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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:
2016-09-14 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:
2010
Deviations:
yes
Remarks:
Deviation in sample preparation without any presumed effect on the study
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2012
Deviations:
yes
Remarks:
Deviation in sample preparation without any presumed effect on the study
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-(4-morpholinyl)ethansulfonic acid hydrate
Cas Number:
1266615-59-1
Molecular formula:
C6H13NO4S * xH2O
IUPAC Name:
2-(4-morpholinyl)ethansulfonic acid hydrate
Test material form:
solid: particulate/powder

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. Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 17.9 - 21.0 g
- Fasting period before study: unknown
- Housing: group caging (4 mice/cage), Type II. Polypropylene / polycarbonate
- Diet: ad libitum, ssniff® Rat/Souris-Elevage E complete diet for rats and mice
- Water: ad libitum, tap water
- Acclimation period: 14 days

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

Study design: in vivo (LLNA)

Vehicle:
other: Pluronic
Concentration:
2.5, 5, 10%
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: yes (using seven recommended solvents)
- Irritation: no irritation was observed for the three concentration 2.5, 5 and 10% in a preliminary test
- Systemic toxicity: no toxicity was observed for the three concentration 2.5, 5 and 10% in a preliminary test
- Ear thickness measurements: no changes in ear thickness were observed for the three concentration 2.5, 5 and 10% in a preliminary test
- Erythema scores: all 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: stimulation index of one concentration > 3

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the three respective formulations (substance dissovled in Pluronic) were applied to the dorsal surface of each ear using a pipette
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
none

Results and discussion

Positive control results:
Stimulation index (SI) of (w/v) hexyl cinnamic aldehyde: 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:
2.3
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
10%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Disintegration per minute (per mouse (average)):
vehicle control: 772.8
2.5%: 513.0
5%: 1759.3
10%: 894.5

DETAILS ON STIMULATION INDEX CALCULATION: ratio of DPM/mouse of test concentrations and vehicle control

EC3 CALCULATION: not applicable

CLINICAL OBSERVATIONS: no irritation or other effects observed

BODY WEIGHTS: no treatment related effects

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present assay, the test item at the maximum feasible concentration of 10 % and also at concentrations of 5 % or 2.5 % (w/v) was shown to have no skin sensitization potential in the Local Lymph Node Assay.
Executive summary:

A study according OECD TG 429 was performed to evaluate the skin sensitising potential of the test item. 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 (alpha-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 1.2, 2.3 and 1.7 at test item concentrations of 10 %, 5 % and 2.5 % (w/v), respectively. No significant dose-response relationship was observed. Accordingly, the substance was considered to be not a skin sensitizer.