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Administrative data

Description of key information

There is no study on skin sensitisation available with the target substance. In a Local Lymph Node Assay performed with the structural analogue substance Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate no skin sensitising potential was reported.

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:
key study
Study period:
October 10, 2016 - February 2, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
30 May 2008, amendment L193/3 20 July 2012
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
TOXI-COOP ZRT. 8230 Balatonfüred, Arácsi út 97. (1103 Budapest, Cserkesz u. 90), Hungary
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
Ola Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90. Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 18.7 – 22.3 g
- Housing: Type II polypropylene/polycarbonate, grouped caging
- Diet: ad libitum, ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: ad libitum, tap water
- Acclimation period: 14 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: 1 % (w/v) Pluronic®PE 9200 (Plu)
Concentration:
The test item was formulated in Plu and evaluated at concentrations of 50 %, 25 % or 10 % (w/v). Due to its viscosity the test item was not suitable for application on the ears of animals at 100 % (w/v) concentration (i.e. as the undiluted form).
No. of animals per dose:
5
Details on study design:
RANGE FINDING TEST:
The pre-experiment on formulation evaluation and the Dose Range Finding test were not performed in compliance with the GLP-Regulations.
- Compound solubility: Due to its viscosity the test item was not suitable for application on the ears of animals at 100 % (w/v) concentration (i.e. as the undiluted form). The test item was appropriately miscible with the vehicle 1% (w/v) Pluronic®PE 9200 (Plu).
- Irritation: none
- Systemic toxicity: none
- Ear thickness measurements: no changes compared to results prior first treatment
- Erythema scores: 0

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: Exposure to at least one concentration (non-irritating, non-toxic) 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 (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, of the positive control substance or of the negative controls. 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 were no treatments on Days 4, 5 and 6. On Day 6 each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS containing 20 μCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe.
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised. A single cell suspension of lymph node cells of each individual animal was prepared. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid and loaded into a β-scintillation counter for the measurement of incorporated radioactivity. 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 % (w/v) TCA.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The measured DPM values corrected with the mean background value were used for statistical analysis of the proliferation data. Statistical analysis was performed by SPSS/PC+ (4.0.1) software package.
The heterogeneity of variance between the groups treated with the test item or the vehicle control (Plu) 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 result of this analysis the inter-group comparison was performed using Mann-Whitney U-test to assess the significance of inter-group differences. Significance of the positive control response was evaluated by t-test versus the relevant vehicle control (AOO).
Significance of the dose-response was evaluated by linear regression made with Microsoft Excel Software.
Positive control results:
The positive control group animals were treated with 25 % HCA solution concurrent to the test item treated groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. The positive control substance induced the appropriate, statistically significant stimulation compared to the relevant control (p < 0.01, t-test). The calculated SI value was 21.8. The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed sensitivity and validity of the assay.
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control for test item (Plu)
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control for the positive control
Key result
Parameter:
SI
Value:
21.8
Test group / Remarks:
positive control
Key result
Parameter:
SI
Value:
2.7
Test group / Remarks:
50% test item
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
25% test item
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
10% test item
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
308.3
Variability:
118
Test group / Remarks:
vehicle control for test item (Plu)
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
453.5
Variability:
375.4
Test group / Remarks:
vehicle control for the positive control
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
9 905.3
Variability:
2752.2
Test group / Remarks:
positive control
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
821.9
Variability:
864.9
Test group / Remarks:
50% test item
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
202.9
Variability:
52.7
Test group / Remarks:
25% test item
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
438.5
Variability:
36.6
Test group / Remarks:
10% test item
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Visually larger lymph nodes compared to the relevant vehicle controls (Aceton:Olive Oil or Plu) were observed in the positive control group (5/5 animals) and in the 50 % (w/v) dose group (1/5 animals). Visual appearance of the lymph nodes were normal in both negative (vehicle) control groups and in the test item treated groups (except 1 animal in the 50 % (w/v) dose group). In the 50 % (w/v) dose group individual DPM values observed for two animals significantly differed from the values observed for the other three animals. This resulted in an SI value of 2.7 which is close to the threshold value of 3. It is considered that differences in individual sensitivity of the animals may result in this deviation. It cannot be concluded obviously that these increased values are real indication of sensitization potential of the test item as proliferation values observed for the other animals in this dose group were comparable with the relevant control values. It is supported by result of the statistical analysis where no statistical significance was observed at the 50 % (w/v) dose group and also by lack of a significant dose-response relationship. Although statistical significance was observed in the 10 % (w/v) dose group (p < 0.05) no biological relevance was considered since proliferation values observed in the higher (25 %, w/v) dose group did not differ significantly from the control values and the group DPM value (calculated from the individual DPM values) of the 10 % (w/v) dose group was within the historical vehicle control range.
DETAILS ON STIMULATION INDEX CALCULATION
DPM was measured for each animal. The results were expressed also as disintegration per node (DPN) (DPM divided by the number of lymph nodes). The mean DPM and DPN values and associated error terms were calculated for each treatment group. The stimulation index (SI = the mean DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A SI value of 3 or greater is an indication of a positive result. All calculations were made by Microsoft Excel Software. Based on the results EC3 value (dose calculated to induce a stimulation index of 3) was not calculated.
CLINICAL OBSERVATIONS
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or other local effects were observed in any treatment groups.
BODY WEIGHTS
Body weights decrease by > 5 % were observed in the following treatment groups: vehicle control for the positive control (Aceton, 1/5 animals, 7 % decrease) and the 10 % (w/v) dose group (1/5 animals, 8 % decrease). The observed effect on the body weights was considered neither significant nor treatment related.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present assay, the substance tested at the maximum feasible concentration of 50 % and also at concentrations of 25 % or 10 % (w/v) as formulations in a suitable vehicle (aqueous 1 % (w/v) Pluronic®PE 9200) was shown to have no skin sensitization potential in the Local Lymph Node Assay.
Executive summary:

