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Diss Factsheets

Administrative data

Description of key information

Weight of evidence: Skin sensitising, 2019

1. Skin sensitisation: skin sensitising(EC3 = 14.1%), female mice, OECD TG 429, 2001

2. Skin sensitisation: sensitising, Guinea Pig, Open Epicutaneous Test, 2005

Supporting data:

3. Sensitisation data (humans) : no sensitisation at 15% in vehicle in 97/97 humans, HRIPT, 2006

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-07-2005 to 26-08-2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
other: CTFA Safety Testing Guidelines
Version / remarks:
The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, D. C. 20036; "Guidelines for Evaluating Photodermatitis", 1991.
Qualifier:
according to guideline
Guideline:
other: Klecak G., Geleick H. and Frey J.R. (1977). Screening of fragrance materials for allergenicity in the guinea pig. J. Soc. Cosmet. Chem. 28: 53-64.
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: DERMATOTOXICOLOGY, Ed. F.N. Marzulli & H.l. Maibach, 1982. Hemisphere Publ. Co., Chapter 9, Author: G. Klecak. pp. 213-219
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: November 2002 ; signature: March 2003
Type of study:
open epicutaneous test
Justification for non-LLNA method:
Study conducted prior to Regulation (EC) 1907/2006 and/or Regulation (EC) 1272/2008 publication and implementation.
Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
CRL:(HA)BR, SPF
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 5-6 weeks.
- Weight at study initiation: 336 – 372 g
- Housing: individually in stainless steel suspended cages
- Diet (e.g. ad libitum): Standard maintenance/breeding diet for guinea pigs from a recognised supplier (details in the full study report).
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions; identical to the test. One group (group 4) had no acclimatisation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3 °C
- Humidity (%): 30 – 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour light (with music) / 12-hour dark

IN-LIFE DATES: From: To: 29-06-2005 to 04-08-2005
Route:
epicutaneous, open
Vehicle:
polyethylene glycol
Concentration / amount:
- Topical: 0% test material (control group)
Day(s)/duration:
Day 1 - 26
Route:
epicutaneous, open
Vehicle:
polyethylene glycol
Concentration / amount:
- Topical: 25% test material
Day(s)/duration:
Day 1 - 26
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, open
Vehicle:
polyethylene glycol
Concentration / amount:
- Topical: 50% test material
Day(s)/duration:
Day 1 - 26
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, open
Vehicle:
polyethylene glycol
Concentration / amount:
- Topical: 75% test material
Day(s)/duration:
Day 1 - 26
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, open
Vehicle:
unchanged (no vehicle)
Concentration / amount:
- Topical: 100%
Day(s)/duration:
Day 1 - 26
Adequacy of induction:
highest technically applicable concentration used
No.:
#1
Route:
epicutaneous, open
Vehicle:
polyethylene glycol
Concentration / amount:
- Challenge: 5%
Day(s)/duration:
day 29
No.:
#2
Route:
epicutaneous, open
Vehicle:
polyethylene glycol
Concentration / amount:
- Challenge: 10%
Day(s)/duration:
day 29
No.:
#3
Route:
epicutaneous, open
Vehicle:
polyethylene glycol
Concentration / amount:
- Challenge: 15%
Day(s)/duration:
day 29
No.:
#4
Route:
epicutaneous, open
Vehicle:
polyethylene glycol
Concentration / amount:
- Challenge: 25%
Day(s)/duration:
day 29
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Test group: 6 ; Control group: 6
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. By cutaneous route: Tested concentrations: 100%, 75%, 50%, and 25% (w/w). Cutaneous reactions were evaluated approximately 24 and 48 hours after the application.
At 24 and 48 hours: No non-irritating dose was observed. The minimal irritating concentration of the concentrations tested was 25%. Final concentrations for definitive testing based on preliminary irritation study:
- Induction: 0.1 mL of test item, undiluted (100%) and diluted to 25%, 50% and 75% in PEG 300.
- Control: 0.1 mL PEG 300 only.
- Challenge: All animals previously treated in induction, as well as control animals, were treated in the same procedure as the induction period, but with a 5%, 10%, 15% or 25% solution in PEG 300.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: epidermal, daily (5 times per week) for 4 weeks.
- Exposure period: Days 1-26.
- Test groups: I: Control group (PEG 300); II: Test Group 1 (induced with 25% solution); III: Test Group 2 (induced with 50% solution); IV: Test Group 3 (induced with 75% solution); V: Test Group 4 (induced with undiluted test item).
- Control group: PEG 300 (vehicle only).
- Site: epidermal application (clipped right flank);
- Frequency of applications:
- Duration: Daily application (5 days/week) over days 1-26.
- Concentrations: Topical induction: 0.1 mL test item, undiluted and diluted to 25%, 50% and 75% in PEG 300.
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered.

