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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:
21 April 2010 - 06 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ebecryl® P116 radiation curing resins- Substance type: Clear colourless liquid- Physical state: liquid- Analytical purity: 99% (wt/wt)- Composition of test material, percentage of components: Tripropylene glycol, esters with acrylic acid, reaction products with diethylamine- Main constituents:65-85% Diadduct TPGDA-DEA29-20% Mono-adduct TPGDA-DEA12-5% TPGDA-AA-DEA22-10% TPGMA-DEA- Lot/batch No.: JBGK0064T- Expiration date of the lot/batch: 29 July 2010- Stability under storage conditions: Stable- Storage condition of test material: At room temperature protected from light- Test substance handling: Use amber-coloured glassware or wrap container in thin -foil- Specific Gravity: 0.99- pH: Neutral- Stability at higher temperatures: Yes, maximum temperature keep not above 40°C

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Harlan, Horst, The Netherlands.- Age at study initiation: Young adult animals (approx. 10 weeks old) - Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.- Housing: 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. During the acclimatization period the accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm). - 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: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. - 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. Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.ENVIRONMENTAL CONDITIONS- Temperature (°C): 18.9 - 22.9ºC- Humidity (%): 37 - 84% Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.- Air changes (per hr): approximately 15 air changes per hour- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.IN-LIFE DATES: From: 21 April 2010 to 06 May 2010

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0-10-25-50% The highest test substance concentration was selected from the preliminary irritation study.
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:- Compound solubility: The vehicle was selected based on trial formulations performed at NOTOX and on test substance datasupplied by the sponsor.- Irritation: 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.Two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids).The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. 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.- Lymph node proliferation response: Not determined in the preliminary irritation study.MAIN STUDYANIMAL ASSIGNMENT AND TREATMENT- Name of test method: Local Lymph Node Assay- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) iscalculated 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, based on the test guideline and recommendations done by ICCVAM (Reference 1).The results were evaluated according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labeling and packaging of substances and mixtures. Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3) (Reference 2).Ref 1. National Institute of Environmental Health Sciences, The Murine Lymph Node Assay: A test method for assessing the allergic contact dermatitis potential of chemicals/compounds. Independent peer review by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods (NICEATM), NIH publication; No 99-4494, February 1999.Ref 2. Basketter DA, Lea LJ, Dickens A, Briggs, D, Pate I, Dearman RJ and Kimber I. A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J Appl Toxicol 1999;19:261-266.ANIMAL ASSIGNMENTThree groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.\\\ and another group of five animals was treated with the positive control substance.TREATMENT PREPARATION AND ADMINISTRATION:Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.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 the vehicle was administered instead of the test substance. 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 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.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.Radioactivity measurements - Day 7Precipitates 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.Toxicity: At least once daily.Body weights: On Days 1 (pre-treatment) and 6.Necropsy: No necropsy was performed according to protocol. 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.Grading Irritation Reactions:Erythema and eschar formation:0: No erythema1: Slight erythema (barely perceptible)2: Well-defined erythema 3: Severe erythema (beef redness) to slight eschar formation (injuries in depth) Oedema formation:0: No oedema 1: Slight oedema (barely perceptible)2: Moderate oedema3: Severe oedema
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-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
>= 3
Test group / Remarks:
The SI values calculated for the substance concentrations 10, 25 and 50% were 10.5, 22.8 and 31.3 respectively.
Parameter:
other: disintegrations per minute (DPM)
Value:
1 800
Test group / Remarks:
Controls
Parameter:
other: disintegrations per minute (DPM)
Value:
18 914
Test group / Remarks:
Groups treated with test substance concentrations 10%
Parameter:
other: disintegrations per minute (DPM)
Value:
41 000
Test group / Remarks:
groups treated with test substance concentrations 25%
Parameter:
other: disintegrations per minute (DPM)
Value:
56 342
Test group / Remarks:
Groups treated with test substance concentrations 50%

Any other information on results incl. tables

The slight irritation of the ears as shown by all animals treated at 10% and the slight up to well defined irritation of the ears as shown by the animals treated at 25 and 50% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

All auricular lymph nodes of the test substance treated animals were considered increased in size. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

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.

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

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Under the test conditions, the test substance elicited an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be 5.87%Based on these results:- according to the recommendations made in the test guidelines, Ebecryl® p116 radiation curing resins would be regarded as skin sensitizer.- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), Ebecryl® p116 radiation curing resins should be classified as skin sensitizer (Category 1).- according to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures, Ebecryl® p116 radiation curing resins should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.
Executive summary:

A mouse local lymph node assay was conducted to determine the sensitizing potential of amine synergist according to OECD Guideline 429, EU Method B.42 and EPA OPPTS 870-2600. Amine synergist elicited an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI = 3) was between 0 and 10%. This result suggests that the substance may be a skin sensitizer (Beerens-Heijnen CGM, 2010). If a trend analysis is conducted on the 10, 25 and 50% concentration data, an overall EC3 of 5.87% is found. This would correspond to a category 1B skin sensitization according to CLP criteria (EC 1271/2008).