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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 13 to June 29, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
6.2. Test system
Species: Mouse, CBA strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).
Source: Charles River France, L’Arbresle Cedex, France.
Number of animals 20 females (nulliparous and non-pregnant), five females per group.
Age and bodyweight Young adult animals (approx. 11 weeks old) were selected. Body
weight variation was within +/- 20% of the sex mean.
Identification Tail mark with marker pen.
Health inspection A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was
paid to the ears, which were intact and free from any abnormality.
Reliability check The results of a reliability test with Hexylcinnamaldehyde, performed not more than 6 months previously, are summarized in
the appendix. Similar procedures were used in the reliability test and in this study.
6.3. Animal husbandry
Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 21.1 – 23.9ºC), a relative humidity of 30-70% (actual range: 43 - 78%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Accommodation
Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
Acclimatization period
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).
Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water
Free access to tap water.

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
with test substance concentrations of 10, 25 or 50% w/w
No. of animals per dose:
5
Details on study design:
6.4. Preliminary irritation study
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2) at the highest concentration.
The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified.
Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids). Based on trial formulations performed at NOTOX, Dimethyl formamide (Merck, Darmstadt, Germany) was selected as vehicle.
Two young adult animals were selected (8-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.
Main study
Initially, three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle.
The initial main study was considered invalid based on abnormalities observed among animals at necropsy. Raw data of the initial main study were included in the study files, but results of the initial main study were not used for interpretation and are not included in this report.
The main study was repeated and the data of the second main study are reported.
6.5.2. Induction - Days 1, 2 and 3
The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing.
The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.
6.5.3. Excision of the nodes - Day 6
All animals:
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 (GE Health care, Buckinghamshire, UK).
After approximately five hours, all animals were killed by intraperitoneal injection 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.
6.5.4. Tissue processing for radioactivity - Day 6
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) stored in the
refrigerator until the next day.

6.5.5. 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. 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).
6.6. Observations
Mortality/Viability Twice daily.

Toxicity At least once daily.

Body weights On Days 1 (pre-treatment) and 6.

Irritation On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.



Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group 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 results:
The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 10, 25 and 50% were 1.4, 1.1 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 697, 569 and 488 respectively. The mean DPM/animal value for the vehicle control group was 495.
Parameter:
SI
Value:
1.4
Test group / Remarks:
Substance concentration 10%
Parameter:
SI
Value:
1.1
Test group / Remarks:
substance concentration 25%
Parameter:
SI
Value:
1
Test group / Remarks:
substance concentration 50%

1. Preliminary irritation study

The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in the table. Based on the results, the highest test substance concentration selected for the main study was a 50% concentration.

2. Main study

2.1. Skin reactions / Irritation All animals at 50% showed slight or well-defined erythema of both ears and four animals at 25% showed slight erythema of one or both ears. No erythema of the ears was observed for one animal at 25%, all animals at 10% and control animals. No oedema was observed in any of the animals examined. The irritation of the ears as shown by the animals, was considered not to have a toxicologically significant effect on the activity of the nodes.

2.2. 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.

2.3. Body weights

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

2.4. Radioactivity measurements Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 697, 569 and 488 respectively. The mean DPM/animal value for the vehicle control group was 495. 7.2.5. Toxicity and mortality No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

2.5. Toxicity and mortality No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Since there was no indication that the test substance could elicit an SI ≥ 3 when tested up to 50%, E-200 was considered to be a non skin sensitizer.
Executive summary:

The purpose of this study was to evaluate whether the test substance induces contact hypersensitivity in mice after three epidermal exposures of the CBA mice. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, three groups of five experimental animals were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 (v/v)). Three days after the last exposure, all animals were injected with 3 H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All animals at 50% showed slight or well-defined erythema of both ears and four animals at 25% showed slight erythema of one or both ears. No erythema of the ears was observed for one animal at 25%, all animals at 10% and control animals. No oedema was observed in any of the animals examined. The irritation of the ears as shown by the animals, was considered not to have a toxicologically significant effect on the activity of the nodes. 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. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 697, 569 and 488 respectively. The mean DPM/animal value for the vehicle control group was 495. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.4, 1.1 and 1.0 respectively.

Since there was no indication that the test substance could elicit an SI ≥ 3 when tested up to 50%, E-200 was considered to be a non skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 50%.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

One skin sensitation study conducted in the Mouse (Local Lymph Node Assay) was available to assess the Contact Hypersensitivity to test substance. In this report, three groups of five experimental animals were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Three days after the last exposure, all animals were injected with

3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 697, 569 and 488 respectively. The mean DPM/animal value for the vehicle control group was 495. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.4, 1.1 and 1.0 respectively. Since there was no indication that the test substance could elicit an SI ≥ 3 when tested up to 50%, Epikote P was considered to be a non skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 50%.


Migrated from Short description of key information:
Test substance is considered to be no skin sensitizer based on the available data.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results test substance does not have to be classified for skin sensitisation according to CLP (Regulation EC No.1272/2008).