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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-03 to 2016-04-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid

In vivo test system

Test animals

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: at arrival: SPF; during the test: Good conventional
- Age at study initiation: Young adult mice, 8 weeks old (at start of the main test)
- Weight at study initiation: 13.6 – 18.0 g; The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: Grouped caging (5 animals/cage); Type II. polypropylene/polycarbonate; Laboratory bedding; 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: 14 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.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
10 %, 5 %, 2.5 %, 1 % (w/v)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The test item was a liquid and was found to be adequately miscible with the selected vehicle at a concentration of 50 % (w/v) and below.
- Systemic toxicity: In the 100 % (w/v) dose group mortality (2/2 animals) was observed on Day 2, although both animals were symptom-free on Day 1. Similarly, mortality (2/2 animals) was observed also in the 50 % (w/v) dose group on Day 3. In this dose group animals were symptom-free on Day 1, but the following symptoms were observed on Day 2. The body weights (measured for dead animals) were significantly decreased in both groups. In the 25 % (w/v) dose group no mortality or decreased body weights were observed, but signs of systemic toxicity were visible on Days 2, 3 and 4.
- Ear thickness measurements: As an indication of irritation significantly increased (≥ 25 %) ear thickness values were observed in the 25 % (w/v) dose group on Day 6 (2/2 animals). The maximum increase was 55 %. In the 10 % (w/v) dose group 25 % increase was observed in case of one animal on Day 6, while no significantly increased ear thickness was observed for the other animal.
- Erythema scores: No significant (scored as ≥ 3) erythema was observed in any dose groups.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- 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 and of the vehicle. 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.
The heterogeneity of variance between the groups treated with the test item and the vehicle control (PG) 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.

Results and discussion

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. 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 21.7. 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.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
126.3
Test group / Remarks:
Dose group 10 %
Key result
Parameter:
SI
Value:
100.7
Test group / Remarks:
Dose group 5 %
Key result
Parameter:
SI
Value:
62.9
Test group / Remarks:
Dose group 2.5 %
Key result
Parameter:
SI
Value:
6.4
Test group / Remarks:
Dose group 1 %
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Vehicle control for test item: PG
Key result
Parameter:
SI
Value:
21.7
Test group / Remarks:
Positive control: 25 % HCA in AOO
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Vehicle control for positive control: AOO
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: Visually larger lymph nodes compared to the relevant vehicle controls (AOO or PG) were observed in the positive control group and in all test item treated groups. Additionally, in the 10 % and 5 % (w/v) dose groups the lymph nodes were even more increased than in the positive control group. Visual appearance of the lymph nodes was normal in both negative (AOO or PG) control groups. Significant lymphoproliferation (SI ≥ 3) was observed for the test item at all test concentrations. The corresponding stimulation index values were 126.3, 100.7, 62.9 and 6.4 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. The observed effect was considered dose related.

DETAILS ON STIMULATION INDEX CALCULATION: SI = the mean DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group.

EC3 CALCULATION: In this test no SI value below 3 was observed, hence no calculation of the EC3 value was possible.

CLINICAL OBSERVATIONS: No mortality or symptoms of systemic toxicity were observed in any treatment group. Ear thickness was monitored in the 10 % and 5 % (w/v) dose groups. Significantly increased values (> 25 % increase compared to the initial values) were observed in the 10 % (w/v) dose group (4/5 animals): the maximum increase was 40 %. Although increased ear thickness values were measured in the 5 % dose groups also, the increase was less than 25 %: the maximum value was 23.8 % in this dose group (observed for 3/5 animals). No erythema or other local effects were observed in the 10 % and 5 % (w/v) dose groups. No sign of irritation (indicated by an erythema score ≥ 3) or other local effects were observed in the other treatment groups.

BODY WEIGHTS: No significant, treatment related effect on the body weights was observed in any treatment group.

Any other information on results incl. tables

Table 1 Mean DPM and DPN± SD

Test group

Mean DPM ± SD

Mean DPN ± SD

Vehicle control for positive control: AOO

799.7 ± 343.2

400 ± 171.6

Positive control: 25% HCA in AOO

17386.5 ± 6086.7

8693 ± 3043.3

Vehicle control for test item: PG

404.1 ± 243.3

202 ± 121.6

Test item 10 % in PG

51034.1 ± 27492.0

25517 ± 13746.0

Test item 5 % in PG

40690.3 ± 12985.4

20345 ± 6492.7

Test item 2.5 % in PG

25408.7 ± 8194.9

12704 ± 4097.4

Test item 1 % in PG

2585.1 ± 1628.6

1293 ± 814.3

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the present Local Lymph Node Assay, the test item tested at 10 %, 5%, 2.5% and 1% (w/v) concentrations as formulations (apparently solutions) in a suitable vehicle (PG) showed to have skin sensitization potential. Based on the EC3 value of <1% the test item was considered a strong or extreme skin sensitizer in this LLNA.
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. Adverse effects (systemic toxicity and/or irritation) were observed at high test concentrations (100 %, 50 % and 25 %, w/v), hence the test item was examined in the main test at 10 %, 5 %, 2.5 % and 1 % (w/v) concentrations as formulations in PG. Appropriate positive control, furthermore two negative control groups (dosed with the vehicles of the test and positive control groups, respectively) were employed. The positive control item (α-Hexylcinnamaldehyde [HCA]; 25 % (w/v) in Acetone: Olive oil 4:1 (v/v) mixture [AOO]) induced the appropriate stimulation over the control (SI = 21.7), thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other signs of systemic toxicity were observed in any treatment group. As a sign of a potential irritation significantly increased ear thickness values (≥ 25 % increase compared to the initial values) were observed in the 10 % (w/v) dose group (4/5 animals) although no significant erythema (scored as ≥ 3) or any other local effect was observed in any test group during the test. Although increased ear thickness values were observed also in the 5 % (w/v) dose group the increase was less than 25 % (compared to the initial values) in each case. The maximum value was 23.8 % in this dose group (observed for 3/5 animals). Ear thickness was not monitored in the other test groups. Significant lymphoproliferation (SI ≥ 3) was observed for the test item at all test concentrations. The corresponding stimulation index values were 126.3, 100.7, 62.9 and 6.4 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. The measured individual DPM values corrected with the mean background value were statistically evaluated. Statistically significant increase of the proliferation values was observed in the positive control group by t-test versus AOO control (p < 0.01). Statistically significant differences compared to the relevant vehicle control (PG) were observed in all test item treated groups (p < 0.01, evaluated by Mann-Whitney U-test). Dose-related response was observed although linearity of the dose-response relationship was not statistically significant in the tested concentration range (p = 0.09, r = 0.91, linear regression using the SI values). Based on results of the ear thickness measurement it is possible that irritation interfered with the results at 10 % (w/v) concentration (and probably at 5 % (w/v) concentration also). On the other hand no adverse effect was observed at 2.5 % and 1 % (w/v) concentrations.Although ear thickness was not monitored in these dose groups, based on the values measured in the 10 % and 5 % (w/v) dose groups a decreasing trend can be expected at lower concentrations. Accordingly the dose-response analysis (using all SI values) was considered equivocal but biologically relevant. Based on the above the measured DPM values (especially at 2.5 % and 1 % (w/v) test concentrations) were 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, the significantly increased lymphoproliferation observed at 1 % (w/v) concentration and above and the dose-related response are considered evidence that the test item is a skin sensitizer. Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) which is normally calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method. In this test no SI value below 3 was observed (hence no calculation of the EC3 value was possible), but based on the recent test results an EC3 value of < 1 % (w/v) is considered for the test item.