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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:
05 March 2014 - 11 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008 (Amendment 06 July 2012 Commission Regulation (EU) No 640/2012)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

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

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: Young adult mice, 11-12 weeks
- Weight at study initiation: 19.1 -23.5 g
- Housing: Grouped caging in small groups
- Diet: ssniff® SM R/M-Z+H, Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod: Light; 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
5, 2.5, 1, 0.5 %
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: DMF
- Irritation: At a concentration of 100% Erythema Scores of 2 were observed after the 3rd day of treatment. An increase of ear thickness ≥ 25 % was observed at 50, 25 and 10 %

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 criteria 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:
Animals in the treatment groups were treated with the relevant vehicles (DMF or AOO), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations 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, of the positive control substance (positive control group) or of the vehicles (AOO or DMF as negative control groups).
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. 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.
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised. After preparation of draining auricular lymph nodes, single cell suspension of lymph node cells were prepared by several washing and centrifugation processes.
For the measurement of incorporated radioactivity the vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. 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) trichloroacetic acid (TCA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was performed by SPSS/PC+ (4.0.1) software package.
The measured DPM values corrected with the mean background value were used for statistical analysis of the proliferation data. The heterogeneity of variance between the groups treated with the test item and the relevant vehicle control (DMF) 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 substance induced the appropriate, statistically significant stimulation compared to the control (SI = 12.2; p < 0.01, T-test versus AOO control). 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:
1
Test group / Remarks:
Vehicle positive control
Key result
Parameter:
SI
Value:
12.2
Test group / Remarks:
Positive control (HCA in AOO)
Key result
Parameter:
SI
Value:
11.8
Test group / Remarks:
Test item 5% in DMF
Key result
Parameter:
SI
Value:
5.2
Test group / Remarks:
Test item 2.5 % in DMF
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
Test item 1 % in DMF
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
Test item 0.5 % in DMF
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Vehicle control of test item: DMF
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
3 163.2
Test group / Remarks:
Vehicle positive control (AOO)
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
38 558.6
Test group / Remarks:
Positive control (HCA in AOO)
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
22 019
Test group / Remarks:
Test item 5% in DMF
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
9 732.8
Test group / Remarks:
Test item 2.5 % in DMF
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
2 193.2
Test group / Remarks:
Test item 1 % in DMF
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
2 201.6
Test group / Remarks:
Test item 0.5% in DMF
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
1 865
Test group / Remarks:
Vehicle control test item (DMF)

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, SIKA Hardener AI tested at up to the maximum applicable (non-toxic, non-irritant) concentration of 5 % (w/v) was shown to have skin sensitization potential. Based on the EC3 value of 1.68% SIKA Hardener AI was considered a moderate skin sensitizer in this LLNA.
Executive summary:

The Local Lymph Node Assay (LLNA) was conducted according to the current OCED guideline 429. Individual approach was used in this test.

Selection of test item concentrations based on the results of a formulation evaluation and also results of a preliminary irritation/toxicity test .The test item was formulated in N,N-Dimethylformamide (DMF).

Due to significant irritation observed in the preliminary test at concentrations of 100 % (the undiluted test item), 50 %, 25 % and 10 % (w/v) the test item was examined in the main test at concentrations of 5 %, 2.5 %, 1 % and 0.5 % (w/v).

 

In the main test, 35 female CBA/Ca mice were allocated to seven groups of five animals each:

- four groups received SIKA Hardener AI at 5 %, 2.5 %, 1 % and 0.5 % (w/v, respectively) in DMF,

- one control group (used as negative control for the test item treated groups) received the vehicle of the test item (DMF) only,

- one control group (used as negative control for the positive control substance treated group) received the vehicle of the positive control substance (AOO) only,

- the positive control group received 25 % (w/v) α-Hexylcinnamaldehyde (HCA) in AOO.

 

Each substance was applied on the external surface of each ear (25 μL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. Auricular lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI).

The positive control substance induced the appropriate, statistically significant stimulation compared to the relevant control (SI = 12.2; p < 0.01, T-test versus AOO control), thus confirming the validity of the assay.

 

No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity was observed in any treatment group. No sign of irritation or any other local effect was observed in any treatment group.

Visually larger lymph nodes than the relevant vehicle controls (AOO or DMF) were observed in the positive control group and also in the 5 % (w/v) dose group. Visual appearance of the lymph nodes was normal in the vehicle (both AOO and DMF) control groups and in the other test item treated groups.

Significant lymphoproliferation (indicated by an SI value > 3) was observed for the test item at concentrations of 5 % and 2.5% (w/v). The observed SI values were 11.8, 5.2, 1.2 and 1.2 at treatment concentrations of 5 %, 2.5 %, 1 % and 0.5 % (w/v), respectively.

The measured DPM values corrected with the mean background value were statistically evaluated. Statistically significant differences compared to the relevant vehicle control (DMF) were observed at the 5 % and 2.5 % (w/v) (p < 0.01, Mann Whitney U-test). Statistically significant dose-related response was observed (evaluated by linear regression, p = 0.006, r = 0.99).

Since the test was valid and no sign of systemic toxicity or significant irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.

 

According to evaluation criteria, the significantly increased proliferation values observed at 5 % and 2.5 % (w/v) concentrations and the significant dose-related response are considered evidence that SIKA Hardener AI is a skin sensitizer.

The EC3 (dose calculated to induce a stimulation index of 3) was calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method. The calculated EC3 value of SIKA Hardener AI was 1.68 % in this LLNA. Using the calculated EC3 values SIKA Hardener AI can be ranked among moderate skin sensitizers in this LLNA according to the published data for classification of contact allergens.