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Diss Factsheets

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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
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:
Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]
EC Number:
940-594-7
Molecular formula:
C52H86O4S3 , C37H58O3S2
IUPAC Name:
Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]
Test material form:
liquid
Details on test material:
Intended use: Antioxidant for plastics
Appearance: Brown liquid
Storage conditions: Room temperature protected from light
Supplier: Sponsor
Lot number: 102Y1
Expiry date: 30 April 2014
Purity 98.8%

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Ltd.
- Females (if applicable) nulliparous and non-pregnant: no
- Age at study initiation:eight to ten weeks of age
- Weight at study initiation:16.5 to 20.8 g
- Housing:Animals were housed inside a barriered rodent facility (Building F21, Room 061/062).
- Diet (e.g. ad libitum):The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum):Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes
- Acclimation period:six days prior to the start of the study
:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr):The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light):Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.
- IN-LIFE DATES: From: Day 1 To: Day 6

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25% and 50% v/v in acetone: olive oil 4:1 v/v (AOO) and as supplied
No. of animals per dose:
4 mice
Details on study design:
MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Assessment of Skin Sensitization Potential using the Local Lymph Node Assay in the Mouse (Pooled treatment group approach)
- Criteria used to consider a positive response:Results for each treatment group were expressed as the Stimulation Index (SI). This was derived by dividing the mean dpm/mouse for each treated group and the positive control group by the mean dpm/mouse in the vehicle control group. If the SI is 3 or more, the test substance is regarded as a skin sensitizer. The positive control group is expected to give an SI of 3 or more to demonstrate the validity of the study.

TREATMENT PREPARATION AND ADMINISTRATION:

The test substance, AO-26, was prepared for administration as a series of graded concentrations in the vehicle, by direct dilution. It was also administered as supplied. The test substance was used without adjustment for purity and all formulations were prepared on the day of dosing at the required concentrations. The absorption of the test substance was not determined. Determination of the homogeneity, stability and purity of the test substance or test substance formulations were not undertaken as part of this study. Detailed records of test substance usage were maintained. The amount of test substance necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Groups of four mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 microL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette. Further groups of four mice received the vehicle alone or the positive control substance in the same manner. The sham control mice were handled in the same manner, however no liquid was applied to the ears.

Five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 microL of phosphate buffered saline containing 3H-methyl Thymidinea (3HTdR: 80 microCi/mL) giving a nominal 20 microCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI for the positive control substance hexyl cinnamic aldehyde (HCA), was 10.7 which demonstrates the validity of this study.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
41.7
Parameter:
SI
Value:
2.8
Test group / Remarks:
25%
Parameter:
SI
Value:
3.1
Test group / Remarks:
50%
Parameter:
SI
Value:
7.1
Test group / Remarks:
As supplied
Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: There were no deaths and no signs of ill health or toxicity were observed during this study. Greasy fur on the head was noted following dose or vehicle administration and dose residue on the ears was noted following dose administration, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.

BODY WEIGHTS: A loss in bodyweight was noted for two mice receiving 50% v/v or dosed with the positive control over the study period, however, a small loss in bodyweight is not uncommon in young laboratory mice and is not considered to be an effect of treatment.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
AO-26 is regarded as a potential skin sensitizer. The EC3 value was calculated to be 41.7% v/v.
Executive summary:

The study was performed to assess the skin sensitization potential of AO-26 using the local lymph node assay (LLNA). The study comprised three treated groups, each comprising four female mice receiving

AO-26 at concentrations of 25 or 50% v/v or as supplied. Similarly constituted groups received the vehicle (acetone: olive oil 4:1v/v), positive control substance (25% v/v hexyl cinnamic aldehyde) or acted as a sham control. The mice were treated by daily application of 25 L of the appropriate concentration of test material or control (vehicle or positive), to the dorsal surface of both ears for three consecutive days. The sham control mice were handled in the same way, however no liquid was applied to the ears. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by -scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index

(SI). The test substance is regarded as a sensitizer if at least one concentration of the chemical has a SI of three or more.

Results

The SI obtained for 25 or 50% v/v or as supplied were 2.8, 3.1 and 7.1 respectively which indicates that AO-26 showed the potential to induce skin sensitization. The EC3 value was calculated to be 41.7% v/v. The SI for the positive control substance hexyl cinnamic aldehyde was 10.7, which demonstrates the validity of this study.

Conclusion

AO-26 is regarded as a potential skin sensitizer. The EC3 value was calculated to be 41.7% v/v.