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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

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Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
28th August 2001 to 10th September 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study carried out according to internationally recognised guidelines and performed according to GLP; assigned reliability score of 2 on the basis that the test substance is being used for read-across.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
(humidity readings in the animal room (52-81%) exceeded the 70% limit value recommended by the OECD Guideline. This deviation is not expected to have a major impact on test results)
GLP compliance:
yes
Remarks:
GLP compliance statement
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: at least 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)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1-24.8 ºC
- Humidity (%): 52-81 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5th September 2001 To: 10th September 2001
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1%, 2.5%, 5%, 10% and 20% final concentrations in acetone/olive oil (AOO 4:1).

Controls included the vehicle and a known sensitiser, isoeugenol, at 0.5%, 1% and 5%.
No. of animals per dose:
Five (six for vehicle control)
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A substance is considered a sensitiser 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 mice were restrained by hand, and 25 µl of test or control article was applied daily for three consecutive days to the dorsum of each ear using a calibrated Finnpipette. The animals were allowed to rest without dosing on Days 4 and 5. The mice were observed daily for signs of toxicity and mortality

On Day 6, individually numbered mice (labeled by tail markings) were injected in the lateral tail vein with 0.25 ml containing 2 µCi of I-125 labeled luDR and 10.5 M FuDR (Sigma) in phosphate buffered saline (PBS). Approximately 5 hours later, the mice were euthanized by CO2 asphyxiation, and the auricular lymph nodes were excised. The nodes were dissociated using the frosted ends of glass slides. The cell suspension was washed with Hanks' balanced salt solution (HBSS) and then with PBS prior to being resuspended in 5% trichloroacetic acid (TCA; 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
Statistics:
The natural log transformed DPM values for each compound were compared against vehicle by first performing a Bartlett's Chi-Square test for variance homogeneity. If this was found to be non-significant, a one-way analysis of variance was used using dose. If this was found to be significant, then a Dunnett's t-test was performed using an alpha of 0.05. If the Bartlett'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 of the 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 a good fit. This is in agreement with the mean EC-3 value of isoeugenol of 1.2 +/- 0.6 % (Basketter & Cadby, Contact Dermatitis 2004 Jan;50(1):15-7). A potency value of 160 µg/cm2 was calculated. These data would classify isoeugenol as a moderate sensitizer (potency value between 100 and 1000 µg/cm2) and are consistent with previously reported results; this demonstrate the capability of the test laboratory to identify positive dermal sensitizers.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
All the calculated SI values for the test material were smaller than 3, for that reason no reliable EC-3 could be calculated (EC-3 > 20 %). (See Table 2 in "Remarks on results including tables and figures"). Calculation of the EC-3 potency value assumed a conversion of 1 ml = 1 g and was based on an exposure area of 1 cm2 per mouse ear and the application of 25 µL. The EC-3 potency value for isoeugenol was determined to be 320 µg/cm2. With an EC-3 greater than 20% for ST 23 C 01, the potency value of the test article can only be estimated to be greater than 5,000 µg/cm2.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM measurements for the four first different concentrations of test material (1%, 2.5 %, 5%, 10%) were similar to DPM measurement of vehicle. DPM measurements for the 5th and highest concentration tested (20%) was roughly 2.5 times higher than vehicle. (See Table 1 in "Remarks on results including tables and figures". )

Irritation

All animals appeared healthy and showed no signs of irritation at the dosing site

Table 1: DPM measurements

 Treatment (% of test material)  Numbers of animals  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
 Acetone/olive oil (4:1)  6  31.1  17.7  7.2
 Isoeugenol 0.5%  5  49.5  29.3  13.1
 Isoeugenol 1%  5  132.2  122.7  54.9
 Isoeugenol 5%  5  757.8  244.9  109.5

Table 2: Stimulation Index following exposure to test and 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%  4.3  1.8
 Isoeugenol 5%  24.4  3.5
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of this study, the positive control (isoeugenol), with an EC-3 of 0.68% and a potency value of 160 µg/cm2 , would be classified as a moderate sensitizer (potency value between 100 and 1000 µg/cm2) (Gerberick, et al, 2001 American Journal of Contact Dermatitis 42:344-348). This is consistent with previously reported results. With all SI values less than 3 for ST 23 C 01, a reliable EC-3 potency value could not be calculated. LLNA results showed ST 23 C 01 to be nonsensitizing at all concentrations tested in this study (1%-20%).
Executive summary:

The purpose of this study is to evaluate the sensitisation potential of ST 23 CO 1 by measuring its ability to stimulate proliferation of lymphocytes within the auricular lymph nodes of the mouse.

Mice were treated daily for three consecutive days by direct epicutaneous application of 25 µL of test or control article to the dorsum of each ear. The test article was tested at 1%, 2.5%, 5%, 10%, 20% final concentration in acetone/olive oil (4:1). The controls includes the vehicle and a known sensitiser, isoeugenol, tested at 0.5%, 1% and 5% in acetone/olive oil (4:1). The mice were observed daily and no irritation or other signs of toxicity were noted. Three days after the final auricular application, the animals were injected with 125-I labelled IuDR to label proliferating cells. 125-I incorporation was quantified using a gamma counter.

A substance is considered a sensitizer if at least one concentration of the test material results in a statistically significant 3-fold or greater stimulation index (SI). The highest dose (20%) of the test article (ST 23 CO 1) had an SI = 2.7.None of the doses tested had an SI greater than, or equal to 3. 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. Since no concentration tested resulted in an SI> 3, no reliable EC-3 value could be calculated for ST 23 C0 1. Based on the results from this study, the positive control isoeugenol would be classified as a moderate sensitizer (potency value between 100-1000 µg/cm2) (Gerberick et al., 2001). This is consistent with previously reported results.The test compound, ST 23 C0 l, was non-sensitizing at the doses tested (1%-20%)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The test substance was non-sensitizing at the doses tested (1%-20%).

On the basis that the test substance (Ebanol) is being used to support MTCP Crude on the basis of read-across, MTCP Crude is also considered to be non-sensitising.


Migrated from Short description of key information:
Sensitisation: Non-Sensitizing in mouse LLNA (OECD 429)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Due to negative results in the LLNA tests the substance (Ebanol) is not classified.

On the basis that the test substance (Ebanol) is being used to support MTCP Crude on the basis of read-across, MTCP Crude is also considered to be unclassified for sensitisation.