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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:
From 2017-12-06 to 2018-01-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
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:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): JNJ-559728-AAA (T001250)
- Physical state: solid (powder)
- Appearance: White powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16DB2043
- Expiration date of the lot/batch: 25 April 2020 (retest date)
- Purity: 99% (base titration - assay)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: No data. Homogeneity was assessed by visual inspection of the formulations

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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 was not applicable, since the test method required a logical concentration range rather than specific dose levels to be dosed.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Remarks:
CBA/J strain, inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: young adults (approx. 10 weeks old)
- Weight at study initiation: 19.6 - 23.7 grams
- Housing: Group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS- J. Rettenmaier & Söhne GmbH + Co. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd., USA) 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 Spezialdiaten 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C; actual daily mean: 22 to 23 °C
- Humidity (%): 40 to 70%; actual daily mean: 42 to 46%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
pre-screen test: 25 and 50% w/w (10 and 25% w/w at later stage based on the results of the initially treated animals)
main study: 0, 2, 10 and 25% w/w
No. of animals per dose:
Pre-screen test: 2 females per group, 4 groups (1 excluded from results)
Main study: 5 females per group; 4 groups
Details on study design:
Rationale vehicle:
The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at Charles River Den Bosch. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v) (test item did not dissolve), N,N-dimethylformamide (test item did not dissolve), methylethylketone (test item did not dissolve), propylene glycol (thin paste/turbid solution) and dimethylsulfoxide (turbid solution). Propylene glycol appeared visually as the most homogenous formulation and was therefore used as vehicle in this study.

PRE-SCREEN TESTS:
- Systemic toxicity: At a 25% and 10% test item concentration no signs of toxicity or excessive irritation were noted.
- Ear thickness measurements: At a 50% test item concentration, variation in ear thickness did not meet the selection criteria. At a 25% test item concentration, variation in ear thickness was within
the selection criteria. At a 10% test item concentration, variation in ear thickness for one animal exceeded the selection criteria while the other animal did not exceed the selection criteria. Since only one animal at 10% and no animals at 25% did not meet the selection criteria, 25% was selected as the highest concentration to be used in the main study.
- Erythema scores: 0 = no erythema in all animals

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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 (2011) (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. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3).
Classification of results:
SI value UN-GHS 2015; 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

TREATMENT PREPARATION AND ADMINISTRATION:
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistics performed

Results and discussion

Positive control results:
At concentrations 5%, 10% and 25% SI values of the positive control item were 1.4, 2.2, and 3.5 respectively. An EC3 value of 19.2% was calculated using linear interpolation. The calculated EC3 value was in the accepable range of 4.8 and 19.5%. The results of the 6 monthly reliability checks of the recent years were 13.2, 14.1, 17.3, 9.8, 17.8, 18.0, 14.7 and 13.2%
Based on the results, it was concluded that the Local Lymph Node Assay as performed in the laboratory is an appropriate model for testing contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.5
Variability:
+/- 0.4
Test group / Remarks:
Based on 5 animals in 2% w/w in propylene glycol group
Parameter:
SI
Value:
1.7
Variability:
+/- 0.4
Test group / Remarks:
Based on 5 animals in 10% w/w in propylene glycol group
Parameter:
SI
Value:
3.6
Variability:
+/- 1.3
Test group / Remarks:
Based on 5 animals in 25% w/w in propylene glycol group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
0% w/w group: mean DPM/animal: 610 ± 46
2% w/w group: mean DPM/animal: 937 ± 237
10% w/w group: mean DPM/animal: 1026 ± 240
25%w/w group: mean DPM/animal: 2205 ± 792

DETAILS ON STIMULATION INDEX CALCULATION
The SI values calculated for the item concentrations 2, 10 and 25% were 1.5, 1.7 and 3.6, respectively.

EC3 CALCULATION
These results indicate that the test item could elicit 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 20.3% was calculated.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation:
No irritation of the ears was observed in any of the animals examined.
White test item remnants were present on the dorsal surface of the ears of all animals treated at 10% and 25% between Days 1 and 3, 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 of the main study.
- Macroscopy of the auricular lymph nodes and surrounding area:
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. 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.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
These results indicate that the test item could elicit 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 20.3% was calculated.

Based on these results:
-According to the recommendations made in the test guidelines (including all amendments), T001250 would be regarded as skin sensitizer.
-According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), T001250 should be classified as skin sensitizer (Category 1B).
- According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), T001250 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.