Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 30 April to 21 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
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
Test material form:
solid - liquid: suspension
Details on test material:
Batch No.: 902/332991/B/X/4/1
Purity: 96.9%

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: individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water (e.g. ad libitum): Free access to mains tap water
- Acclimation period: at least five days

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

IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
25%, 10% , 5% w/w
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
- Irritation: No signs of systemic toxicity, visible local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
- Lymph node proliferation response:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer 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 will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION: Groups of five mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in propylene glycol. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 μL of the positive control item, Phenylacetaldehyde (>90%), at a concentration of 2.5% v/v in propylene glycol was applied to the dorsal surface of each ear.
Positive control substance(s):
other: Phenylacetaldehyde
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non-homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)

Results and discussion

Positive control results:
Phenylacetaldehyde (>90%), gave a Stimulation Index of greater than 3 when tested at a concentration of 2.5% v/v in propylene glycol.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Vehicle control group: na 5% w/w in propylene glycol: 1.37 10% w/w in propylene glycol: 1.06 25% w/w in propylene glycol: 1.19
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean dpm/Animal: Vehicle control group: 2318.22 5% w/w in propylene glycol: 3180.36 10% w/w in propylene glycol: 2453.06 25% w/w in propylene glycol: 2767.60

Any other information on results incl. tables

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

Body weight change of the test animals between Day 1 and Day 6 was comparable to those observed in the corresponding control group animals over the same period.

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 a non-sensitizer under the conditions of the test.