[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2008 - 07 July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

325740-68-9

Cas Number:

325740-68-9

IUPAC Name:

325740-68-9

Details on test material:

- Name of test material (as cited in study report): SPP100 B7
- Substance type: Highly viscous brownish/yellowish oil
- Physical state: liquid
- Analytical purity: 94.9%
- Impurities (identity and concentrations): not indicated
- Composition of test material, percentage of components: not indicated
- Isomers composition: not indicated
- Purity test date: not indicated
- Lot/batch No.: C0031
- Expiration date of the lot/batch: 31 January 2009
- Stability under test conditions:
Stability SPP 100 B7 under storage conditions: Stable
Stability in vehicle Methyl ethyl ketone Not indicated
- Storage condition of test material: At room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France.
- Age at study initiation: Approximately 10 weeks.
- Weight at study initiation: 21 to 25 gram (at initiation of treatment).
- Housing: Specific Pathogen Free area (animal room nu.s 18 and 19). Individual housing in 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 su[lied again after scoring of the ears on Day 3.
- 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.
- Other:
The Mouse, CBA strain, inbred, SPF-Quality, was selected as it is an acceptable animal species to various regulatory authorities as the recommended test system (e.g. OECD, EC, EPA) and background data are available at this facility.
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.
Animals were identified by tail mark with marker pen.
Results of analysis for each batch of 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): 21.4 to 23.5° C.
- Humidity (%): 39 to 93%. Temporary deviations from the maximum level of relative humidity (70%) occurred, but laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): Approximately 15 per hour.
- Photoperiod (hrs dark / hrs light): Fluorescent light for a 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 25 June 2008 To: 07 July 2008

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Remarks:

Merck, Darmstadt, Germany

Concentration:

1 Vehicle: Methyl ethyl ketone
2 10% test item concentration
3 25% test item concentration
4 50% test item concentration

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Methyl ethyl ketone (Merck, Darmstadt, Germany) was selected as suitable vehicle 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 item concentration to be used in the main study.
A series of two test item concentrations was tested: 25 and 50%. 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 (approximately 11 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. In order to facilitate scoring, the ears were cleaned of residual test substance with tap water. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation. No lymph nodes were excised.
- Lymph node proliferation response: Not determined in 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 (National Institute of Environmental Health Sciences, 1999).
The results were evaluated according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the EC criteria for classification and labeling 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 Communities).
The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation (Basketter, 1999).

TREATMENT PREPARATION AND ADMINISTRATION:
Preparation for administration: Formulations (w/w) were prepared within 4 hours prior to dosing and were homogenised to visually acceptable levels. Open epidermal application.

Four groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3
For the experimental animals, the dorsal surface of both ears was epidermally treated (25 μl/ear) with the test item concentration, approximately the same time each day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated as described for the experimental animals, except that, instead of the test substance, the vehicle or positive control item was administered.

Treatment - 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 (GE Health care, Buckinghamshire, UK).
After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital Euthesate® (0.2 mL/animal) (Ceva Sante Animale BV, Naaldwijk, 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 4º C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) at 4º C during the night.

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 comes first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

In-life examinations: Data collection was performed individually.
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weight: On Days 1 (pre-treatment) and 6.
Irritation of the ears: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded according to the numerical scoring system (see 'Any other information on materials and methods incl. tables). Furthermore, a description of all other (local) effects was recorded.
Food consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced.

Positive control substance(s):

other: The results of a reliability test with Alpha-hexylcinnamicaldehyde, performed not more than 6 months previously, are summarized in the attached document 'Reliability check'.

Statistics:

Not performed.

Results and discussion

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. (Attached document 'Reliability check')

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 10, 25 and 50% were 1.0, 1.2 and 4.9 respectively (Figure 1 and Table 4 of attached document 'Figures and tables').

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Median DPM/animal values are reported due to possible outlier responses for individual animals within groups. Median DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 370, 461 and 1812 respectively. The median DPM/animal value for the vehicle control group was 371. (Tables 3 and 4 of attached document 'Figures and tables')

Any other information on results incl. tables

Preliminary irritation study (Table 1 of attached document 'Figures and tables')

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

Main Study (Table 2 of attached document 'Figures and tables')

Slight to well-defined erythema of one or both ears was noted in two animals at 10% and all animals at 25 and 50%. 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. The majority of nodes were considered normal in size, except for one or both nodes of four animals at 50% which were considered enlarged in size. No macroscopic abnormalities of the surrounding area were noted.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

No mortality occurred 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:

The results indicate that the test substance could elicit an SI > 3. The data showed a dose-response and an EC3 value of 37.2% was calculated.

Based on these results:
- according to the recommendations made in the test guidelines, SPP100 B7 would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), SPP100 B7 should be classified as skin sensitizer (Category 1).
- according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), SPP100 B7 should be labeled as: may cause sensitization by skin contact (R 43).