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Diss Factsheets

Administrative data

Description of key information

Based on the EC3 value of 37 % the test item was considered a weak skin sensitizer in the LLNA.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-10-09 to 2012-11-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: 11 weeks (at start of experiment)
- Weight at study initiation: 17.7 to 21.5 g
- Housing: Grouped caging (5 animals/cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 10 October 2012 To: 18 October 2012
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50 %, 25 % and 10 % (w/v)
No. of animals per dose:
5 per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Soluble in vehicle
- Irritation: dry, hard, exfoliating skin and loss of hair was observed at the base of ears at test concentration of 100 % (the undiluted test item) although no erythema was observed
- Lymph node proliferation response: not measured
- Ear thickness: significantly increased ear thickness (≥ 25 %) was observed at the treatment site (ears) at 100 % test concentration

MAIN STUDY
ANIMAL ASSIGNMENT, TREATMENT AND PROLIFERATION ASSAY
- Name of test method: Skin Sensitization Study: Local Lymph Node Assay (Individual Approach)
- Criteria used to consider a positive response: The test item is considered as a skin sensitizer, if the following criteria are fulfilled:
1) That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
2) The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was administered at three different concentrations in the selected vehicle (AOO) according to the results of the dose range finding test.
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, of the positive control substance (positive control group) or of the vehicle (negative control group). The formulations were applied with a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
On Day 6 each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (1xPBS, diluted from 10x concentrate with purified water) containing 20 μCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the lymph nodes were removed using forceps. Once removed, the lymph nodes of individual mice were collected separately in a Petri dish containing a small amount (1 - 2 mL) of PBS to keep the nodes wet before processing.
A single cell suspension (SCS) of lymph node cells (LNCs) of each individual animal was prepared and collected in disposable tubes by a gentle mechanical disaggregation of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4°C. After centrifugation, the majority of the supernatant was aspirated off, leaving 1 - 2 mL supernatant above each pellet. The pellets were gently agitated and the LNCs were resuspended in 10 mL of PBS. This washing procedure was repeated twice. This procedure was repeated for lymph nodes of each individual animal.
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloracetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2 - 8 °C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4 °C and decanting the supernatants and the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and stored at room temperature protected from light until measurement.
For the measurement of incorporated radioactivity the vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expresses the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % TCA.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was performed by SPSS/PC+ (4.0.1) software package.
The measured DPM values corrected with the mean background value were used for statistical analysis of the proliferation data. The heterogeneity of variance between the groups treated with the test item or the positive control and the vehicle control was checked by Bartlett's test. Since significant heterogeneity was detected, the normal distribution of data was examined by Kolmogorow-Smirnow test followed by the non-parametric method of Kruskal-Wallis One-Way analysis of variance. As a results of this analysis the inter-group comparisons was performed using Mann-Whitney U-test to asses the significance of inter-group differences.
Positive control results:
The positive control substance induced a statistically significant stimulation compared to the control (SI = 10.0; p < 0.01).
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Negative control (AOO)
Key result
Parameter:
SI
Value:
10
Test group / Remarks:
Positive control (25 % HCA)
Key result
Parameter:
SI
Value:
4.4
Test group / Remarks:
Test item 50 %
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
Test item 25 %
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
Test item 10 %
Parameter:
EC3
Value:
> 10 - < 100
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Visual appearance of the lymph nodes was larger than the control (AOO) in the positive control group and in the 50 % dose group. Visual appearance of the lymph nodes was normal in the negative (vehicle) control group and in the other dose groups (25 % and 10 %). Significant lymphoproliferation (SI ≥ 3) was observed at concentration of 50 %. No significant lymphoproliferation was observed at concentration of 25 % or 10 %. The corresponding stimulation index values were 4.4, 1.8 and 1.5 at treatment concentrations of 50 %, 25 % or 10 %, respectively. Significance of the dose-response was evaluated by linear regression using the SI values. The dose-response observed was not statistically significant (p = 0.18; r value = 0.96).

EC3 CALCULATION
The EC3 (dose calculated to induce a stimulation index of 3) was calculated by using two methods: by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method and by using equation of the regression curve. The calculated EC3 value of Incozol 4 based on linear interpolation was 37 %, while 34 % was calculated based on the regression curve in this LLNA. Using both calculated EC3 values Incozol 4 can be ranked among weak sensitizers (10 ≤ EC3 ≤ 100) in this LLNA according to the published data for classification of contact allergens.

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No visible sign of significant irritation (indicated by an erythema score ≥ 3) or any other local effect was observed in any treatment groups.

BODY WEIGHTS
No significant, treatment related effect on the body weights was observed in any treatment group.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the present Local Lymph Node Assay, Incozol 4 tested at concentrations of 50 % , 25 % and 10% as formulations in a suitable vehicle (AOO) was shown to have sensitization potential. Based on the EC3 values Incozol 4 was considered a weak sensitizer in this LLNA.
Executive summary:

The aim of this study was to determine the skin sensitization potential of the test item in the Local Lymph Node Assay (LLNA). Individual approach was used in this test.

Selection of test item concentrations based on the results of a formulation evaluation and also results of preliminary irritation/toxicity tests according to the relevant guidelines. The test item was a viscous liquid hence applicability of the undiluted test item (100 % concentration) and miscibility in a suitable vehicle was evaluated. Based on the preliminary test results, the maximum attainable concentration of 100 % (undiluted test item) could not be used due to local irritation. For the main test the test item was formulated in the selected vehicle (Acetone: Olive oil 4:1 (v/v) mixture, AOO) at concentrations of 50 %, 25 % and 10 % in a final volume of 1 mL.