B. CHALLENGE EXPOSURE
- No. of exposures: 1 initial challenge
- Day(s) of challenge: day 29 (topical challenge)
- Exposure period: Days 1-26 (induction) ; Day 29 (topical challenge). The treated sites were assessed for challenge reactions 24, 48 and 72 hours after challenge procedure.
- Test groups: 1 control group, and 4 test groups, all challenged.
- Control group: 1; vehicle only application instead of induction with test item.
- Site: One flank (clipped)
- Concentrations: 5%, 10%, 15%, 25% in PEG 300.
- Evaluation (hr after challenge): 24, 48 and 72 hours after challenge (Day 30, 31, 32).
The control group were treated as described for the experimental group except that induction exposure had been with the vehicle rather than the test item.

OTHER: Mortality, toxicity and body weights along with irritation were examined as part of the study
Challenge controls:
negative control group consisting of 6 females, consistent with the main study (the difference being that instead of the test item only the vehicle was administered during induction)
Positive control substance(s):
yes
Remarks:
Alpha-hexylcinnamaldehyde (HCA)
Positive control results:
A reliability check was performed (presented in the full study report) to check the sensitivity of the test system and the reliability of the experimental techniques used. The study used the same conditions as the main study using alpha-hexylcinnamaldehyde (induced topically with 5%, 10% and 15% solutions in PEG 300, and challenged with 0.1 - 10% challenge concentrations) as positive control.
The highest challenge concentration of 10% chosen as well as the concentration of 5% were irritant as revealed by the positive challenge results obtained with the control group. The threshold for induction of skin sensitisation was approximately 5%. No sensitisation was induced with 1%, 0.3% and 0.1%. The challenge threshold was dependent on the induction concentration used. The declining frequency of positives indicated that the threshold was achieved at 3%.
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
0% (control group); challenge concentration 5%
No. with + reactions:
0
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
0% (control group); challenge concentration 10%
No. with + reactions:
0
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
0% (control group); challenge concentration 15%
No. with + reactions:
0
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
0% (control group); challenge concentration 25%
No. with + reactions:
0
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 25%; challenge concentration 5%
No. with + reactions:
2
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 25%; challenge concentration 10%
No. with + reactions:
4
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 25%; challenge concentration 15%
No. with + reactions:
3
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 25%; challenge concentration 25%
No. with + reactions:
5
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 50%; challenge concentration 5%
No. with + reactions:
2
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 50%; challenge concentration 10%
No. with + reactions:
2
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 50%; challenge concentration 15%
No. with + reactions:
3
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 50%; challenge concentration 25%
No. with + reactions:
2
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 75%; challenge concentration 5%
No. with + reactions:
3
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 75%; challenge concentration 10%
No. with + reactions:
3
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 75%; challenge concentration 15%
No. with + reactions:
5
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 75%; challenge concentration 25%
No. with + reactions:
5
Total no. in group:
6
Clinical observations:
No clinical observations noted.
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 100%; challenge concentration 5%
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
One animal was humanely terminated on day 1, due to emaciation and diarrhoea. No clinical observations were noted in the survivors
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 100%; challenge concentration 10%
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
One animal was humanely terminated on day 1, due to emaciation and diarrhoea. No clinical observations were noted in the survivors
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 100%; challenge concentration 15%
No. with + reactions:
4
Total no. in group:
5
Clinical observations:
One animal was humanely terminated on day 1, due to emaciation and diarrhoea. No clinical observations were noted in the survivors
Reading:
1st reading
Hours after challenge:
72
Group:
test chemical
Dose level:
induction concentration 100%; challenge concentration 25%
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
One animal was humanely terminated on day 1, due to emaciation and diarrhoea. No clinical observations were noted in the survivors
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
PC induction concentration 10%; PC challenge concentration 10%
No. with + reactions:
5
Total no. in group:
6
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item was found to be a skin sensitiser.
Executive summary:

