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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:
4 Aug 2018 - 8 Sept 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The CBA/J mouse was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHW) to assess skin sensitization.

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
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-methylenebis[N-sec-butylaniline]
EC Number:
226-122-6
EC Name:
4,4'-methylenebis[N-sec-butylaniline]
Cas Number:
5285-60-9
Molecular formula:
C21H30N2
IUPAC Name:
4,4'-methylenebis[N-sec-butylaniline]
Test material form:
liquid
Details on test material:
Appearance: Slight yellow liquid
Storage conditions: At room temperature protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Remarks:
Inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
- Source:Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation of dosing: 19.2 to 23.0 g.
- Housing: Animals were group housed in labeled Makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 44 - 75
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12/12
- Psychological/environmental enrichment: paper and shelters except when interrupted by study procedures/activities.

IN-LIFE DATES: From: 24 Aug 2018 - 17 Sept 2018

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TEST
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. Two test item concentrations were tested; a 50% and 100% concentration. Based on the results of the initially treated animals, two additional animals were treated in a similar manner with two lower concentrations (10% and 25%) at a later stage.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10-11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed.
Two young adult animals (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 prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The SI is the ratio of the DPM/animal compared to DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION
Test substance preparation:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/ composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at Charles River and on test substance data supplied by the sponsor. The vehicle was chosen from the vehicles specified in the test guideline.

ADMINISTRATION
Induction- days 1, 2 and 3:The dorsal surface of both ears was topically treated (25 μL/ear) with the test item
Excision of nodes - day 6: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine, after five hours were euthanized and the draining limph node was excised.
Tissue processing for radioacitivity - day 6: A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze, washed and frozen until the next day.
Radioactivity measurements - day 7: Radioactivity measurements were performed using a Packard scintillation counter (2910TR)

OBSERVATIONS
Mortality/Viability: Twice daily.
Body weights: On day 1 (pre-dose) and day 6 (prior to necropsy).
Clinical signs: Once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on days 1-6 (on days 1 - 3 within 1 hour after dosing) according to a numerical scoring system (see table 1 below). Furthermore, a description of all other (local) effects was recorded according to guidelines.

TERMINAL PROCEDURES
No necropsy was performed, since all animals survived until the end of the observation period.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
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.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
3.4
Test group / Remarks:
25%
Cellular proliferation data / Observations:
- Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 437, 530 and 1010 DPM, respectively.
- The mean DPM/animal value for the vehicle control group was 299 DPM.
- The SI values calculated for the test item concentrations 5, 10 and 25% were 1.5, 1.8 and 3.4, respectively.

Any other information on results incl. tables

Results  Pre-screen Test

At a 100% and 50% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria. At a 25% and 10% test item concentration, no signs of systemic toxicity were noted and no to very slight irritation was observed and therefore the 25% concentration was selected as highest concentration for the main study.

Skin Reactions / Irritation

The very slight erythema of the ears as shown by three animals treated at 25% on Days 2 or 3 was considered not to have a toxicologically significant effect on the activity of the nodes. 

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

All of the auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 25%, which were considered to be enlarged. 

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
A LLNA test was performed for 4,4’-methylenebis-N-sec-butylaniline. The test substance could elicit a SI ≥ 3. The data showed a dose response and an EC3 value of 21% was calculated. The test substance has been found to be skin sensitizer and has to be classified as skin sensitizer category 1B according to the GHS, and according to the Regulation (EC) No 1272/2008 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.
Executive summary:

An LLNA skin sensitization study was performed according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 5, 10 or 25% w/w. The very slight erythema of the ears as shown by three animals treated at 25% on Days 2 or 3 was considered not to have a toxicologically significant effect on the activity of the nodes. All of the auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 25%, which were considered to be enlarged. 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 5, 10 and 25% were 437, 530 and 1010 DPM, respectively. The mean DPM/animal value for the vehicle control group was 299 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.5, 1.8 and 3.4, respectively. Reliable negative controls included, and the six-month reliability check with a positive control  demonstrate the reliability of the model for contact hypersensitivity.

Since 4,4’-methylenebis-N-sec-butylaniline could elicit a SI ≥ 3 and according to the recommendations made in the test guidelines (including all amendments), 4,4’-methylenebis-N-sec-butylaniline would be regarded as skin sensitizer and because the EC3 value is > 2%, it has to be classified as skin sensitizer category 1B according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments). Moreover, according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), 4,4’-methylenebis-N-sec-butylaniline should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.