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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:
20 May 2019 - 18 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
Qualifier:
according to
Guideline:
other: EC No 640/2012, Part B: "Skin Sensitization: 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
Specific details on test material used for the study:
Identification: Esacure 3644
Physical Description: Light yellow solid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Test Facility test item number: 209751/A
Test item handling: The test item solutions should be prepared in amber glassware or wrapped in aluminum foil to protect them from light
Substance Name: Reaction mass of 5,7-dimethoxy-3-(4-(sec-C10-C13-alkyl)-benzoyl)-2H-chromen-2-one
EC Number: 949-790-7
EC Name: 5,7-dimethoxy-3-[4-(sec-C10-C13-alkyl)-benzoyl]-2H-chromen-2-one

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST SYSTEM
Species: Mouse
Strain: CBA/J
Condition: Inbred, SPF-Quality
Source: Janvier, Le Genest-Saint-Isle, France
Number of Animals: 20 Females (nulliparous and non-pregnant). Five females per group.
Age at the Initiation of Dosing: Young adult animals (approximately 11 weeks old) were selected.
Weight at the Initiation of Dosing: 19.8 to 25.6 g.

ENVIRONMENTAL ACCLIMATION
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

HOUSING
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

ENVIRONMENTAL CONDITIONS
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 42 to 58%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

FOOD
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

WATER
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ANIMAL ENRICHMENT
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

VETERINARY CARE
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 25 and 60% test item concentration, no signs of systemic toxicity were noted and no irritation was observed. Therefore, a 60% concentration was selected as highest concentration for the main study.
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TEST
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; a 25% and 60% concentration. The highest concentration was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

INDUCTION - DAYS 1, 2 AND 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

EXCISION OF THE NODES - DAY 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

TISSUE PROCESSING FOR RADIOACTIVITY - DAY 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS - DAY 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).


MORTALITY/MORIBUNDITY CHECKS
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

POSTDOSE OBSERVATIONS
Postdose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

BODY WEIGHTS
Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy).

IRRITATION
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing), according to the following numerical scoring system.
Furthermore, a description of all other (local) effects was recorded.
Erythema and eschar formation:

No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

TERMINAL PROCEDURES
No necropsy was performed, since all animals survived until the end of the observation period.

Results and discussion

Positive control results:
For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.7
Test group / Remarks:
10% test concentration
Parameter:
SI
Value:
1.4
Test group / Remarks:
25% test concentration
Parameter:
SI
Value:
1.4
Test group / Remarks:
60% test concentration
Cellular proliferation data / Observations:
Skin reactions/ irritation
The very slight erythema of the ears as shown by animals treated at 60% on Days 2 and/or 3 was considered not to have a toxicologically significant effect on the activity of the nodes.
White test item remnants were present on the dorsal surface of the ears of test item treated animals between Days 1 and 5, which did not hamper scoring of the skin reactions.

Systemic Toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic Examination of the Lymph Nodes and Surrounding Area
The auricular lymph nodes of the control animals and the animals treated at 60% were considered normal in size and the auricular lymph nodes of the animals treated at 10% and 25% were considered to be slightly enlarged compared to normal. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 60% were 887, 703 and 731 DPM, respectively. The mean DPM/animal value for the vehicle control group was 508 DPM. The SI values calculated for the test item concentrations 10, 25 and 60% were 1.7, 1.4 and 1.4, respectively.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 60%, Esacure 3644 was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 60%.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity (see Appendix 3).

Based on these results, Esacure 3644 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
Executive summary:

Test item concentrations selected for the main study were based on the results of a pre-screen test.  At a 25 and 60% test item concentration, no signs of systemic toxicity were noted and no irritation was observed.  Therefore, a 60% concentration was selected as highest concentration for the main study.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 60% w/w on three consecutive days, by open application on the ears.  Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v) (AcOO)).  Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.  After precipitating the DNA of the lymph node cells, radioactivity measurements were performed.  The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The auricular lymph nodes of the control animals and the animals treated at 60% were considered normal in size and the auricular lymph nodes of the animals treated at 10% and 25% were considered to be slightly enlarged compared to normal.  No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 60% were 887, 703 and 731 DPM, respectively.  The mean DPM/animal value for the vehicle control group was 508 DPM.  The SI values calculated for the test item concentrations 10, 25 and 60% were 1.7, 1.4 and 1.4, respectively.

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 60%, Esacure 3644 was considered not to be a skin sensitizer.  It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 60%.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, Esacure 3644 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.  The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).