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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:
12 April 2013 - 28 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
yes
Remarks:
as the dose formulations of the main test were delivered in stoppered plastic tubes, they were stirred using vortex prior to application.
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
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: the animals of the preliminary test were approximately 11 weeks old on the day of treatment, and the animals of the main test were approximately 8 weeks old.
- Mean body weight at study initiation: the animals of the preliminary test had a mean body weight of 22.5 g (range: 21.7 g to 23.7 g) and the animals of the main test had a mean body weight of 20.1 g (range: 18.5 g to 22.3 g).
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 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: 15 May 2013 to 27 May 2013

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
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.
No. of animals per dose:
- preliminary test: 4 nulliparous and non pregnant females,
- main test: 28 nulliparous and non pregnant females.
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, at approximately the same time each day, a dose-volume of 25 µL of the control or dose formulation 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, and to facilitate ear thickness measurement, 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.
The dose formulations were stirred manually throughout the dosing procedure.
Positive control substance(s):
other: a-hexyl cinnamaldehyde (HCA)
Statistics:
no

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group (SI = 6.68).

In vivo (LLNA)

Results
Key result
Parameter:
EC3
Value:
6
Cellular proliferation data / Observations:
No unscheduled deaths and no clinical signs indicative of systemic toxicity were observed during the observation period. Body weight of animals was unaffected by the test item treatment.
At the concentration of 25%, erythema was observed on day 3 and on day 4/4 and in 2/4 females, respectively. Dryness of ear skin was noted in 3/4 and 4/4 females at the concentrations of 10 and 25%, respectively on day 6.
No notable increase in ear thickness was observed at 1, 2.5, 5 and 10%. But a significant increase in ear thickness of 50.50% was observed in females treated at the concentration of 25% on day 6.
The threshold positive value of 3 for the SI was reached in the positive control group (SI = 6.68). The experiment was therefore considered valid.
A significant dose-related lymphoproliferation (SI > 3) was noted at 10 and 25%.

Based on the positive SI value observed at concentrations of 10% and 25% (7.12 and 7.72 respectively) , the test item should be considered as a skin sensitizer. As irritation was also observed at concentration of 25%, it might be possible to conclude that an excessive local irritation may be involved in false positive lymphoproliferation responses at this concentration.
Nevertheless, in the absence of local irritation at 10%, the significant lymphoproliferative response observed at this concentration was attributed to delayed contact hypersensitivity. The EC3value is equal to 6%.

Any other information on results incl. tables

Table of results (main study):

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

1

I

1.01

Test item

2.5

I

1.21

Test item

5

I

1.93

Test item

10

I

7.12

Test item

25

III

7.72

HCA

25

-

6.68

-: not recorded,

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

III: irritant (increase in ear thickness = 25%),

HCA: a-hexyl cinnamaldehyde.

 

 

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the experimental conditions of this study, the test item gave a positive result in the murine Local Lymph Node Assay, indicative of skin sensitization properties.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). This study was conducted in compliance with OECD Guideline No. 429 and the principles of Good Laboratory Practices.

 

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 thickness of 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 recorded once during the acclimation period and then on days 1 and 6. On completion of the observation period, the animals were sacrificed then 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 ears on days 1, 2 and 3 at concentrations of 1, 2.5, 5, 10 or 25% 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, a-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 of 3H-TdR.

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

 

Results

No unscheduled deaths and no clinical signs indicative of systemic toxicity were observed during the observation period. Body weight of animals was unaffected by the test item treatment.

At the concentration of 25%, erythema was observed on day 3 and on day 4/4 and in 2/4 females, respectively. Dryness of ear skin was noted in 3/4 and 4/4 females at the concentrations of 10 and 25%, respectively on day 6.

No notable increase in ear thickness was observed at 1, 2.5, 5 and 10%. But a significant increase in ear thickness of 50.50% was observed in females treated at the concentration of 25% on day 6.

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 6.68). The experiment was therefore considered valid.

A significant dose-related lymphoproliferation (SI > 3) was noted at 10 and 25%.

 

Based on the positive SI value observed at concentrations of 10% and 25% (7.12 and 7.72 respectively) , the test item should be considered as a skin sensitizer. As irritation was also observed at concentration of 25%, it might be possible to conclude that an excessive local irritation may be involved in false positive lymphoproliferation responses at this concentration.

Nevertheless, in the absence of local irritation at 10%, the significant lymphoproliferative response observed at this concentration was attributed to delayed contact hypersensitivity. The EC3value is equal to 6%.

 

Conclusion

Under the experimental conditions of this study, the test item gave a positive result in the murine Local Lymph Node Assay, indicative of skin sensitization properties. According to the EC3value obtained, the test item should be considered as a moderate sensitizer.

 

According to the criteria of CLP Regulation,the test item should be classified as skin sensitizer (category 1 and sub-category 1B) and assigned the signal word "warning" and the hazard statement "H317: May cause an allergic skin reaction".