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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
01 June 2012 - 10 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material: Ethoxylated bisphenol A diacrylate

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: approximately 11 weeks old (for the animals of the preliminary test) and approximately 8 weeks old (for the animals of the main test) on the beginning of treatment
- Mean body weight at study initiation: the animals of the preliminary test mean body weight of 21.6 g (range: 19.7 g to 22.6 g) and the animals had a mean body weight of 20.0 g (range: 18.8 g to 21.2 g).
- Fasting period before study: no
- Housing: the animals were housed by group of two (preliminary test) or four (main test) in polycarbonate cages. In the main test, the animals were individually housed in disposable crystal polystyrene cages (22.0 cm x 8.5 cm x 8.0 cm).
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 6 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.

IN-LIFE DATES: 20 June 2012 to 02 July 2012.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10, 25, 50 and 100%
No. of animals per dose:
4 females per dose.
Details on study design:
RANGE FINDING TESTS:
The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
- the vehicle should be selected on the basis of producing a homogeneous preparation suitable for application of the test item,
- the concentrations were selected from the concentration series 100% (when test item can be sampled by a pipette), 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc.,
- the maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an > 25% increase of the ear thickness.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test item was prepared at the chosen concentrations in the vehicle.
The positive control was dissolved in AOO at the concentration of 25% (v/v).
- 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)
Statistics:
No

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group: The SI of HCA (25%) was 8.36.
This experiment was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Group 4 (5%): 1.05 Group 5 (10%): 0.99 Group 6 (25%): 2.07 Group 7 (50%): 1.53 Group 8 (100%): 1.81
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per group: Group 3 (Vehicle): 2600 Group 4 (5%): 2737 Group 5 (10%): 2581 Group 6 (25%): 5381 Group 7 (50%): 3987 Group 8 (100%): 4717

Any other information on results incl. tables

No unscheduled deaths and no clinical signs were observed during the observation period.

No local reactions were observed in any animals. No notable increase in ear thickness was observed at any tested concentrations.

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

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA).

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

 

Methods

To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 10, 25, 50 or 100% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thicknessof both ears of each animal was measured and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and thenon days 1 and 6. On completion of the observation period, the animals were sacrificedthen discarded without macroscopic post-mortem examination.

 

In the main test, five groups of four female mice received the test item by topical route to the dorsal surface of both earson days 1, 2 and 3 at concentrations of 5, 10, 25, 50 or 100% under a dose‑volume of 25 µL. One negative control group of four females received the vehicle (acetone/olive oil (4/1; v/v)) under the same experimental conditions. Additionally, one positive control group of four females received the positive control, α‑hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions.

From day 1 to day 3, then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6.

After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of3H-TdR.

The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

 

Results

Mortality and clinical signs

No unscheduled deaths and no clinical signs were observed during the observation period.

 

Local irritation

No local reactions were observed in any animals.

No notable increase in ear thickness was observed at any tested concentrations.

 

Proliferation assay

The SI of the positive control was > 3; this experiment was therefore considered valid.

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


Conclusion

The test item gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 8.36). The experiment was therefore considered valid.

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

5

I

1.05

Test item

10

I

0.99

Test item

25

I

2.07

Test item

50

I

1.53

Test item

100

I

1.81

HCA

25

-

8.36

-: not recorded.

I: non-irritant (increase in ear thickness < 10%).