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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of diphenyl ether and 9-methylene nonadecane
EC Number:
943-303-1
Molecular formula:
Variable, substance is a UVCB
IUPAC Name:
Reaction products of diphenyl ether and 9-methylene nonadecane
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest -Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Inbred SPF quality
- Age at study initiation: 10 weeks
- Weight at study initiation: Within 20% of sex mean
- Housing: Group housed in makrolon cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: Checked as part of health inspection

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12 hours light 12 hours dark

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
50 or 100% concentration (preliminary phase)
0, 25, 50 and 100% concentration (main phase)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Soluble in vehicle
- Irritation: Slight irritation was noted for the majority of animals between Days 2 and 4.
- Systemic toxicity: None
- Ear thickness measurements: Less than 25% variation
- Erythema scores: Not measured

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI >/= 3

TREATMENT PREPARATION AND ADMINISTRATION:

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the Nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine.
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue Processing for Radioactivity - Day 6:
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter: 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.

Radioactivity Measurements - Day 7:
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis not required in study design

Results and discussion

Positive control results:
Showed that the model was acceptable for the testing of contact hypersensitivity

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.3
Test group / Remarks:
25% test item concentration
Parameter:
SI
Value:
1.6
Test group / Remarks:
50% test item concentration
Parameter:
SI
Value:
2.2
Test group / Remarks:
100% test item concentration
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION: The SI values are calculated as a ratio between the individual dpm values, compared to those in control animals.

CLINICAL OBSERVATIONS: No irritation was observed in any of the animals. The scaliness observed in the majority of vehicle dosed animals on Days 2 and 3 was considered to not have a toxicologically significant effect on the activity of the nodes.

BODY WEIGHTS: There were no effects on bodyweights or bodyweight gain.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 100%, the test substance was not considered to be a skin sensitizer.