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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Remarks:
WIL Research Europe B.V., Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Celitement
- Chemical name: Calcium hydrosilicate
- Physical state: White powder
- pH-value: 10.5
- Expiration date of the lot/batch: 12/2015
- Stability under test conditions: Hydration reaction with water
- Storage condition of test material: Room temperature protected from light, in original container in evacuated desiccator

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: Mean female body weight 22 gram.
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: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
10, 25, 50 (% w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Two test substance concentrations were tested; a 25% and 50% concentration. The test system, procedures and techniques were identical to those used in the main study except that the application method may have been different 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.

MAIN STUDY
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.
- Induction: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, and repeated on day 2 and 3. 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 substance.
Excision of the nodes: On 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 approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with 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: 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 4°C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.
- Radioactivity measurements: On 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. Radioactive 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).
- 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 the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.
- 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 individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
In this study, performed in April 2014, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Hexylcinnamaldehyde. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.4 and 4.7 respectively. An EC3 value of 17.3% 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 16.9, 14.4, 16.5, 14.5, 13.4 and 14.1%.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 10, 25 and 50% were 0.8, 0.9 and 1.0, respectively
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 234, 272 and 305 DPM, respectively. The mean DPM/animal value for the vehicle control group was 309 DPM.

Any other information on results incl. tables

Pre-screen test

No irritation and no signs of systemic toxicity were observed in any of the animals examined. White test substance remnants were present on the dorsal surface of the ears of all animals (Days 1, 2 and/or 3), which did not hamper scoring of the skin reactions. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

Skin reactions / Irritation

No irritation of the ears was observed in any of the animals examined. White test substance remnants were present on the dorsal surface of the ears of all animals at 25% (Days 1-3) and 50% (Days 1-4), 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. 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 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.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions (OECD 429, GLP) there was no indication that the test substance elicited a SI ≥ 3 when tested up to 50%, the test substance was not considered to be a skin sensitizer.
Executive summary:

According to OECD guideline 429 and GLP, the test substance was examined for skin sensitization using the Local Lymph Node Assay. Test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears (25μL/ear). Five vehicle control animals were similarly treated, but with vehicle alone (Propylene glycol). Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. No irritation of the ears was observed in any of the animals examined. White test substance remnants were present on the dorsal surface of the ears of all animals at 25% (Days 1-3) and 50% (Days 1-4), which did not hamper scoring of the skin reactions. 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. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 234, 272 and 305 DPM, respectively. The mean DPM/animal value for the vehicle control group was 309 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 0.8, 0.9 and 1.0, respectively. Since there was no indication that the test substance elicited a SI ≥ 3 when tested up to 50%, the test substance was not considered to be a skin sensitizer.