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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:
2015-10-21 to 2016-01-11
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
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane
EC Number:
924-669-1
Cas Number:
192526-20-8
Molecular formula:
No data available
IUPAC Name:
Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane
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: at arrival: SPF; during test: Good conventionally
- Age at study initiation: Young adult mice, 9-10 weeks old (at start of the preliminary test); Young adult mice, 10-11 weeks old (at start of the main test)
- Weight at study initiation: 17.8 - 21.9 g; The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: Grouped caging (5 animals/cage); Cage type: 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:
acetone/olive oil (4:1 v/v)
Concentration:
10 %, 5 %, 2.5 % and 1 % (w/v)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The test item was a viscous liquid hence miscible with an appropriate vehicle was evaluated. The test item was adequately miscible with Acetone: Olive oil 4:1 (v/v) mixture (AOO) at a maximum achievable concentration of 75 % (w/v).
- Irritation: As a sign of significant irritation significantly increased (≥ 25 %) ear thickness values were observed in the 75 %, 50 % and 25 % (w/v) dose groups on Day 3 and Day 6. It should be noted that that test item residuals were observed -as a film dried onto surface of the ears- in the 75 % and 50 % (w/v) dose groups on Day 6 (after terminal killing). It is considered that the ear thickness values measured in these dose groups (especially on Day 6) could be affected by the test item residuals.
- Systemic toxicity: No mortality, significant, treatment related effect on body weights or any other sign of systemic toxicity were observed.
- Erythema scores: No significant erythema (scored as ≥ 3) was observed in any dose group during the DRF test.

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 (AOO) 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 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 (an SI ≥ 3) compared to the relevant (AOO) control: the calculated SI value was 12.0. 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:
24.2
Test group / Remarks:
Test item 10 % in AOO
Key result
Parameter:
SI
Value:
21.2
Test group / Remarks:
Test item 5 % in AOO
Key result
Parameter:
SI
Value:
21
Test group / Remarks:
Test item 2.5 % in AOO
Key result
Parameter:
SI
Value:
6.2
Test group / Remarks:
Test item 1 % in AOO
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Negative control (vehicle): AOO
Key result
Parameter:
SI
Value:
12
Test group / Remarks:
Positive control: 25 % HCA in AOO
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Visually larger lymph nodes compared to the vehicle control (AOO) were observed in the positive control group and in all test item treated groups. Visual appearance of the lymph nodes was normal in the negative (vehicle) control group. Outlier DPM values were considered in the 10 % (w/v) dose group (animal no. 16) and in the 2.5 % (w/v) dose group (animal no. 21) therefore these values were excluded from the evaluation. Significant lymphoproliferation (SI ≥ 3) was observed for the test item at all test concentrations. The corresponding stimulation index values (calculated without outliers) were 24.2, 21.2, 21.0 and 6.2 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively.

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. As a sign of a potential irritation significantly (≥ 25 %) increased ear thickness values were observed in both monitored dose groups: in the 10 % (w/v) dose group (5/5 animals, with a maximum increase of 40 %) and also in the 5 % (w/v) dose group (1/5 animals, 25 % increase). Ear thickness was not monitored in the other test groups. No significant erythema (scored as ≥ 3) or any other local effect was observed in any treatment groups.

BODY WEIGHTS
No significant, treatment related effect on the body weights was considered 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

Positive control: 25% HCA in AOO

13758.6 ± 1337.9

6879 ± 668.9

Negative (vehicle) control: AOO

1142.8 ± 590.4

571 ± 295.2

Test item 10 % in AOO

27624.5 ± 4049.6

13812 ± 2024.8

Test item 5 % in AOO

24174.6 ± 2300.6

12087 ± 1150.3

Test item 2.5 % in AOO

23993.0 ± 9696.7

11997 ± 4848.4

Test item 1 % in AOO

7120.4 ± 3373.3

3560 ± 1686.7

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.

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 concentrations of 10 %, 5 %, 2.5% and 1 % (w/v) concentrations as formulations (solutions) in a suitable vehicle (AOO) showed to have 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. Individual approach was used in this test. The vehicle and maximum dose selection was performed according to the relevant guidelines. The test item was a viscous liquid and adequately miscible with Acetone: Olive oil 4:1 (v/v) mixture (AOO) at a maximum achievable concentration of 75 % (w/v). Since AOO is the most preferred vehicle by the relevant guidelines no other vehicles were evaluated. Based on results of the Dose Range Finding Test, where adverse effect (irritation) was observed at concentrations of 75 %, 50 % or 25 % (w/v), the test item was examined in the main test at 10 %, 5 %, 2.5 % and 1 % (w/v) concentrations as formulations in AOO. Appropriate positive control group, furthermore a negative (vehicle) control group were employed. The positive control item (α-Hexylcinnamaldehyde [HCA]; 25 % (w/v) in AOO) induced the appropriate stimulation over the control (SI = 12.0), 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 sign 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 value) were observed in the 10 % and in the 5 % (w/v) dose groups (5/5 or 1/5 animals, respectively) although no significant erythema (scored as ≥ 3) or any other local effect was observed in any test group during the test. (Ear thickness was not monitored in the other test groups.) Outlier DPM values were considered in the 10 % and in the 2.5 % (w/v) dose groups (1/5 animals in each). Although no technical reason was revealed, these values were excluded from the evaluation. Significant lymphoproliferation (SI ≥ 3) was observed for the test item at all test concentrations. The corresponding stimulation index values (calculated without outliers) were 24.2, 21.2, 21.0 and 6.2 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. The measured DPM values corrected with the mean background value were statistically evaluated. Statistically significant differences compared to the vehicle control (AOO) were observed at all test concentrations (p < 0.01 at 5 % and 1 % (w/v) or p < 0.05 at 10 % and 2.5 % (w/v) concentrations, evaluated by Mann-Whitney U-test). Dose-related response was considered although linearity of the dose-response relationship was not statistically significant (linear regression, p = 0.26, r = 0.74). It is possible that irritation interfered with the results at 10 % and 5 % (w/v) concentrations hence the dose-response analysis was equivocal. On the other hand no adverse effect or outlier value was observed at 1 % (w/v) concentration, hence the measured DPM values 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) is 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.

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