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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:
from 08 November 2012 to 20 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD Guideline 429 and EU Method B.42.

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Remarks:
2.5% v/v
Concentration:
Preliminary screening test:
25 µL of the test item at a concentration of 25% w/w in propylene glycol. This concentration was selected as maximum concentration suitable for dosing in solubility trials.

Main test:
Test item at concentrations of 25%, 10% or 5% w/w in propylene glycol. These concentrations were selected as, at 25%, no excessive irritation was noted.
No. of animals per dose:
four animals
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the solubility of the test item in different vehicles was determined on the basis of maximising the concentration and solubility whilst producing a solution/suspension suitable for application. The vehicles tested were: acetone/olive oil (4:1), dimethyl formamide, butanone, dimethyl sulphoxide, acetone, ethanol/distilled water (7:3), 1% pluronic L92 in distilled water and propylene glycol. The concentrations tested were 50% (0.5 g test item + 0.5 g vehicle) and 25 % (1 g of 50% dilution made up to 2 g).
- Irritation: A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at a concentration of 25% w/w in propylene glycol, to the dorsal surface of each ear for three consecutive days (day 1, 2, 3). The mouse was observed twice daily on days 1, 2, and 3 and once daily on days 4, 5 and 6. Local skin irritation was scored daily. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in propylene glycol.
- Lymph node proliferation response: The thickness of each ear was measured using an Oditest micrometer, pre-dose on Day 1, post dose on day 3 and on day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods days 1 and 3 and days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: After the acclimatisation the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card.
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). The test item is regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation is classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION: For the purpose of the study, the test item was freshly prepared as a suspension in propylene glycol. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. All vehicles tried at 50% were unsuitable for the LLNA test. The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
Positive control substance(s):
other: Phenylacetaldehyde
Statistics:
No data

Results and discussion

Positive control results:
DPM: 70878.16
Stimulation index: 18.93
Result: positive

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Test item stimulation index: 0.74 (5% w/w in propylene glycol) 1.90 (10% w/w in propylene glycol) 1.97 (25% w/w in propylene glycol)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 3744.41 (vehicle) 2768.51 (5% w/w in propylene glycol) 7127.13 (10% w/w in propylene glycol) 7364.06 (25% w/w in propylene glycol)

Any other information on results incl. tables

Solubility:

The vehicle suitable for dosing was propylene glycol.

Clinical signs and mortality data:

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight:

Bodyweight changes of the test animals between day 1 and day 6 were comparable to those observed in the corresponding control group animals over the same period.

Ear thickness measurements and ear thickness changes, local skin irritation:

There was no increase in ear thickness (> 25%) in any of the test or control animals on days 3 and 6. No signs of irritation were seen in any of the animals throughout the test.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be non-sensitiser under the conditions of the test according to CLP/GHS criteria.