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Administrative data

Description of key information

Local Lymph Node Assay:

The test item tested at the maximum achievable concentration of 100 % (w/v, as the undiluted form) and at 50%, 25% and 10 % (w/v) concentrations as formulations (apparently solutions) in a suitable vehicle (PG) showed to have no skin sensitization potential.

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:
2016-03-08 to 2016-05-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
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
- Microbiological status of animals, when known: SPF (at arrival); Good conventional (during the test).
- Age at study initiation: Young adult mice 8 weeks old (at start of the DRF test); Young adult mice, 8-9 weeks old (at start of the main test).
- Weight at study initiation: 16.7 – 19.5 g; The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing during acclimatization period: Grouped caging in small groups
- Housing during the test: Grouped caging (5 animals/cage), Cage type: Type II. polypropylene/polycarbonate, laboratory bedding,
- Housing/Enrichment: Mice are group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
- Diet: Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum. The food is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are maintained in TOXI-COOP ZRT.’s archive.
- Water: Animals received tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are maintained in TOXI-COOP ZRT.’s archive.
- Acclimation period: 21 or 7 days
- Indication of any skin lesions: Only healthy animals were used.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 %
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Vehicle:
propylene glycol
Concentration:
test item concentration: 100 %, 50 %, 25 %, 10 % (w/v)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
The pre-experiment on formulation evaluation and the Dose Range Finding (DRF) test were not performed in compliance with the GLP-Regulations and are excluded from the Statement of the Study Director, but the raw data of these tests will be archived under the study code of present study. The DRF was conducted in an identical experimental manner to the main assay except there was no assessment of lymph node proliferation and fewer animals were used. The maximum test concentration was selected according to results of preliminary formulation evaluation. The test item was a liquid and found to be suitable for application in its undiluted form (100 %, w/v). According to this four groups of 2 CBA/Ca mice were treated with the undiluted test item (100 %) and the 50 %, 25 % and 10 % (w/v) formulations prepared with the selected vehicle (PG). All formulations were adequately applicable on the ears of animals. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to the first treatment (Day 1) and prior to termination (Day 6). Both ears of each mouse were observed for erythema and scored. Ear thickness measurement was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or an increase of ≥ 25 % of ear thickness observed on any day of measurement. No mortality, significant, treatment related effect on body weights or any other sign of systemic toxicity were observed. 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) or any other local effect was observed.

MAIN STUDY
Animals in the treatment groups were treated with physiological saline solution (PS, naive control), with the vehicles (PG or AOO), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test was administered at four different concentrations according to the results of the DRF test.

ANIMAL ASSIGNMENT AND TREATMENT
- Randomization: The animals were set in order of their body weights. The animals were randomly assigned to control and test groups using a randomization scheme. The randomization was checked by computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the homogeneity and deviations between the groups.
- Name of test method: LLNA
- Criteria used to consider a positive response: The test item is considered as a skin sensitizer, if the following criterion is fulfilled:
That 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 (PS, PG or AOO). 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.
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.
Significance of the positive control response was evaluated by t-test versus the relevant vehicle control (AOO).
Significance of the response observed at 100 % (w/v) test concentration was evaluated by t-test versus the relevant control (naive, PS).
Significance of the response observed for the test item formulations (50 %, 25 % or 10 %, w/v) was checked by Bartlett's test followed by Kruskal-Wallis One-Way analysis.
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 (formulated in AOO) 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 stimulation compared to the relevant control (AOO). Statistically significant increase of the proliferation values was observed in the positive control group by t-test versus AOO control (p < 0.01). The calculated SI value was 14.9. 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:
0.7
Test group / Remarks:
Dose group 100%
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
Dose group 50%
Key result
Parameter:
SI
Value:
3.2
Test group / Remarks:
Dose group 25%
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
Dose group 10%
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Naive control: PS
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Vehicle control: PG
Cellular proliferation data / Observations:
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:
Loss of body weights (≥ 5% decrease) was observed in the 100 % (w/v) dose group (1/5 animals; 7 % decrease), in the 50 % (w/v) dose group (1/5 animals; 6 % decrease) and in the 25 % (w/v) dose group (1/5 animals, 5 % decrease). Similar effect on the body weights was observed in the AOO treated group (3/5 animals, 5 %, 7% or 6 % decrease) and in the PG treated group (1/5 animals, 6 % decrease). The mean values did not decrease significantly in any of these dose groups hence the effect was considered neither significant nor treatment related.

