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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:
key study
Study period:
21-09-2012 to 10-10-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
(2E)-2-methyl-3-(4-methylphenyl)prop-2-en-1-ol
EC Number:
700-548-7
Cas Number:
56138-10-4
Molecular formula:
C11H14O
IUPAC Name:
(2E)-2-methyl-3-(4-methylphenyl)prop-2-en-1-ol
Details on test material:
- Physical state: Waxy solid at temperature < 23°C and liquid at temperature > 23°C
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen
- Other: white waxy solid at temperature < 23°C and clear colourless liquid at temperature > 23°C

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J strain, inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: approximately 12 weeks old
- Weight at study initiation: 21- 23 grams; Body weight variation was within +/- 20% of the sex mean.
- Housing: Group housed, in labelled Makrolon cages sterilised sawdust as bedding material, paper and shelters as cage enrichment. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet: Free access to pelleted rodent diet (certified, recognised supplier)
- Water: mains tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (actual: 20.1 to 23.4°C)
- Humidity (%): 40 to 70 (actual: 41 – 68%)
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 21-09-2011 To: 10-10-2011

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
- Preliminary test: Initially, two test substance concentrations were tested; at 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids). At a 50% test substance concentration, very slight erythema was noted on Days 2 to 4. At a 100% test substance concentration, very slight erythema was noted on Days 2, 4 and 5 and well defined erythema was noted on Day 3. In addition, both animals at 100% showed hunched posture and/or ptosis on Days 3 and 4. One animal at 25% showed a 26% increase in ear thickness of the right ear on Day 3 compared to Day 1 pre-dose values. The other animal at 25% and both animals at 50% showed variations in ear thickness less than 25% from Day 1 pre-dose values during the observation period. Based on the pre-screen results, the highest test substance concentration selected for the main study was a 50% concentration.
- Main test: 0% (vehicle control), 10, 25 and 50 %. Test concentrations were determined from the results of the preliminary test. Based on the results, in order to achieve more information, an additional group of animals was treated with a 10% test substance concentration together with a concurrent vehicle control group.
No. of animals per dose:
Preliminary test: Two per concentration: 50% and 100%.
Main test: 5 mice per dose group 0% (vehicle control), 10%, 25% and 50%
Details on study design:
RANGE FINDING TESTS:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation at most (maximum grade 2 and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied. Two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids). The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge on prior to dosing on Day 1, 1-hour post application of test item on Days 1, 2 and 3 and on Days 4, 5 and 6.

MAIN STUDY
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 substance may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle (and a further group for the additional dose as control).
- Induction: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.
- Node excision: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine. After approximately five hours, all animals were terminated by intraperitoneal injection (0.2 mL/animal) with Euthasol 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.
- Tissue processing and radioactivity measurements: A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day. Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactive measurements were performed using a scintillation counter. Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
- Mortality/Viability: Twice daily.
- Bodyweights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
- Clinical Observations: Once daily on days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
- Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing). Numerical system documented in the full study report. Furthermore, a description of all other (local) effects was recorded.
- Ear Thickness: Measurements were conducted in the pre-screen test and in the main study. A digital thickness gauge was used to measure the ear thickness of each ear prior to dosing on Day 1, 1-hour post application of test substance on Days 1, 2 and 3 and on Days 4, 5 and 6 in order to monitor for any changes in ear thickness.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Analysis was conducted according to Basketter DA et al., A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999;19:261-266.

Results and discussion

Positive control results:
In a separate 'positive control study' performed according to OECD TG 429 during March 2011. the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde. The positive control was tested at concentrations 5, 10 and 25% in Acetone/Olive oil (4:1 v/v). The highest concentration tested showed a Stimulation Index (SI) of 6.6 ± 1.0 (at 25% v/v) and met the criteria for a 'positive' result. An EC3 value of 14.4% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.8, 13.9, 16.0, 11.9, 16.9 and 10.7%.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
EC3
Remarks:
%
Value:
5.2
Variability:
C.I. 1.3% - 9.2%
Remarks on result:
other: See table below
Parameter:
SI
Value:
5
Variability:
± 1.4
Test group / Remarks:
10% in acetone:olive oil (4:1)
Parameter:
SI
Value:
10.7
Variability:
± 2.5
Test group / Remarks:
25% in acetone:olive oil (4:1)
Parameter:
SI
Value:
13
Variability:
± 3.1
Test group / Remarks:
50% in acetone:olive oil (4:1)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: See tables.

DETAILS ON STIMULATION INDEX CALCULATION: See tables.

