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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was done in a GLP facility to OECD guidelines

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Butyronitrile
EC Number:
203-700-6
EC Name:
Butyronitrile
Cas Number:
109-74-0
Molecular formula:
C4H7N
IUPAC Name:
butyronitrile
Constituent 2
Reference substance name:
NBN
IUPAC Name:
NBN
Details on test material:
Identification n-Butyronitrile
CAS Number 109-74-0
Description Clear colourless liquid
Batch TXTXOL
Purity/Composition 99.891 W%
Test substance storage At room temperature in the dark
Stability under storage conditions: Stable
Expiry date 21 February 2014 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)
6.1.2. Study specific test substance information
Specific Gravity / Density 0.7924
pH (1% in water, indicative range) 8.8-8.4 (determined at WIL Research Europe B.V. )

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Test system
Species Mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).

Source: Janvier, Le Genest-Saint-Isle, France

Number of animals: 20 females (nulliparous and non-pregnant), five females per group.

Age and body weight 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: At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.

Reliability check: The results of a reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures is summarized in the appendix of this report. For both scientific and animal welfare reasons, no concurrent positive control group was added to the study.

Animal husbandry

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

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. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.

Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) and tap water. Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.



Study design: in vivo (non-LLNA)

Positive control substance(s):
not specified

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 10, 25 and 50%
No. of animals per dose:
5
Details on study design:
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. Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3
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.

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 (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 - 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) and 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. 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. Final Report
The scintillation counter was programmed to automatically subtract background and convert Counts
Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
not specified

Results and discussion

Positive control results:
The results of a reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures is summarized in the appendix of this report. For both scientific and animal welfare reasons, no concurrent positive control group was added to the study

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 2.0, 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 861, 405 and 462 DPM respectively. The mean DPM/animal value for the vehicle control group was 441 DPM.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
The SI values calculated for the substance concentrations 10, 25 and 50% were 2.0, 0.9 and 1.0 respectively. Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%, n-Butyronitrile was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 50%. The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity. Based on these results, n-Butyronitrile would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance 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 (2011) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.
Executive summary:

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. 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 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. The majority of auricular lymph nodes were considered normal in size, except for the enlarged nodes in three animals of the 25% group. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 861, 405 and 462 DPM respectively. The mean DPM/animal value for the vehicle control group was 441 DPM.

 

The SI values calculated for the substance concentrations 10, 25 and 50% were 2.0, 0.9 and 1.0 respectively. Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%, n- Butyronitrile was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 50%. The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity. Based on these results, n-Butyronitrile would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance 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 (2011) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures