source 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 target substance.

- A Guinea-Pig Maximisation test (GMPT, Toxicol, 1989, Rel.2) was performed according to the EU test method B.6 and in compliance with GLP. The source substance, diluted in liquid paraffin at 5% (v/v), was administered by injection for intradermal induction. Topical induction was performed with the source substance as supplied, 7 days after intradermal injections. For the challenge, the source substance was tested at 100% and 50% v/v in ethanol. Following the challenge application, one animal in the test group exhibited responses at the site challenged with the undiluted source substance. There were no responses apparent in the control group. Under the test conditions, the source substance is not classified as skin sensitizer. This study performed according to the previous version of the EU Method B.6 (1981), therefore positive control data were not required. Based on current requirements, the sensitivity and the reliability of the method cannot be ascertained.

Based on the whole data, it can be concluded that, based on data on the source substance, the target substance is not a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data on the source substance, no additional self-classification is proposed for the target substance according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

No data was available for respiratory sensitisation. However, this substance is not a skin sensitizer, therefore according to Figure R.7.3 -2 of the Chapter R.7 (V 4.1 - October 2015) the chemical is not considered as a respiratory sensitizer.