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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:
2018-07-25 to 2018-08-20
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
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Official Journal of the European Union No. L142, May 2008, including most recent amendments
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Diethyl ethoxymethylenemalonate
EC Number:
201-725-7
EC Name:
Diethyl ethoxymethylenemalonate
Cas Number:
87-13-8
Molecular formula:
C10H16O5
IUPAC Name:
diethyl ethoxymethylenemalonate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: JC170405
- Expiration date of the lot/batch: 2018-10-27 (retest date)
- Purity test date: 2018-08-21
- Purity: 99.4% (GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

OTHER SPECIFICS:
correction factor : 1.00

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 20 female (nulliparous and non-pregnant) mice, CBA/J strain, inbred, SPF-Quality from Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: within +/- 20% of the sex mean
- Housing: group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material . Paper and shelters were supplied as cage-enrichment. On day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days before the start of treatment, under laboratory conditions. Health inspection at least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.
- Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 48 to 70%.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pre-Screen Test: 0.5, 1, 2, 5, 50 and 100% (w/w)
Main Study: 0.1, 0.2 and 0.5% (w/w)
No. of animals per dose:
5 females per group; 4 groups
Details on study design:
RANGE FINDING TESTS:
- 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 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
- The test system, procedures and techniques were identical as those used in the main study except that the animals were aproximately 10-12 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals 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.
- Based on the results of the initially treated animals, eight additional animals (two animals per test item concentrations) were treated in a similar manner with four lower concentrations (0.5%, 1%, 2% and 5%) at a later stage.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT - INDUCTION days 1, 2, 3
- 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 vehicle.
- The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day for three consecutive days. 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.
- Criteria used to consider a positive response: a Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated 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 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, including all amendments.The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation.
Classification of results: UN-GHS 2007; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer -
SI ≥ 3 Cat 1 Skin sensitizer H317: May cause an allergic skin reaction
EC3 value ≤ 2%: sub-category 1A
EC3 value ≥ 2%: sub-category 1B

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
- After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. 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 approximately 3 mL 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) 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 as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter. 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).

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item preparations (w/w) were prepared within 4 hours prior to each dosing.
- No adjustment was made for specific gravity of the vehicle.
- Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI values calculated for the item concentrations 5, 10 and 25% were 1.1, 2.0 and 5.5 respectively. An EC3 value of 14.3% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8 and 19.2%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.5
Variability:
± 0.3
Test group / Remarks:
based on 5 animals of the 0.1% group
Parameter:
SI
Value:
3.6
Variability:
± 0.6
Test group / Remarks:
based on 5 animals of the 0.2% group
Parameter:
SI
Value:
5
Variability:
± 0.7
Test group / Remarks:
based on 5 animals of the 0.5% group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
mean DPM ± SEM:
0% w/w group: mean DPM ± SEM: 651 ± 98
2% w/w group: mean DPM ± SEM: 1633 ± 166
25% w/w group: mean DPM ± SEM: 2352 ± 378
50% w/w group: mean DPM ± SEM: 3242 ± 428
SEM = Standard Error of the Mean

DETAILS ON STIMULATION INDEX CALCULATION: The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group.

EC3 CALCULATION: An EC3 value of 0.15% was calculated

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: No irritation of the ears was observed in any of the animals examined.
- Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- Macroscopy of the auricular lymph nodes and surrounding area: All auricular lymph nodes of the animals of the control group, two animals treated at 0.2% and all animals treated at 0.5% were considered normal in size. The auricular lymph nodes of all animals treated at 0.1% and three animals treated at 0.2% were considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Any other information on results incl. tables

Pre-screen test

At a 100% test item concentration animals were killed in extremis on Day 3.

At 50%, 5%, 2% and 1% test item concentrations signs of systemic toxicity were noted and/or variations in ear thickness during the observation period exceeded 25% from Day 1 pre-dose values.

At a 0.5% test item concentration no signs of systemic toxicity were noted and no irritation was observed and variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values, except for one ear of one animal on Day 3, which was 27% thickened (22% the other ear on Day 3, both ears 11% on Day 6), this was considered to be an incidental finding as ear thickness was not increased for the other ear and both ears of the other animal and was well below 25% on Day 6.

Based on these results, the highest test item concentration selected for the main study was a 0.5% test item concentration.

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
These results show that the test item elicits a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 0.15% was calculated.
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:
- according to the recommendations made in the test guidelines (including all amendments), JNJ-39005525-AAA (EMME) would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), JNJ-39005525-AAA (EMME) should be classified as skin sensitizer (Category 1A).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), JNJ-39005525-AAA (EMME) should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.