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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 2015-09-16 to 2015-10-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Paris Cedex, July 2010
Deviations:
no
Qualifier:
according to
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:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
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:
- Physical state: 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: Janssen Pharmaceutica N.V; 0020726249
- Expiration date of the lot/batch: 13 November 2016 (retest date)
- Purity correction factor: 1.23

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not indicated
- Solubility of the test substance in the solvent/vehicle: N,N-Dimethylformamide >200 g/L

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 is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.


In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 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: 19.2 - 23.6 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
- 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
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: N,N-Dimethyl formamide
Concentration:
Pre-screen test: 25 and 50% (w/w)
Main Study: 0, 2, 25 and 50% (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 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (50% for solids).
- The test system, procedures and techniques were identical as those used in the main study except 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.

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 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 (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: 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
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data

Results and discussion

Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.7, 3.0 and 9.1 respectively. An EC3 value of 10% 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 16.5, 14.5, 13.4, 14.1, 17.3 and 9.8%.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.9
Variability:
+/- 0.2
Test group / Remarks:
Based on 5 animals in 2% w/w in N,N-Dimethyl formamide group
Parameter:
SI
Value:
3.8
Variability:
+/- 0.9
Test group / Remarks:
Based on 5 animals in 25% w/w in N,N-Dimethyl formamide group
Parameter:
SI
Value:
2.5
Variability:
+/- 0.5
Test group / Remarks:
Based on 5 animals in 50% w/w in N,N-Dimethyl formamide group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM +/- SEM
0% N,N-Dimethyl formamide group: 636 +/- 80
2% N,N-Dimethyl formamide group: 591 +/- 135
25% N,N-Dimethyl formamide group: 2446 +/- 470
50% N,N-Dimethyl formamide group: 1590 +/- 278
SEM = Standard Error of the Mean

EC3 CALCULATION : An EC3 value (the estimated test item concentration that will give a SI =3) of 18.3% was calculated.

CLINICAL OBSERVATIONS:
Skin Reactions/Irritation: No irritation of the ears was observed in any of the animals examined. White staining of test item remnants on the dorsal surface of the ears of the animals treated at 25% and 50% (between Days 1-4) did not hamper scoring for erythema.
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: One animal treated at a concentration of 2% showed one auricular lymph node that
was considered reduced. At a concentration of 25% one animal showed enlarged lymph nodes and at a concentration of 50% two animals showed enlarged lymph nodes. The other lymph nodes 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. An EC3 value (the estimated test item concentration that will give a SI =3) of 18.3% was calculated.

Based on these results:
- according to the recommendations made in the test guidelines (including all amendments), T003680 would be regarded as skin
sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), T003680 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),
T003680 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.