The study was performed using an Open Epicutaneous Test method under GLP. The method was consistent with Klecak G. et. al., (1977). Screening of fragrance materials for allergenicity in the guinea pig. J. Soc. Cosmet. Chem. 28: 53-64, test to assess the skin sensitisation potential of the test item. The concentrations selected for the main study were based on the results of a preliminary study. In the main study, induction was via daily topical application of the test item diluted with polyethylene glycol to 0% (control), 25%, 50%, 75%, and 100% for 5 days/week between days 1 -26. A treatment group of 6 per concentration and a control group of 6, respectively were on day 26, challenged with 5%, 10%, 15% or 25% test item in PEG 300 vehicle. Skin reactions were evaluated 24 to 72 hours after challenge treatment. No clinical signs were reported, with the exception of one animal in the 100%-induction group, which was euthanised on day 1 due to emaciation and diarrhoea. Bodyweight gain in the treated group was comparable to controls, and all survivors gained bodyweight during the study. The maximum score in control was zero. All test groups showed positive results in at least 2 animals. Under the conditions of this study, the test item is considered to be a contact skin sensitiser.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15-04-2004 to 28-04-2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Method performed to guideline, well documented and performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: April 2002 ; signed: May 2002
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: approximately 8 to 12 weeks old (prior to acclimatisation)
- Weight at study initiation: 16 – 24 grams
- Housing: Individually housed, in Makrolon type-2 cages with standard softwood bedding
- Diet: Free access to rodent diet (certified, recognised supplier)
- Water: mains tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 to 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark (at least 8 hours music during light period).

IN-LIFE DATES: From: 21-04-2004 To: 28-04-2004
Vehicle:
other: ethanol:water, 7:3 (v/v)
Concentration:
0% (vehicle control), 1%, 10%, 30%, and 50% (w/v)
Alpha-hexylcinnamaldehyde (positive control), 5%, 10% and 25% (w/v) in acetone:olive oil, 4:1 (v/v) (for non-concurrent positive control ; conducted within 6 months of the study ; details in the full study report)
No. of animals per dose:
Main test: 4 females 0% (vehicle control) and 4 females per test item dose group 1%, 10%, 30%, and 50%
Details on study design:
RANGE FINDING TESTS:
Not applicable. The rationale for the test item dose was based on study sponsor selection, which typically would incorporate all available knowledge on the test item potential local/systemic toxicity prior to testing.

MAIN STUDY
- Compound solubility: Fully soluble in vehicle ethanol:water, 7:3 (v/v) in solubility checks performed within the study.
- Irritation: On the second application day, a slight ear erythema was observed at both dosing sites in all females of Group 4 (30 %) and Group 5 (50%), persisting for a total of four days. In addition, on the second and the third application days, a slight ear swelling was observed at both dosing sites in all mice of Group 5 (50%) and Group 4 (30 %), separately, persisting for the remainder of the in-life phase of the study and for a total of three days.
- Systemic toxicity: None observed.
- Ear thickness measurements: Not conducted.
- Erythema scores: See above. Erythema scores were equal to 1.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Following excision of the nodes. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Five groups of four animals were treated with one test item concentration per group. One group of five animals was treated with vehicle.
- Induction: The dorsal surface of both ears was topically treated (25 μL/ear) with the test item concentration, at approximately the same time on each day with monitoring for local and systemic toxicity during dosing. A rest period was allowed on days 4 and 5. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

- Node excision: Each animal was injected via the tail vein with 0.25 mL of 83.4 μCi/mL 3HTdR. After approximately five hours, all animals were terminated. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 oc for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of 'Ultima Gold' scintillation liquid and thoroughly mixed. The radioactivity was measured using a scintillation counter. Background lebels were also measured in two 1 mL aliquots of 5% trichloroacetic acid.
- Tissue processing and radioactivity measurements: See above. Results from each cell suspension counted on the counter were recorded in disintigrations per minute (DPM). After the DPM values had been calculated, the mean "blank" DPM was subtracted from each individual DPM to obtain corrected DPM values. The mean corrected DPM and standard error of the mean (SE) were determined for each group. The stimulation index (SI) was then calculated by dividing the treatment-group mean DPM by the control (vehicle)-group mean DPM.

Observations:
- Mortality/Viability: Twice daily
- Bodyweights: On Day 1 (pre-dose) and Day 6 (post termination)
- Clinical Observations: Once daily
- Irritation: Once daily.
- Ear Thickness: Not conducted.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables in the study report. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation

EC3 = (a-c) [(3-d)/(b-d)] + c

where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c. d) are respectively the co-ordinates of the two pair of data lying immediately below and above the S.l. value of 3 on the local lymph node assay dose response plot.
Positive control results:
The non-concurrent positive control (alpha-hexylcinnamaldehyde) had an EC3 = 11.7% (w/v) (conducted within 6 months of the study ; details provided in the full study report)
Parameter:
EC3
Remarks:
%
Value:
14.1
Variability:
C.I. -%
Remarks on result:
other: See table below
Parameter:
SI
Value:
1.2
Test group / Remarks:
1% in ethanol:water (7:3)
Parameter:
SI
Value:
1.8
Test group / Remarks:
10% in ethanol:water (7:3)
Parameter:
SI
Value:
7.7
Test group / Remarks:
30% in ethanol:water (7:3)
Parameter:
SI
Value:
14.7
Test group / Remarks:
50% in ethanol:water (7:3)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: See tables.

