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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:
24 Jul 2017 to 26 Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
Name as cited in study report: N, N, N', N'-tetrakis(2, 3-epoxypropyl)-m-xylene-α, α'-diamine
Batch: 70518-X
Expiry date: 25 May 2018
Appearance: Yellowish liquid
Test item handling: Handle in glove box (nitrogen environment)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approximately 10 weeks old) at the initiation of dosing
- Weight at study initiation: 19.3 to 23.6 g at initiation 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. These housing conditions were maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. 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.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water: Municipal tap-water was freely available to each animal via water bottles.
- Acclimation period: for at least 5 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 0.2, 0.5 and 1% (w/w)
No. of animals per dose:
5
Details on study design:
PREPARATION OF TEST ITEM
- 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.

PRE-SCREEN TEST 1:
Initially, 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 to those used in the main study except that the animals were approximately 8-11 weeks (at initiation of treatment), shelters used were either disposable paper corner home (MCORN 404, Datesand Ltd, USA) or plastic round shelters (ø 9cm, height 5 cm, Datesand Ltd, USA) 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.

PRE-SCREEN TEST 2:
- Based on the results of the initially treated animals, eight additional animals were treated in a similar manner with four lower concentrations (10%, 5%, 2% and 1%) at a later stage.

MAIN STUDY
Based on tolerability of the test item, the highest test item concentration selected for the main study was 1%. 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 killed 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 (2800TR). 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).

IN-LIFE PROCEDURES, OBSERVATIONS AND MEASUREMENTS
- 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.
- Clinical 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). Terminal body weights were collected from animals found dead or euthanized moribund after Day 1.
- Irritation: Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing).
- Necropsy: 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. Doses of 0, 5, 10 and 25 % resulted in SI of 1.0 ± 0.3, 1.2 ± 0.3, 2.3 ± 0.6 and 5.6 ± 1.8, respectively.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.4
Test group / Remarks:
0.2%
Parameter:
SI
Value:
1.5
Test group / Remarks:
0.5%
Parameter:
SI
Value:
2.3
Test group / Remarks:
1%
Key result
Parameter:
EC3
Value:
> 1
Cellular proliferation data / Observations:
PRE-SCREEN EXPERIMENT 1
At a 50% and 100% test item concentration animals were found dead or killed in extremis on Day 3.

PRE-SCREEN EXPERIMENT 2
At a 10%, 5% and 2% test item concentration, clinical signs were noted and variations in ear thickness during the observation period exceeded 25% from Day 1 pre-dose values. At a 1% test item concentration, very slight erythema was noted on Days 2 and 3 and variation in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

MAIN TEST
- No irritation was observed in any of the animals.
- 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.
- The majority of auricular lymph nodes were considered normal in size, except for one node in one animal treated at 1%, which was considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals

Applicant's summary and conclusion

Interpretation of results:
other: not sensitizing
Remarks:
in accordance with CLP (1272/2008 and its updates)
Conclusions:
Under the experimental conditions of the study, the test item has no sensitising potential in mice after dermal application of a concentration up to and including 1% (The highest dose tested, based on tolerability of the test item in pre-screen tests)
Executive summary:

The skin sensitisation potential of the test substance has been tested using a Local Lymph Node Assay according to OECD 429 and GLP principles. Test concentrations were determined based on two pre-screen tests. In the first pre-screen test, performed at dose levels of 50% and 100% animals were found dead or killed in extremis on Day 3. In the subsequent test at 10%, 5% and 2% test item, clinical signs were noted and variations in ear thickness during the observation period exceeded 25% from Day 1 pre-dose values. At a 1% test item concentration, very slight erythema was noted on Days 2 and 3 and variation in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on tolerability of the test item, the highest test item concentration selected for the main study was 1%. For the main test concentrations of 0.2, 0.5 and 1% were applied in a vehicle of acetone/olive oil (4:1 v/v). During the main test no irritation, no mortality and no clinical signs of systemic toxicity were observed in any of the animals. The body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The majority of auricular lymph nodes were considered normal in size, except for one node in one animal treated at 1%, which was considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. At none of the doses tested, the SI was increased a 3-fold, and consequently the EC3 is greater than 1%. The six-month reliability check with the positive control Alpha-hexylcinnamaldehyde indicated that the model is appropriate model for testing for contact hypersensitivity. Based on the results, the substance was not considered to be a skin sensitizer.