In the main test, 25 female CBA/Ca mice were allocated to five groups of five animals each:

- three groups received the test item at 50 %, 25 % or 10 %,

- the negative control group received the vehicle (AOO) only,

- the positive control group received 25 %α-Hexylcinnamaldehyde (HCA).

Each substance was applied on the external surface of each ear (25μL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. Auricural lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI).

The positive control item (25 % HCA in AOO) induced the appropriate, statistically significant stimulation compared to the control (SI = 10.0; p < 0.01 Mann-Whitney U-test versus the vehicle (AOO) control), thus confirming the validity of the assay.

No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity was observed in any treatment group. No visible sign of significant irritation or any other local effect was observed in any treatment group.

Visually larger lymph nodes than the control was observed in the positive control group and in the 50% dose group. Visual appearance of the lymph nodes was normal in the negative (vehicle) control group and in the other dose groups (25 % and 10 %).

Significant lymphoproliferation (SI ≥ 3) was observed at test item concentration of 50 %. No significant lymphoproliferation (SI ≥ 3) was observed at concentrations of 25 % or 10 %. The corresponding stimulation index values were 4.4, 1.8 and 1.5 at treatment concentrations of 50 %, 25% or 10 %, respectively. The observed proliferation values were statistically significant in the 50 % and in the 25 % dose groups (Mann-Whitney U-test was performed using the individual DPM values corrected with the mean background value) compared to the vehicle control (AOO). Dose-related response was observed although it was not statistically significant (linear regression, p = 0.18; r value = 0.96).

Since the test was valid and no signs of systemic toxicity or irritation were observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

According to evaluation criteria of the relevant guidelines, although the dose-response observed was not statistically significant the SI value above 3 observed at the maximum applied concentration of 50 % was considered evidence that the test item has sensitization potential.

The EC3 (dose calculated to induce a stimulation index of 3) was calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method and by using equation of the regression curve. The calculated EC3 value of the test item based on linear interpolation was 37 %, while 34 % was calculated based on the regression curve in this LLNA. Using both calculated EC3 values the test item can be ranked among weak sensitizers in this LLNA according to the published data for classification of contact allergens.

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

The aim of this study was to determine the skin sensitization potential of the test item in the Local Lymph Node Assay (LLNA). Individual approach was used in this test.

Selection of test item concentrations based on the results of a formulation evaluation and also results of preliminary irritation/toxicity tests according to the relevant guidelines. The test item was a viscous liquid hence applicability of the undiluted test item (100 % concentration) and miscibility in a suitable vehicle was evaluated. Based on the preliminary test results, the maximum attainable concentration of 100 % (undiluted test item) could not be used due to local irritation. For the main test the test item was formulated in the selected vehicle (Acetone : Olive oil 4:1 (v/v) mixture, AOO) at concentrations of 50 %, 25 % and 10 % in a final volume of 1 mL.

In the main test, 25 female CBA/Ca mice were allocated to five groups of five animals each:

- three groups received the test item at 50 %, 25 % or 10 %,

- the negative control group received the vehicle (AOO) only,

- the positive control group received 25 % α-Hexylcinnamaldehyde (HCA).

Each substance was applied on the external surface of each ear (25 μL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. Auricural lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI).

The positive control item (25 % HCA in AOO) induced the appropriate, statistically significant stimulation compared to the control (SI = 10.0; p<0.01 Mann-Whitney U-test versus the vehicle (AOO) control), thus, confirming the validity of the assay.

No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity was observed in any treatment group. No visible sign of significant irritation or any other local effect was observed in any treatment group.

Visually larger lymph nodes than the control was observed in the positive control group and in the 50 % dose group. Visual appearance of the lymph nodes was normal in the negative (vehicle) control group and in the other dose groups (25 % and 10 %).

Significant lymphoproliferation (SI ≥ 3) was observed at test item concentration of 50 %. No significant lymphoproliferation (SI ≥ 3) was observed at concentrations of 25 % or 10 %. The corresponding stimulation index values were 4.4, 1.8 and 1.5 at treatment concentrations of 50 %, 25% or 10 %, respectively. The observed proliferation values were statistically significant in the 50 % and in the 25 % dose groups (Mann-Whitney U-test was performed using the individual DPM values corrected with the mean background value) compared to the vehicle control (AOO). Dose-related response was observed although it was not statistically significant (linear regression, p = 0.18; r value = 0.96).

Since the test was valid and no signs of systemic toxicity or irritation were observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

According to evaluation criteria of the relevant guidelines, although the dose-response observed was not statistically significant the SI value above 3 observed at the maximum applied concentration of 50 % was considered evidence that the test item has sensitization potential.

The EC3 (dose calculated to induce a stimulation index of 3) was calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method and by using equation of the regression curve. The calculated EC3 value of the test item based on linear interpolation was 37 %, while 34 % was calculated based on the regression curve in this LLNA. Using both calculated EC3 values the test item can be ranked among weak sensitizers in this LLNA according to the published data for classification of contact allergens.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the study available the test item is classified for skin sensitization Cat. 1B, H317 (May cause an allergic skin reaction.) according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.