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Description of key information

Skin sensitisation

LLNA, in vivo OECD 429, GLP: negative

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:
2019-09-03 to 2019-10-16
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:
adopted 22 July 2010
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)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYEI, 22 May, 2019
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/Ca Ola Hsd mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:11-12 weeks old
- Weight at study initiation: 19.9 – 23.5 g
- Housing: Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
- Diet (e.g. ad libitum): ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water (e.g. ad libitum): tap water from watering bottles ad libitum
- Acclimation period: 14 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light from 6.00 a.m. to 6.00 p.m.
Vehicle:
other: Dichloromethane
Concentration:
Based on the preliminary test results: 25 %, 10 %, 5 % and 2.5 % (w/v)
No. of animals per dose:
5 animals/dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: According to the Sponsor’s request Dichloromethane as vehicle was evaluated. The maximum soluble concentration in this vehicle was 25 % (w/v).
- Irritation: No local effect was observed in any dose group during the test. The effect on the ear thicknesses observed in the 25 % (w/v) dose group was not considered as obvious sign of a significant irritation
- Systemic toxicity: No mortality, significant, treatment related effect on body weights or any other sign of systemic toxicity were observed.
- Ear thickness measurements: An increase of > 25 % of ear thickness (compared to the initial value) was observed in the 25 % (w/v) dose group for one of the two animals on Day 3. No significantly increased ear thicknesses were observed on Day 6 in this group or in the 10 % (w/v) dose group on any day of measurements.
- Erythema scores: No erythema was observed in any dose group during the test.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item is considered as a skin sensitiser, if:
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 (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.


TREATMENT PREPARATION AND ADMINISTRATION:
The test item was administered at four different concentrations selected 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, the positive control substance or the vehicles (a-Hexylcinnamaldehyde (HCA) or Dichloromethane) using a pipette, on the dorsal surface of each ear. After the treatment animals were returned to their cages. 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.
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 (Dichloromethane) 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 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 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
Significant lymphoproliferative response (SI >= 3) was noted for HCA (SI = 16.0). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.
Parameter:
SI
Value:
1.8
Test group / Remarks:
25 % (w/v)
Parameter:
SI
Value:
0.8
Test group / Remarks:
10 % (w/v)
Parameter:
SI
Value:
1.4
Test group / Remarks:
5 % (w/v)
Parameter:
SI
Value:
1.8
Test group / Remarks:
2.5 % (w/v)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Since no signs of systemic toxicity or significant irritation were observed, no animal or treatment group was excluded from the evaluation.
Visually larger lymph nodes compared to the vehicle control (AOO) were observed in the positive control group only. Appearance of the lymph nodes was normal in the vehicle control groups and in the test item treated groups.

DETAILS ON STIMULATION INDEX CALCULATION
No significant lymphoproliferative response (indicated by an SI >= 3) compared to the relevant vehicle control (Dichloromethane) was noted for the test item at the tested concentrations. The observed stimulation indices were 1.8, 0.8, 1.4 and 1.8 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. A stimulation index of 16.0 was observed for the positive control.

CLINICAL OBSERVATIONS
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of significant irritation (indicated by an erythema score >=3 and/or an increase of >=25 % of ear thickness observed on any day of measurement in the measured groups) was observed during the test. No other local effect was observed during the test.

BODY WEIGHTS
No significant, treatment related effects on body weights were observed during the test. On the other hand, body weight decrease by > 5 % was observed in the following groups: AOO control (3 of the 5 animals; 8 %, 6 % or 8 % decrease, respectively); positive control (1 of the 5 animals; 8 % decrease), 25 % (w/v) dose group (1 of the 5 animals; 6 % decrease) and 5 % (w/v) dose group (1 of the 5 animals; 9% decrease). This observation was not considered significant as the mean body weights did not decrease significantly in any dose group and no dose relevance was observed.
Interpretation of results:
other: EU GHS criteria not met
Conclusions:
Under the conditions of the present assay, the test item tested at the maximum attainable concentration of 25 % (w/v, based on solubility) and also at concentrations of 10 %, 5 % or 2.5 % (w/v) formulations (apparently solutions) in a suitable vehicle (Dichloromethane) was shown to have no skin sensitisation potential in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to evaluate the skin sensitisation potential of the test item following dermal exposure in the Local Lymph Node Assay, according to OECD 429 under GLP compliance. Individual approach was used in this LLNA.

The maximum dose selection was performed according to the relevant guidelines and based on results of the formulation evaluation and the Dose Range Finding test (DRF).

