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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:
26 September 2012 to 07 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-Propanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-, monosodium salt
EC Number:
700-631-8
Cas Number:
102601-34-3
Molecular formula:
C9 H19 N O4 S . Na
IUPAC Name:
1-Propanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-, monosodium salt

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, 1103 Budapest, Cserkesz u. 90, Hungary
- Age at study initiation: 10 weeks
- Weight at study initiation: 17.5-20.8 g
- Housing: Grouped caging (4 animals/cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): no information
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: Aqueous 1 % Pluronic®PE9200 (aqueous 1% Pluronic)
Concentration:
10%, 25%, 37.5% (w/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: well soluble up to 37.5%
- Irritation: no (erythema < 3)
- Lymph node proliferation response: assessed through ear thickness, which was < 25%

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: randomisation scheme (not specified)
- Criteria used to consider a positive response:
The test item is considered as a skin sensitizer, if the following criteria are fulfilled:
- That 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.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Based on the preliminary test results the test item was examined in the main test as formulations in aqueous 1 % Pluronic®PE9200 (aqueous 1 % Pluronic). The formulations in this vehicle were homogenous (apparently solutions). The test item was weighed and formulations in aqueous 1 % Pluronic prepared daily on a weight: volume basis at concentrations of 37.5 %, 25 % and 10 % (w/v) in a final volume of 1 mL using calibrated volumetric vials and intensive mechanical agitation. Formulations were freshly prepared just before the treatments.
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance (positive control group) or the vehicles (aqueous 1 % Pluronic or AOO as negative control groups) using a pipette, on the dorsal surface of each ear. After the treatments 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.

Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (1 x PBS, diluted from 10 x concentrate with purified water) containing 20 μCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistics were applied. However, the data processing is described here:
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPN (DPM divided by the number of pooled lymph nodes).
The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Based on the results EC3 value (dose calculated to induce a stimulation index of 3) of the test item could not be calculated.

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) α-Hexylcinnamaldehyde, ≥ 95 % (HCA) solution (dissolved 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 = 5.3). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
Test item concentration: 37.5 %
Key result
Parameter:
SI
Value:
2.7
Test group / Remarks:
Test item concentration: 25 %
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
Test item concentration: 10 %
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Visually larger than the control lymph nodes was observed in the positive control group only. Appearance of the lymph nodes was normal in both negative control groups (aqueous 1 % Pluronic and AOO) and in the test item treated groups.

DETAILS ON STIMULATION INDEX CALCULATION
No significant (SI ≥ 3) lymphoproliferative response was noted for the test item at the tested concentrations. The observed stimulation index values were 1.7, 2.7 and 1.7 at test item concentrations of 37.5 %, 25 % and 10 %, respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No significant dose-related response was observed (p = 0.97; r value = 0.05).

EC3 CALCULATION
Based on the results EC3 value (dose calculated to induce a stimulation index of 3) of the test item could not be calculated.

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 any other local effect was observed in any treatment groups.

BODY WEIGHTS
No significant, treatment related effect on body weights was observed during the test.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In a Local Lymph Node Assay (acc. to OECD Test Guideline 429), the test item tested at the maximum concentration of 37.5 % (w/v) based on solubility and at concentrations of 25 % and 10 % as formulations in an appropriate vehicle (aqueous 1 % Pluronic) was shown to have no sensitization potential.
Executive summary:

The skin sensitization potential of the test item following dermal exposure was assessed in the Local Lymph Node Assay. Selection of test item concentrations based on the results of a formulation evaluation and also results of preliminary irritation/toxicity test according to the relevant guidelines. The maximum concentration based on solubility was 37.5 % (w/v) in aqueous 1 % Pluronic®PE9200 (aqueous 1 % Pluronic). Based on the preliminary test results the test item was examined in the LLNA at concentrations of 37.5 %, 25 % and 10 % as formulations in the selected vehicle (aqueous 1 % Pluronic). In the main test 24 female CBA/Ca mice were allocated to 6 groups of four animals each: - three groups received the test item at three different concentrations of 37.5 %, 25 % and 10 %, - the group used as negative control for the test item treated groups received the vehicle of the test item (aqueous 1% Pluronic) only, - the positive control group received α-Hexylcinnamaldehyde (HCA) at concentration of 25 %, - the group used as negative control for the positive control group received the vehicle of the positive control substance (AOO) only. 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 item (25 % HCA in AOO) induced the appropriate stimulation over the control (SI = 5.3), thus confirming the validity of the assay. No mortality was observed during the study. No significant, treatment related effect on body weight or any other signs of systemic toxicity were observed in any treatment group during the test. No signs of irritation or any other local effect were observed at the treatment site (ears) in any treatment group. Larger lymph nodes than the control was observed in the positive control group only. Visual appearance of the lymph nodes was normal in the negative control groups (both aqueous 1% Pluronic and AOO) and in the test item treated groups. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (aqueous 1% Pluronic) was noted for the test item at the tested concentrations.The stimulation index values were 1.7, 2.7 and 1.7 at test item concentrations of 37.5 %, 25 % and 10 %, respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No significant dose-related response was observed. Since the test was valid and no sign of systemic toxicity or irritation was observed, the proliferation values obtained are 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 lack of a significantly increased proliferation up to the maximum attainable concentration of 37.5 % (w/v) based on solubility and the lack of a significant, dose-related response are considered evidence that the test item is considered to be not a skin sensitizer.

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