Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 August 2010 - .....................................
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

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
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Details on test material:
- Name of test material: distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products
- Physical state: yellow liquid
- Purity: not indicated (complex composition)
- Lot/batch No.: LR_OXO_2010-07-12
- Date of analysis: 12 July 2010
- Expiration date of the lot/batch: July 2011
- Storage conditions of test material: at +4°C and under nitrogen gas.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of the treatment period, the animals of the preliminary test were approximately 10 weeks old and the animals of the main test were approximately 9 weeks old
- Weight at study initiation: 22.6 +/- 0.8 g
- Housing: housed individually in disposable crystal polystyrene cages
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter) contained in bottles
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: From: 20 August 2010 To: 17 September 2010.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1, 2.5, 5, 10 and 25%
No. of animals per dose:
4 females per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at the
maximum tested concentration of 50%.
- Irritation: dryness of the skin of the ears was noted at concentrations of 50% (on day 6) and 100% (on days 3 and 6). An increase in ear thickness
was recorded at the concentrations of 50 and 100%, showing the irritant potential of the test item at these concentrations. The highest concentration retained for the main test was therefore 25%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay (base on the design adopted by ICCVAM (Interagency Coordination Committee on the
Validation of Alternative Methods, ICCVAM 1999) and ECETOC (Monograph No. 78 Skin sensitization Testing for the Purpose of Hazard Identification
and Risk Assessment, September 2000), with the addition of the evaluation of local irritation.
- Criteria used to consider a positive response: Stimulation indice >= 3.

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: the test item was prepared at the chosen concentrations in AOO by successive dilutions.
The dosage form preparations were homogenized by vortex.
The reference item was dissolved in AOO at the concentration of 25% (v/v).
Fresh dosage form preparations were made extemporaneously of the day of each administration and the dosage form preparations were stored at
room temperature prior to use.
- Administration: on days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both
ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each
application.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Group 2: test item 1%: 0.67 Group 3: test item 2.5%: 0.83 Group 4: test item 5%: 0.98 Group 5: test item 10%: 1.05 Group 6: test item 25%: 1.62
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM per group: Group 1: Vehicle: 2479 Group 2: test item 1%: 1655 Group 3: test item 2.5%: 2047 Group 4: test item 5%: 2432 Group 5: test item 10%: 2601 Group 6: test item 25%: 4004 DPM per node: Group 1: Vehicle: 309.88 Group 2: test item 1%: 206.88 Group 3: test item 2.5%: 255.88 Group 4: test item 5%: 304.00 Group 5: test item 10%: 325.13 Group 6: test item 25%: 500.50

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products, did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

The aim of this study was to evaluate the potential of the test item distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel.

This study was conducted in compliance with the principles of Good Laboratory Practice.

 

Methods

 

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

In the main test, twenty eight female CBA/J mice were allocated to seven groups:

.           five treated groups of four animals receiving the test item distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products at the concentration of 1, 2.5, 5, 10 or 25% in a mixture acetone/olive oil (4/1; v/v) (vehicle),

.           one negative control group of four animals receiving the vehicle,

.           one positive control group of four animals receiving the reference item,a‑hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in the vehicle.

 

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2

and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI).

 

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

 

Results

Choice of vehicle

The test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at the maximum tested concentration of 50%.

Consequently, the concentrations selected for the preliminary test were 10, 25, 50 and 100%.

 

Preliminary test

Since the test item was irritant in the preliminary test at the concentrations of 50 and 100%, the highest tested concentration retained for the main test was 25%

 

Main test

Clinical signs and mortality

Neither mortality nor clinical signs were observed during the study.

 

Local irritation

No cutaneous reactions and no notable increase in ear thickness were observed at any of the tested concentrations.

 

Proliferation assay

As all acceptance criteria were met, this experiment was therefore considered valid.

The results are presented in the following table:

 

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

1

non-irritant

0.67

Test item

2.5

non-irritant

0.83

Test item

5

non-irritant

0.98

Test item

10

non-irritant

1.05

Test item

25

non-irritant

1.62

HCA

25

-

4.18

No notable lymphoproliferation was noted with the test item at any tested concentration.

 

Conclusion

Under the experimental conditions of this study, the test item distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.