A study according OECD TG 429, EU method B.42 and EPA OPPTS 870.2600 was performed to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. An individual approach was used in this test. The vehicle and maximum dose selection was performed according to the relevant guidelines.

Due to its viscosity the test item was not suitable for application on the ears of animals at 100 % (w/v) concentration. Formulations were prepared with the standard vehicle of aqueous 1 % (w/v) Pluronic®PE 9200 (Plu). The test item was appropriately miscible with the vehicle. In the pre-experiment no adverse effects were observed at test concentrations of 50 %, 25 % or 10 %; w/v), hence the test item was examined in the main test at the maximum feasible concentration (based on solubility) of 50 % and at 25 % or 10 % (w/v) concentrations as formulations in Plu.

Appropriate positive control and two negative control groups (dosed with the vehicles of the test and positive control groups, respectively) were employed.

The positive control item α-Hexylcinnamaldehyde (25 % (w/v) in Acetone: Olive oil 4:1 (v/v) mixture) induced the appropriate, statistically significant stimulation over the control, thus confirming the validity of the assay.

No mortality was observed during the main test. No significant, treatment related effect on body weights was considered during the test. No other signs of systemic toxicity were observed in any treatment group. No visible signs of irritation or other local effects were observed in any treatment group.

No significant lymphoproliferative response indicated by an SI ≥ 3 was observed at the tested concentrations. In the 50 % (w/v) dose group the proliferation values observed for two of five animals were remarkably higher than the values observed for the other animals of the same dose group. This resulted in an SI value of 2.7 which is close to the threshold value of 3. However, these differences may be due to individual sensitivity of the animals. This assumption is supported by proliferation values observed for the other animals in this dose group that were similar to the relevant control values. Therefore, and considering the lack of a dose response relationship, it cannot be concluded that these increased values are real indication of sensitization potential of the test item.

Individual DPM values were statistically evaluated by Mann-Whitney U-test. No statistical significance compared to the relevant control (Plu) was observed in the 50 % (w/v) dose group (outlier values were included in the evaluation) and in the 25 % (w/v) dose group. Although statistical significance was observed in the 10 % (w/v) dose group (p < 0.05) no biological relevance was considered.

In accordance with evaluation criteria of the relevant guidelines the lack of a significantly increased lymphoproliferation up to the maximum attainable concentration of 50 % (w/v, based on solubility) and also the lack of a significant dose-response relationship are considered evidence that the test item is not a skin sensitizer.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Please refer to section 13 for "Read-Across justification".
Reason / purpose:
read-across source
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control for test item (Plu)
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control for the positive control
Key result
Parameter:
SI
Value:
21.8
Test group / Remarks:
positive control
Key result
Parameter:
SI
Value:
2.7
Test group / Remarks:
50% test item
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
25% test item
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
10% test item
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
308.3
Variability:
118
Test group / Remarks:
vehicle control for test item (Plu)
Key result
Parameter:
other: vehicle control for test item (Plu)
Value:
453.5
Variability:
375.4
Test group / Remarks:
vehicle control for the positive control
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
9 905.3
Variability:
2752.2
Test group / Remarks:
positive control
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
821.9
Variability:
864.9
Test group / Remarks:
50% test item
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
202.9
Variability:
52.7
Test group / Remarks:
25% test item
Key result
Parameter:
other: Disintegrations per minute (DPM)
Value:
438.5
Variability:
36.6
Test group / Remarks:
10% test item
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitisation potential was addressed with a read-across approach to the analogue substance Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate:

Skin sensitisation

A study according OECD TG 429, EU method B.42 and EPA OPPTS 870.2600 was performed to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. An individual approach was used in this test. The vehicle and maximum dose selection was performed according to the relevant guidelines.

Due to its viscosity the test item was not suitable for application on the ears of animals at 100 % (w/v) concentration. Formulations were prepared with the standard vehicle of aqueous 1 % (w/v) Pluronic®PE 9200 (Plu). The test item was appropriately miscible with the vehicle. In the pre-experiment no adverse effects were observed at test concentrations of 50 %, 25 % or 10 %; w/v), hence the test item was examined in the main test at the maximum feasible concentration (based on solubility) of 50 % and at 25 % or 10 % (w/v) concentrations as formulations in Plu.

Appropriate positive control and two negative control groups (dosed with the vehicles of the test and positive control groups, respectively) were employed.

The positive control item α-Hexylcinnamaldehyde (25 % (w/v) in Acetone: Olive oil 4:1 (v/v) mixture) induced the appropriate, statistically significant stimulation over the control, thus confirming the validity of the assay.

No mortality was observed during the main test. No significant, treatment related effect on body weights was considered during the test. No other signs of systemic toxicity were observed in any treatment group. No visible signs of irritation or other local effects were observed in any treatment group.

No significant lymphoproliferative response indicated by an SI ≥ 3 was observed at the tested concentrations. In the 50 % (w/v) dose group the proliferation values observed for two of five animals were remarkably higher than the values observed for the other animals of the same dose group. This resulted in an SI value of 2.7 which is close to the threshold value of 3. However, these differences may be due to individual sensitivity of the animals. This assumption is supported by proliferation values observed for the other animals in this dose group that were similar to the relevant control values. Therefore, and considering the lack of a dose response relationship, it cannot be concluded that these increased values are real indication of sensitization potential of the test item.

Individual DPM values were statistically evaluated by Mann-Whitney U-test. No statistical significance compared to the relevant control (Plu) was observed in the 50 % (w/v) dose group (outlier values were included in the evaluation) and in the 25 % (w/v) dose group. Although statistical significance was observed in the 10 % (w/v) dose group (p < 0.05) no biological relevance was considered.

In accordance with evaluation criteria of the relevant guidelines the lack of a significantly increased lymphoproliferation up to the maximum attainable concentration of 50 % (w/v, based on solubility) and also the lack of a significant dose-response relationship are considered evidence that the test item is not a skin sensitizer.

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 available data on skin sensitisation obtained with an analogue substance, the test item is not classified as skin sensitizer according to Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.