EC3 CALCULATION: The EC3 was determined based on the Hillman and/or Exponential methods in the benchmark dose approach.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss noted at 10% was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Any other information on results incl. tables

In the preliminary screening test: At a 50% test substance concentration, very slight erythema was noted on Days 2 to 4. At a 100% test substance concentration, very slight erythema was noted on Days 2, 4 and 5 and well defined erythema was noted on Day 3. In addition, both animals at 100% showed hunched posture and/or ptosis on Days 3 and 4. One animal at 25% showed a 26% increase in ear thickness of the right ear on Day 3 compared to Day 1 pre-dose values. The other animal at 25% and both animals at 50% showed variations in ear thickness less than 25% from Day 1 pre-dose values during the observation period. Based on the pre-screen results, the highest test substance concentration selected for the main study was a 50% concentration.

 

In the main test:

Very slight irritation of the ears as shown by all animals treated at 25 and 50% on Days 2, 3 and/or 4 was considered not to have a toxicologically significant effect on the activity of the nodes. All animals showed variations in ear thickness less than 25% from Day 1 pre-dose values during the observation period.

 

The radioactive disintegrations per minute (dpm) and stimulation index (SI) are given in the table below. The SI values calculated for the substance concentrations 10, 25 and 50% were 5.0, 10.7 and 13.0, respectively. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 5.2% was calculated.

 

Table 1. Results from the definitive test

Group

TS (%)

#1

Number

Size nodes #2

 

DPM / animal #3

mean

mean

 

 

 

left

right

 

DPM ± SEM #4

SI ± SEM

1

0

1

n

n

103

233

±

50

1.0

±

0.3

 

 

2

n

n

410

 

 

3

n

n

244

 

 

4

n

n

190

 

 

5

n

n

219

 

 

 

 

 

 

 

 

 

 

 

 

2

10

6

n

n

1499

1174

±

193

5.0

±

1.4

 

 

7

n

n

596

 

 

8

n

n

1096

 

 

9

n

n

989

 

 

10

n

n

1691

 

 

 

 

 

 

 

 

 

 

 

 

3

25

11

n

n

2287

2486

±

255

10.7

±

1.3

 

 

12

n

n

2200

 

 

13

n

n

3170

 

 

14

n

n

1799

 

 

15

n

n

2971

 

 

 

 

 

 

 

 

 

 

 

 

4

50

16

+

+

12031 #5

3039

±

322

13.0

±

3.1

 

 

17

+

+

3633

 

 

18

n

n

3552

 

 

19

n

n

2399

 

 

20

n

n

2571

 

 

 

 

 

 

 

 

#1. TS = test substance (% w/w).

#2. Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

#3. DPM = Disintegrations per minute

#4. SEM = Standard Error of the Mean

#5. Value rejected for interpretation as outlier according to Grubb's test

Applicant's summary and conclusion

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 is considered to be sensitising to skin with EC3 of 5.2%.
Executive summary:

The study was performed to OECD TG 429, EU Method B.42 and US EPA OPPTS 870.2600 guidelines under GLP to assess the skin sensitisation potential of the test material in the CBA/J strain mouse following topical application to the dorsal surface of the ear. In a preliminary screening test mice were treated by daily application of 25 μl of the test substance at 50% and 100% v/v in acetone/olive oil 4:1 to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice was observed twice daily and local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. Ear thickness measurements were conducted using a digital thickness gauge on prior to dosing on Day 1, 1-hour post application of test item on Days 1, 2 and 3 and on Days 4, 5 and 6. Based on the pre-screen results, the highest test substance concentration selected for the main study was a 50% concentration. A mean ear thickness increase of equal to or greater than 25% and/or well-defined irritation at the most (maximum grade 2) was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation. No irritation or signs of systemic toxicity were observed. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on the preliminary test, the concentrations selected for the main test were 0%, 10%, 25% and 50% v/v. In the main test, three groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). The very slight irritation of the ears as shown by all animals treated at 25 and 50% on Days 2, 3 and/or 4 was considered not to have a toxicologically significant effect on the activity of the nodes. All animals showed variations in ear thickness less than 25% from Day 1 pre-dose values during the observation period. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights were within the range seen for historic control animals. The majority of auricular lymph nodes were considered normal in size when compared to other (control) animals, except for both nodes of two animals treated at 50% test substance concentration that appeared larger in size when compared to other (control) animals. No macroscopic abnormalities of the surrounding area were noted in any of the animals. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1174, 2485 and 3039 DPM respectively. The mean DPM/animal value for the vehicle control group was 233 DPM.The SI values calculated for the substance concentrations 10, 25 and 50% were 5.0, 10.7 and 13.0 respectively. These results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 10%. From the graph of the data (Benchmark dose approach) the point estimate of the EC3 is 5.2% (confidence interval: 1.3% - 9.2%). Under the conditions of this study, the test substance would be considered to be classified under Regulation (EC) No 1272/2008 as skin sensitizer category 1B.