Proliferation Assay:

Visual appearance of the lymph nodes was normal in all negative control groups (AOO, PS or PG). Visually larger lymph nodes compared to the relevant vehicle control (AOO) were observed in the positive control group only. No visually larger lymph nodes compared to the relevant vehicle controls (PS or PG) were observed in the test item treated groups. Borderline result (an SI value of 3.2) was observed at the 25 % (w/v) test concentration. No significant lymphoproliferation (SI ≥ 3) was observed for the test item at the other test concentrations. The corresponding stimulation index values were 0.7, 1.3, 3.2 and 1.4 at treatment concentrations of 100 %, 50 %, 25 % and 10 % (w/v), respectively.

Table 1 Mean DPM and DPN ± SD

Test group

Mean DPM±SD

Mean DPN±SD

Vehicle control for positive control

1168.9 ± 881.8

584 ± 440.9

Positive control: 25% HCA in AOO

17362.9 ± 8810.8

8681 ± 4405.4

Vehicle control for test item: PG

364.1 ± 264.3

182 ± 132.2

Naive control: PS

814.1 ± 711.8

407 ± 355.9

Test item 100 % undiluted

609.7 ± 455.8

305 ± 227.9

Test item 50 % in PG

482.5 ± 208.0

241 ± 104.0

Test item 25 % in PG

1173.7 ± 1511.4

587 ± 755.7

Test item 10 % in PG

516.3 ± 306.9

259 ± 153.4

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present Local Lymph Node Assay, the test item tested at the maximum achievable concentration of 100 % (w/v, as the undiluted form) and at 50%, 25% and 10 % (w/v) concentrations as formulations (apparently solutions) in a suitable vehicle (PG) showed to have no skin sensitization potential.
Executive summary:

The aim of this study was 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 test item was a liquid. Propylene glycol (PG), one of the vehicles preferred by the relevant guidelines, was selected to be used for formulation of the test item according to the Sponsor’s request. The test item was adequately miscible with the vehicle. The maximum dose selection was based on results of a Dose Range Finding (DRF) test performed according to the relevant guidelines.

No adverse effects (systemic toxicity or irritation) were observed at the maximum achievable test concentration of 100 % (w/v) and below (50 %, 25 % or 10 %, w/v), hence the test item was examined in the main test at 100 % (w/v) concentration as the undiluted form and at 50 %, 25 % and 10 % (w/v) concentrations as formulations in PG. Appropriate positive control (25 % (w/v) α-Hexylcinnamaldehyde [HCA] in Acetone: Olive oil 4:1 (v/v) mixture [AOO]) and negative control groups dosed with physiological saline solution (PS, used as negative (naive) control for the undiluted test item) and the vehicles of the test item treated and positive control groups (PG or AOO, respectively) were employed.

