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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:
2016-07-06 to 2016-08-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
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:
1-((2-chlorophenyl)-N-methylcarbonimidoyl)cyclopentan-1-ol
EC Number:
641-894-8
Cas Number:
6740-87-0
Molecular formula:
C13H16ClNO
IUPAC Name:
1-((2-chlorophenyl)-N-methylcarbonimidoyl)cyclopentan-1-ol
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I14LD5177
- Expiration date of the lot/batch: 2016-12-31 (retest date)
- Purity: 96.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

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
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France ; 20 females (nulliparous and non-pregnant); CBA/J strain, inbred, SPF-Qualtiy.
- Age at study initiation: approx. 10 weeks old
- Weight: 19.9 to 24.9 g at study initiation
- Housing: Animals were 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.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to 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): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2, 25 and 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN 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 (see section 0) 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.
- The test system, procedures and techniques were identical as those used in the main study except that the animals were approximately 13 weeks (at initiation of treatment), that the test item preparations were vortexed immediately prior to dosing 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.
- No erythema and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. White test item remnants were present on the dorsal surface of the ears of all animals (between Days 1 and 3), which did not hamper scoring of the skin reactions.
Based on these results, the highest test item concentration selected for the main study was a 50% concentration.

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.

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 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 (diameter 125 μm). 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).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no data

Results and discussion

Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by Charles River Den Bosch. In this study, performed in May 2016, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade (HCA).
The SI values calculated for the item concentrations 5, 10 and 25% were 1.4, 1.5 and 4.3 respectively. An EC3 value of 18.0% 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%.
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:
1
Variability:
+/- 0.3
Test group / Remarks:
Based on 5 animals in 2% w/w in Acetone/Olive oil (4:1 v/v)
Parameter:
SI
Value:
1.1
Variability:
+/- 0.2
Test group / Remarks:
Based on 5 animlas in 25% w/w in Acetone/Olive oil (4:1 v/v)
Parameter:
SI
Value:
1
Variability:
+/- 0.2
Test group / Remarks:
Based on 4 animals in 50% w/w in Acetone/Olive oil (4:1 v/v)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Average DPM/animal
Acetone/Olive oil (4:1 v/v) = 448
2% w/w = 462
25% w/w = 512
50% w/w = 430

DETAILS ON STIMULATION INDEX CALCULATION
- The SI values calculated for the item concentrations 2, 25 and 50% were 1.0, 1.1 and 1.0, respectively.
- One high dose animal showed a DPM value clearly outside the range of the study (outlier, based on Grubbs outlier test). This value was not used for interpretation. Sufficient data was available for evaluation.

EC3 CALCULATION
- Not applicable as the test item did not elicit a SI>3.

CLINICAL OBSERVATIONS:
- No erythema 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 a concentration of 25% and 50% (Days 2 and/or 3), which did not hamper scoring of the skin reactions.
- No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS
- 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
- All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for one node of one high dose animal which was considered enlarged, the DPM value of this animal was considered an outlier. No macroscopic abnormalities of the surrounding area were noted for any of the animals

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 50%, T003641 was not considered to be a skin sensitizer.

Based on the results, T003641 was not regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).