DETAILS ON STIMULATION INDEX CALCULATION: See tables.

EC3 CALCULATION: The mean values and standard deviations were calculated in the body weight tables in the study report. A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
EC3 = 14.1%
Where: a = 10, b = 1.8, c = 30 and d = 7.7

CLINICAL OBSERVATIONS: No clinical signs were observed in any animals of the control group, Group 2 (1 %) or Group 3 (10 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (30 %) and Group 5 (50%), persisting for a total of four days. In addition, on the second and the third application days, a slight ear swelling was observed at both dosing sites in all mice of Group 5 (50%) and Group 4 (30 %), separately, persisting for the remainder of the in-life phase of the study and for a total of three days. The individual clinical signs are included in the study report.

BODY WEIGHTS: On Day 1 (pre-dose) and Day 6 (post termination). The weights were within the range commonly recorded for animals of this strain and age. All females gained bodyweight during the study.

Table 1. Results from the definitive test

Test item concentration % (w/v)


Measurement dpm

Calculation

Result

dpm – BG (a)

Number of lymph nodes

dpm per lymph node (b)

S.I.

--

BG I

12

--

--

--

--

--

BG II

11

--

--

--

--

--

CG 1

1901

1889

8

236

--

1

TG 2

2361

2349

8

294

1.2

10

TG 3

3416

3404

8

426

1.8

30

TG 4

14486

14474

8 *

1809

7.7

50

TG 5

27781

27769

8 *

3471

14.7

* The size of the draining lymph nodes of this group is somewhat larger than that of the control group.

BG = Background (1 mL 5 % trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

(a) = The mean value was taken from the figures BG I and BG 11

(b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes poole

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item was found to be a skin sensitiser. The EC3 value of 14.1% (w/v) was determined.
Executive summary:

The study was performed according to OECD TG 429 using the local lymph node assay method under GLP to assess the skin sensitisation potential of the test item in the CBA/CaOlaHsd strain mouse following topical application to the dorsal surface of the ear. The test item was tested at concentrations of : 1%, 10%, 30% and 50% (w/v) in ethanol/water (7:3) and with a 0% vehicle control group. A non-concurrent positive control consisting of alpha-hexylcinnamaldehyde at 5%, 10% and 25% concentrations (w/v) in acetone:olive oil, 4:1 (v/v) conducted within 6 months of the study was additionally documented in the full study report. Observations were made twice daily. No clinical signs were observed in any animals of the control group, Group 2 (1 %) or Group 3 (10 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (30 %) and Group 5 (50%), persisting for a total of four days. In addition, on the second and the third application days, a slight ear swelling was observed at both dosing sites in all mice of Group 5 (50%) and Group 4 (30 %), separately, persisting for the remainder of the in-life phase of the study and for a total of three days. Five days after the final auricular application, the females were injected intravenously with 3H-methyl thymidine. 3HTdR incorporation was quantified using a beta-scintillation counter. The SI values calculated for the test item concentrations 1%, 10%, 30% and 50% were 1.2, 1.8, 7.7 and 14.7 respectively. The results show that the test item elicited an SI ≥ 3 and resulted in a calculated EC3 value of 14.1% (w/v). The non-concurrent positive control alpha-hexylcinnamaldehyde had an EC3 value of 11.7%. Under the conditions of this study, the test item was found to be a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Key study : in vivo, Open Epicutaneous Test (OET), 2005 : The study was performed using an Open Epicutaneous Test method under GLP. The method was consistent with Klecak G. et. al., (1977). Screening of fragrance materials for allergenicity in the guinea pig. J. Soc. Cosmet. Chem. 28: 53-64, test to assess the skin sensitisation potential of the test item. The concentrations selected for the main study were based on the results of a preliminary study. In the main study, induction was via daily topical application of the test item diluted with polyethylene glycol to 0% (control), 25%, 50%, 75%, and 100% for 5 days/week between days 1 -26. A treatment group of 6 per concentration and a control group of 6, respectively were on day 26, challenged with 5%, 10%, 15% or 25% test item in PEG 300 vehicle. Skin reactions were evaluated 24 to 72 hours after challenge treatment. No clinical signs were reported, with the exception of one animal in the 100%-induction group, which was euthanised on day 1 due to emaciation and diarrhoea. Bodyweight gain in the treated group was comparable to controls, and all survivors gained bodyweight during the study. The maximum score in control was zero. All test groups showed positive results in at least 2 animals. Under the conditions of this study, the test item is considered to be a contact skin sensitiser.

