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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
20 Oct - 01 Nov 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

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 (incl. certificate)
Remarks:
Food and Consumer Product Safety Authority (VWA), Den Haag, The Netherlands
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): Vinasses, residue of fermentation containing biomass of bakers yeast (Saccharomyces cerevisiae)
- Substance type: Dark brown liquid
- Physical state: liquid
- Analytical purity: Not indicated by the sponsor; treated as 100% pure
- Dry matter (d.m.): 54.5% o.m.
- pH: 4.92
- Purity test date: 15 March 2010 until 06 May 2010
- Lot/batch No.: 135710
- Expiration date of the lot/batch: 13 October 2011 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Not indicated
- Stability in vehicle (Propylene glycol): Not indicated
- Solubility in vehicle (Propylene glycol): Not indicated
- Specific Gravity / Density: 1.3 g/cm³

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France.
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- 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) was 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.
- Health inspection: A health inspection was performed prior to treatment, to ensure that the animals were 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): 20.0 – 23.1
- Humidity (%): 41 - 60
- 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: 20 Oct - 01 Nov 2010

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
0, 25, 50, 100% (w/w)
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 data supplied 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. Four young adult animals were selected (in the range of 8 to14 weeks old) and two test substance concentrations were tested; a 50% and 100% concentration. Two animals were treated at each 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 in the main study, with the exceptions that assessment of lymph node proliferation and necropsy were not performed and the animals were group housed in MI cages. Both ears (left and right) were punched in the apical area using a biopsy punch (Stiefel, Ø 6 mm => 0.28 cm2). For each animal both punches were immediately weighed pooled per animal using an analytical balance after which the punches will be discarded.
After the final observation, all animals were sacrificed by intra-peritoneal injection with Euthasol® (AST Farma BV, Oudewater, The Netherlands).
- Lymph node proliferation response: Not determined in the preliminary irritation study.

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, based on the test guideline and recommendations done by ICCVAM (Reference 1).

The results were evaluated according to:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007),
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures,
- EC criteria for classification and labelling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptations to technical progress and amendments of this Directive published in the Official Journal of the European Union).

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 ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

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 and at request of the sponsor.

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 mixed thoroughly using a vortex mixer 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 with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).
Both ears (left and right) were punched in the apical area using a biopsy punch (Stiefel, Ø 6 mm => 0.28 cm2). For each animal both punches were immediately weighed pooled per animal using an analytical balance after which the punches will be discarded.
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. 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: Once daily (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 erythema
1: 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 oedema
3: 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 (which is a non-complex substance) indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document "Reliability check".

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 25, 50 and 100% were 1.0, 1.0 and 0.8 respectively (see Table 6 and Figure 1 in attached document "tables and figures").
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 468, 465 and 381 DPM respectively. The mean DPM/animal value for the vehicle control group was 450 DPM (see Tables 5 and 6 in attached document "tables and figures").

Any other information on results incl. tables

Tables and figures of the Preliminary Irritation study and Main study have been included in the attached document "tables and figures".

Results Preliminary irritation study:

No irritation and no signs of systemic toxicity were observed in any of the animals examined (Table 1). Ear puncture weights (a measure of irritation) were similar among the treated groups (Table 1), which indicated that no (notable) irritation had occurred.

Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

Other results - main study:

No irritation of the ears was observed in any of the animals examined (Table 3). Ear puncture weights (a measure of irritation) were similar among the treated and control groups, which indicated that no (notable) irritation had occurred (Table 4).

 

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size (Table 2).

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 (Table 2).

    

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

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 100%, Vinasses, residue of fermentation containing biomass of bakers yeast (Saccharomyces cerevisiae) was considered not to be a skin sensitizer.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information