According to the Sponsor’s request the test item was formulated in Dichloromethane. The maximum soluble concentration in this vehicle was 25 % (w/v). According to results of the DRF (where no adverse irritation or systemic toxic effect were observed up to this maximum concentration) the test item was examined in the main test as 25 %, 10 %, 5 % and 2.5 % (w/v) formulations (apparently solutions) in Dichloromethane.

Appropriate positive control (a-Hexylcinnamaldehyde, HCA), furthermore two negative control groups dosed with AOO (as vehicle control of the positive control) or Dichloromethane (as vehicle control for the test item formulations) were employed.

The positive control item [25 % (w/v) HCA in AOO] induced significant stimulation over the relevant control (SI = 16.0) thus confirming the validity of the assay.

No mortality or signs of systemic toxicity were observed during the test. No significant, obviously treatment related effect on the body weights was observed in any dose group (although body weight decrease by > 5 % was observed in several cases). Irritation was monitored by erythema scoring in all test groups and with ear thickness measurements in the Dichloromethane and the test item treated groups. No sign of irritation (indicated by an erythema score 3 and/or an increase of 25 % of ear thickness compared to the initial values) was observed. No other local effect was observed at the treatment site (ears) in any treatment group.

No significant lymphoproliferative response (SI 3) compared to the relevant vehicle control (Dichloromethane) was noted for the test item at the tested concentrations. The corresponding stimulation index values were 1.8, 0.8, 1.4 and 1.8 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.26, r2 = 0.55, evaluated by linear regression using the calculated SI values).

According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation up to the maximum attainable concentration of 25 % (w/v, based on solubility in a suitable vehicle) as well as the lack of a significant dose-response relationship is considered as evidence that the test item is not a skin sensitiser.

In conclusion, under the conditions of the present assay, the test item tested at the maximum attainable concentration of 25 % (w/v, based on solubility) and also at concentrations of 10 %, 5 % or 2.5 % (w/v) formulations (apparently solutions) in a suitable vehicle (Dichloromethane) was shown to have no skin sensitization potential in the Local Lymph Node Assay.

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

The aim of this study was to evaluate the skin sensitisation potential of the test item following dermal exposure in the Local Lymph Node Assay, according to OECD 429 under GLP compliance. Individual approach was used in this LLNA.

The maximum dose selection was performed according to the relevant guidelines and based on results of the formulation evaluation and the Dose Range Finding test (DRF).

According to the Sponsor’s request the test item was formulated in Dichloromethane. The maximum soluble concentration in this vehicle was 25 % (w/v). According to results of the DRF (where no adverse irritation or systemic toxic effect were observed up to this maximum concentration) the test item was examined in the main test as 25 %, 10 %, 5 % and 2.5 % (w/v) formulations (apparently solutions) in Dichloromethane.

Appropriate positive control (a-Hexylcinnamaldehyde, HCA), furthermore two negative control groups dosed with AOO (as vehicle control of the positive control) or Dichloromethane (as vehicle control for the test item formulations) were employed.

The positive control item [25 % (w/v) HCA in AOO] induced significant stimulation over the relevant control (SI = 16.0) thus confirming the validity of the assay.

No mortality or signs of systemic toxicity were observed during the test. No significant, obviously treatment related effect on the body weights was observed in any dose group (although body weight decrease by > 5 % was observed in several cases). Irritation was monitored by erythema scoring in all test groups and with ear thickness measurements in the Dichloromethane and the test item treated groups. No sign of irritation (indicated by an erythema score >= 3 and/or an increase of >=25 % of ear thickness compared to the initial values) was observed. No other local effect was observed at the treatment site (ears) in any treatment group.

No significant lymphoproliferative response (SI >= 3) compared to the relevant vehicle control (Dichloromethane) was noted for the test item at the tested concentrations. The corresponding stimulation index values were 1.8, 0.8, 1.4 and 1.8 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.26, r2= 0.55, evaluated by linear regression using the calculated SI values).

According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation up to the maximum attainable concentration of 25 % (w/v, based on solubility in a suitable vehicle) as well as the lack of a significant dose-response relationship is considered as evidence that the test item is not a skin sensitiser.

In conclusion, under the conditions of the present assay, the test item tested at the maximum attainable concentration of 25 % (w/v, based on solubility) and also at concentrations of 10 %, 5 % or 2.5 % (w/v) formulations (apparently solutions) in a suitable vehicle (Dichloromethane) was shown to have no skin sensitisation potential in the Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the result of the in vivo skin sensitisation study following OECD Guideline 429 and GLP, the registered substance is not classified in accordance with Regulation (EC) No 1272/2008.