Key study : in vivo, OECD TG 429, 2004 : The study was performed according to OECD TG 429 using the local lymph node assay method under GLP to assess the skin sensitisation potential of the test item in the CBA/CaOlaHsd strain mouse following topical application to the dorsal surface of the ear. The test item was tested at concentrations of : 1%, 10%, 30% and 50% (w/v) in ethanol/water (7:3) and with a 0% vehicle control group. A non-concurrent positive control consisting of alpha-hexylcinnamaldehyde at 5%, 10% and 25% concentrations (w/v) in acetone:olive oil, 4:1 (v/v) conducted within 6 months of the study was additionally documented in the full study report. Observations were made twice daily. No clinical signs were observed in any animals of the control group, Group 2 (1 %) or Group 3 (10 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (30 %) and Group 5 (50%), persisting for a total of four days. In addition, on the second and the third application days, a slight ear swelling was observed at both dosing sites in all mice of Group 5 (50%) and Group 4 (30 %), separately, persisting for the remainder of the in-life phase of the study and for a total of three days. Five days after the final auricular application, the females were injected intravenously with 3H-methyl thymidine. 3HTdR incorporation was quantified using a beta-scintillation counter. The SI values calculated for the test item concentrations 1%, 10%, 30% and 50% were 1.2, 1.8, 7.7 and 14.7 respectively. The results show that the test item elicited an SI ≥ 3 and resulted in a calculated EC3 value of 14.1% (w/v). The non-concurrent positive control alpha-hexylcinnamaldehyde had an EC3 value of 11.7%. Under the conditions of this study, the test item was found to be a skin sensitiser.

Supporting study : Sensitisation data (humans) - HRIPT, 2006 : The study was conducted as a human repeat insult patch test under occlusive dressing according to: US FDA Regulation: CFR Title 21, Parts 50, 56 and 312 under GLP conditions. The test article (consisting of the test substance at 15% concentration) was tested was to determine the irritation and/or sensitisation potential of the test article after repeated application under occlusive patch test conditions to the skin of human subjects (exclusive panel). A total of 110 volunteer subjects, 20 males and 90 females ranging in age from 18 to 74 years, were empanelled for the test. Applicant assessment indicates that the test protocol could be considered: equivalent or similar to the RFIM human repeated insult patch test protocol cited in: V.T. Politano & A.M. Api, Regulatory Toxicology and Pharmacology 52 (2008) 35–38. 0.2 mL of the test article was applied every Monday, Wednesday and Friday until 9 applications had been made with the use of occlusive dressing to increase test sensitivity applied to the upper arm in the induction phase. The subjects were instructed to remove the patch 24 hours after application. Twenty-four hour rest periods followed the Tuesday and Thursday removals and 48-hour rest periods followed each Saturday removal. Subjects returned to the Testing Facility and the site was scored by a trained examiner just prior to the next patch application.If a subject developed a positive reaction of a level 2 erythema or greater during the Induction phase or if, at the discretion of the Study Director, the skin response warranted a change in site, the patch was applied to a previously unpatched, adjacent site for the next application. If a level 2 reaction or greater occurred at the new site, no further applications were made. However, any reactive subjects were subsequently Challenge patch tested. In the challenge phase: after a rest phase of ca. 2 weeks (no applications) the challenge patch was applied to a previously unpatched (virgin) site. The site was scored at 24 and 72 hours after application. All subjects were instructed to report any delayed skin reactivity that occurred after the final challenge patch reading. 13 out of 110 panellist discontinued for reasons unrelated to the study (all score = 0 prior to discontinuing). 97 out of 97 panellists that concluded the study indicated a maximum score = 0 with no other observations noted. There was no skin reactivity observed at any time during the course of the study.Under the conditions of this study, there was no evidence of sensitisation and of irritation to the test item (which consisted of the test substance at 15% concentration in vehicle).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation category 1B.

 

The weight of evidence indicates that the substance has a low frequency of occurrence in humans and/or low to moderate potency in animals (EC3 >2%) and can be presumed to have the potential to produce sensitisation in humans via the dermal route.