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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:
2016-09-14 to 2016-09-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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:
2-(cyclohexylamino)ethanesulphonic acid
EC Number:
203-115-6
EC Name:
2-(cyclohexylamino)ethanesulphonic acid
Cas Number:
103-47-9
Molecular formula:
C8H17NO3S
IUPAC Name:
2-(cyclohexylamino)ethanesulfonic acid
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/Ca Ola Hsd mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI COOP ZRT. Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF at arrival, Good conventional during test
- Age at study initiation: 9 - 12 weeks
- Weight at study initiation: 17.5 - 21.0 g
- Fasting period before study: no
- Housing: group caging (4 mice/cage)
- Diet: ad libitum, ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: ad libitum, tap water
- Acclimation period: 14 days
- Indication of any skin lesions: Only healthy animals (and not showing any sign of skin lesion) were used.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: Pluronic®PE 9200
Concentration:
2.5, 5, 10 % (w/v) (max. concentration determined by solubility)
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Solubility was assessed in 7 vehicles. The best solubility was achieved in Plu with a maximum concentration of 10 % (w/v). No or not adequate solubility was observed with the other vehicles at this maximum concentration.
- Irritation: No sign of significant irritation were observed.
- Systemic toxicity:No mortality or sign of systemic toxicity was observed during the test. No significant, treatment related effect on the body weights was considered in any dose group. Although body weight was decreased by > 5 % (actually 7 %, in 1 of 2 animals) was observed in the 2.5 % (w/v) dose group the effect was considered not treatment related in the absence of a dose response.
- Ear thickness measurements: No increase of ≥ 25 % of ear thickness observed on any day of measurement.
- Erythema scores: All values were 0 for all concentrations, mice and time points (days 1, 2, 3, 4, 5, 6).

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: animals were assigned randomly achieving body weight homogeneity between groups
- Criteria used to consider a positive response: 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 (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION: Formulations (apparently solutions) were prepared with aqueous 1 % (w/v) Pluronic®PE 9200 (Plu). The test item was weighed and the 10 % (w/v) formulation (stock solution) was prepared with the vehicle in a final volume of 2 mL using volumetric vials and mechanical agitation. Further test concentrations were diluted from the stock solution. All formulations were freshly prepared just before the daily treatments.
25 µL of the three respective formulations (substance dissovled in Pluronic), the positive control substance or the vehicles were applied to the dorsal surface of each ear using a pipette. 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not applicable.

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) HCA solution (formulated 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 = 6.7).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
0.6
Test group / Remarks:
10%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Please refer to Table 1.

DETAILS ON STIMULATION INDEX CALCULATION: the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective vehicle control group

EC3 CALCULATION: not applicable

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 were observed in any treatment group.

BODY WEIGHTS: No significant, treatment related effect on body weights was observed during the test. Body weight decreased by ≥ 5 % (actually 5 % decrease) was observed in one animal only in the 10 % (w/v) dose group but it was considered neither significant nor treatment related because one vehicle control had a similar weight decrease of 4 %.

Any other information on results incl. tables

Table 1: Results

Dose Group Measured DPM/group Group* DPM DPM/Mouse# Stimulation Index Values
Vehicle control for the positive control: AOO 9567 9534 2383.5 1
Positive control: 25 % HCA in AOO 64108 64075 16018.8 6.7
Vehicle control for the test item: Plu 3124 3091 772.8 1
Test item: 25 % in Plu 1994 1961 490.3 0.6
Test item: 10 % in Plu 3392 3359 839.8 1.1
Test item: 5 % in Plu 2080 2047 511.8 0.7

HCA = α-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

Plu = aqueous 1 % (w/v) Pluronic®PE 9200

*Group DPM = measured DPMgroup- average DPMbackground

Average DPMbackground = 33

# Number of animals/group = 4

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item tested at the maximum feasible concentration of 10 % (w/v, based on solubility) and also at concentrations of 5 % or 2.5 % (w/v) in a suitable vehicle was shown to have no skin sensitization potential in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay according to OECD 429. A formulation evaluation and a Dose Range Finding test (DRF) were performed to find an appropriate vehicle and the maximum applicable concentration according to the relevant guidelines. Solubility of the test item in vehicles preferred in the LLNA was evaluated and concentration series of 100 %, 50 %, 25 %, 10 % etc. were used. The maximum attainable concentration (based on solubility) was 10 % (w/v) in aqueous 1 % (w/v) Pluronic®PE 9200 (Plu). According to results of the DRF, where no adverse effect was observed up to this maximum concentration, the test item was examined in the main test as 10 %, 5 % or 2.5 % (w/v) formulations in the selected vehicle of Plu. Appropriate positive control (α-Hexylcinnamaldehyde, HCA), furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed. The positive control item (25 % (w/v) HCA in Acetone:Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation over the relevant control (SI = 6.7) 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 sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (Plu) was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.6, 1.1 and 0.7 at test item concentrations of 10 %, 5 % and 2.5 % (w/v), respectively. No significant dose-response relationship was observed. Accordingly, the test item was considered to be not a skin sensitizer. In conclusion, under the conditions of the present assay, the test item tested at the maximum feasible concentration of 10 % (w/v, based on solubility) and also at concentrations of 5 % or 2.5 % (w/v) as formulations (apparently solutions) in a suitable vehicle (aqueous 1 % (w/v) Pluronic®PE 9200) was shown to have no skin sensitization potential in the Local Lymph Node Assay.