The positive control item induced the appropriate stimulation over the control (SI = 14.9), thus confirming the validity of the assay. No mortality was observed during the main test. No obvious sign of systemic toxicity were observed in any treatment group. Although ≥ 5 % decrease of the body weights were observed in several cases (with a maximum decrease of 7 %) the effect was considered neither significant nor treatment related. No signs of irritation or any other local effect were observed at the treatment site (ears) in any treatment group. Borderline result (an SI value of 3.2) was observed at the 25 % (w/v) test concentration. No significant lymphoproliferation (SI ≥ 3) was observed for the test item at the other test concentrations. The corresponding stimulation index values were 0.7, 1.3, 3.2 and 1.4 at treatment concentrations of 100 %, 50 %, 25 % and 10 % (w/v), respectively. The measured DPM values corrected with the mean background value were statistically evaluated. No statistically significantly increased lymphoproliferation was observed in the 100 % (w/v) dose group (t-test versus PS control). No statistically significant increase of the proliferation values compared to the relevant vehicle control (PG) were observed in the dose groups treated with the 50 %, 25 % or 10 % (w/v) test item formulations. No dose-related response was observed (p = 0.40, r = 0.60, linear regression using the calculated SI values). It was considered that the test item did not cause significant lymphoproliferation at the tested concentrations. Although the SI value of 3.2 observed at 25 % (w/v) test concentration was slightly higher than the threshold value of 3 the effect was neither statistically significant nor biologically relevant since no significantly increased lymphoprolipheration was observed at higher test concentrations. According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation at the maximum achievable concentration of 100 % (w/v) and also the lack of a significant dose-response relationship are considered good evidence that the test item is not a skin sensitizer.

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

LLNA:

The aim of this study was 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 test item was a liquid. Propylene glycol (PG), one of the vehicles preferred by the relevant guidelines, was selected to be used for formulation of the test item according to the Sponsor’s request. The test item was adequately miscible with the vehicle. The maximum dose selection was based on results of a Dose Range Finding (DRF) test performed according to the relevant guidelines.

No adverse effects (systemic toxicity or irritation) were observed at the maximum achievable test concentration of 100 % (w/v) and below (50 %, 25 % or 10 %, w/v), hence the test item was examined in the main test at 100 % (w/v) concentration as the undiluted form and at 50 %, 25 % and 10 % (w/v) concentrations as formulations in PG. Appropriate positive control (25 % (w/v) α-Hexylcinnamaldehyde [HCA] in Acetone: Olive oil 4:1 (v/v) mixture [AOO]) and negative control groups dosed with physiological saline solution (PS, used as negative (naive) control for the undiluted test item) and the vehicles of the test item treated and positive control groups (PG or AOO, respectively) were employed.

The positive control item induced the appropriate stimulation over the control (SI = 14.9), thus confirming the validity of the assay. No mortality was observed during the main test. No obvious sign of systemic toxicity were observed in any treatment group. Although ≥ 5 % decrease of the body weights were observed in several cases (with a maximum decrease of 7 %) the effect was considered neither significant nor treatment related. No signs of irritation or any other local effect were observed at the treatment site (ears) in any treatment group. Borderline result (an SI value of 3.2) was observed at the 25 % (w/v) test concentration. No significant lymphoproliferation (SI ≥ 3) was observed for the test item at the other test concentrations. The corresponding stimulation index values were 0.7, 1.3, 3.2 and 1.4 at treatment concentrations of 100 %, 50 %, 25 % and 10 % (w/v), respectively. The measured DPM values corrected with the mean background value were statistically evaluated. No statistically significantly increased lymphoproliferation was observed in the 100 % (w/v) dose group (t-test versus PS control). No statistically significant increase of the proliferation values compared to the relevant vehicle control (PG) were observed in the dose groups treated with the 50 %, 25 % or 10 % (w/v) test item formulations. No dose-related response was observed (p = 0.40, r = 0.60, linear regression using the calculated SI values). It was considered that the test item did not cause significant lymphoproliferation at the tested concentrations. Although the SI value of 3.2 observed at 25 % (w/v) test concentration was slightly higher than the threshold value of 3 the effect was neither statistically significant nor biologically relevant since no significantly increased lymphoprolipheration was observed at higher test concentrations. According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation at the maximum achievable concentration of 100 % (w/v) and also the lack of a significant dose-response relationship are considered good 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 sensitization, the test item is not classified as sensitising according to Regulation /EC) No 1272/2008 (CLP) as amended for the eighth time, Regulation (EU) 2016/863.