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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:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
yes
Remarks:
Temporary deviations from the minimum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: F-12676, PDC lot 20
- Expiration date of the lot/batch: 03 September 2013 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)
- Purity test date: 99.96%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.
Correction of the purity/composition of the test substance is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be applied.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Dissolved in N,N-Dimethyl formamide

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: Approximately 10 weeks old
- Weight at study initiation: 20.55 g (average)
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). 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. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and 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: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Indication of any skin lesions: Prior to dosing, it was ensured that the animals are without any observable skin lesions and the ears are intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40-70%
- Air changes (per hr): Approximately 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12:12 light:dark cycle
- IN-LIFE DATES: From: 14 November 2012 To: 03 December 2012

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 10, 25, and 50% (w/w)
No. of animals per dose:
5 females/group
Details on study design:
PRE-SCREEN TESTS:
- Irritation: Very slight irritation was noted in all animals on Day 2 (erthema score = 1 in each animal)
- Systemic toxicity: No signs of systemic toxicity were observed in any of the animals examined
- Ear thickness measurements: Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values
- Erythema scores: Score of 1 in each animal (n = 4) on day 2; reversible within 72 hours

MAIN STUDY

ANIMAL 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) 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. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, including all amendments. Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3)

TREATMENT PREPARATION AND ADMINISTRATION:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, 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 substance.

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.

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 stored in the refrigerator until the next day. 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).

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Variability:
± 0.1
Test group / Remarks:
Control
Key result
Parameter:
SI
Value:
1.2
Variability:
± 0.2
Test group / Remarks:
10% (w/w)
Key result
Parameter:
SI
Value:
1
Variability:
± 0.3
Test group / Remarks:
25% (w/w)
Key result
Parameter:
SI
Value:
0.7
Variability:
± 0.1
Test group / Remarks:
50% (w/w)
Key result
Parameter:
EC3
Value:
> 50
Test group / Remarks:
EC3 value (if any) exceeds 50%
Cellular proliferation data / Observations:
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 two animals at 25% (Day 1) and all animals at 50% (Days 1-3), which did not hamper scoring of the skin reactions.

MACROSCOPY
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 in any of the animals.

RADIOACTIVITY MEASUREMENTS
Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 569, 452 and 315 DPM respectively. The mean DPM/animal value for the vehicle control group was 457 DPM.

CLINICAL OBSERVATIONS:
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. The body weight loss noted for one animals at 25% was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the test, the test article showed no evidence of dermal sensitization.
Executive summary:

The dermal sensitization potential of the test article was evaluated in the local lymph node assay (LLNA) with CBA/J strain mice. The study was performed in compliance with OECD GLP ENV/MC/CHEM (98) 17 (revised 1997). The test method was based on OECD Section 4 No. 429 (2010), EC No. 440/2008 B42, and EPA OPPTS 870.2600 (2003). The test article was prepared in N,N-Dimethyl formamide to the appropriate concentrations. A pre-screen test was conducted to select the highest test article concentration of 50%. Female mice (5/treatment) received the control (Dimethyl formamide), 10%, 25%, or 50%, concentrations of the test substance. The corresponding treatment (25 uL/ear) was applied to the dorsal surface of both ears for three consecutive days. Three days after the last exposure, all animals were injected with 0.25 mL sterile phosphate buffered solution containing 3H-methyl thymidine and subsequently euthanized. The auricular lymph nodes were removed to visually estimate the relative size and to observe any abnormalities. The nodes were pooled for each animal to measure the amount of operative DNA by disintegrations per minute (DPM). The stimulation index (SI) was calculated for each group. Consideration was given to the EC3 value. The 6-month reliability check with alpha-hexylcinnamicaldehyde indicated that the LLNA is an appropriate model for evaluating dermal sensitization at the test facility. Observations for mortality (twice daily), body weights (Day 1 and Day 6), clinical signs (once daily), and irritation (once daily) were performed as well. Mean DPM/animal values for the 10%, 25%, and 50%, test article concentrations were 569, 452, and 315 DPM respectively. The control mean DPM/animal value was 457 DPM. The SI values for test concentrations of 10%, 25%, and 50% were 1.2, 1.0, and 0.7 respectively. It was established that the EC3 value (if any) exceeds 50%. No irritation of the ears was observed in any animal. No mortality, clinical signs of toxicity, or significant body weight changes were observed. All auricular lymph nodes of the test and control animals were considered normal in size. Based on the results of the test, the test article showed no evidence of dermal sensitization.