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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:
weight of evidence
Study period:
2004-10-25 to 2004-11-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
yes
Remarks:
-temperary fluctuations of relative humidity above level of 70 %. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
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
- Batch No.of test material: 99/484
- Expiration date of the batch: 2005-06-10

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark.
- Solubility and stability of the test substance in the vehicle: Dimethyl formamide, at least 96 h

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: When required, 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.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Remarks:
inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: ~ 10 weeks
- Weight at study initiation: ± 20 % of the sex mean
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Housing: individually, Macrolon cages (type I)
- Diet : ad libitum, standard pelleted laboratory animal diet (code VRF 1, Altromin, Lage, Germany)
- Water: ad libitum, tap-water
- Acclimation period: at least 5 days
- Preparation of test formulations: the test substance formulations (wlw) were prepared within 4 hours prior to each treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3
- Humidity (%): 41 - 75
- Air changes (per hr): ~ 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
Selected based on trial formulations performed at NOTOX.
Concentration:
25 %, 50 % in vehicle and 100 % (undiluted)
No. of animals per dose:
5 females
Details on study design:
RANGE FINDING TESTS:
- Concentrations: 50 %, 100 % (undiluted)
- No of animals: 1 per dose (total of 2)
- Age of animals: 5 - 14 weeks
- Duration of treatment: Each animal was treated with one concentration on three consecutive days.
- Procedure: Approximately 4 hours after the last exposure, the ear was cleaned of residual test substance with water and the irritation was assessed. Test system, procedure and techniques were identical to those used during days 1 to 3 of the main study unless where specified. Bodyweights were determined on day 3. No necropsy was performed after termination.

MAIN STUDY
INDUCTION
- Frequency of application: once daily for a total of 3 days (days 1, 2 and 3)
- Site of application: The dorsum surface of both ears
- Route of application: epidermal
- Volume applied: 25 µL/ear
- Concentrations: 25 %, 50 % in vehicle and 100 % (undiluted)

Injection of 3H-methyl thymidine (Amersham Pharmacia Biotech, NOTOX Substance 105624)
- Day of injection: 3 days after last treatment (day 6)
- Site of injection: tail vein
- Vehicle: PBS
- Volume injected: 0.25 mL
- Specific radioactivity: 20 µCi/injection

SACRIFICE
- Time schedule: 5 hours after injection of 3H-methyl thymidine
- Method: injection of phenobarbital

TISSUE PROCESSING AND MEASUREMENTS
- Lymph node processed: auricular lymph nodes
- Pooling of lymph nodes: yes
- Measurement of radioactivity: Packard scintillation counter (1900TR) (Counting time was to a statistical precision of ± 0.2 % or a maximum of 5 minutes whichever comes first)

OTHER
The test animals were checked twice daily for mortality/viability and at least once a day for toxicity signs. Bodyweight of the test animals was taken on day 1, prior to treatment and on day 6. On day 3 (3 - 4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded following the Draize numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI values calculated for the substance concentrations 5,10 and 25 % were 1.0, 3.2, and 7.1 respectively. An EC3 value of 9.5 % was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20 %. The results of the 6 monthly HCA reliability checks of the recent years were 8.8, 5.5, 7.3 and 10.3 %.
Based on the results, it was concluded 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:
1.1
Test group / Remarks:
25 %
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
50 %
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
100 %
Parameter:
other: DPM/animal
Value:
411
Test group / Remarks:
25 %
Parameter:
other: DPM/animal
Value:
385
Test group / Remarks:
50 %
Parameter:
other: DPM/animal
Value:
243
Test group / Remarks:
100 %

Any other information on results incl. tables

Group

Induction

Mean DPM ± S.D.

SI ± S.D.

1

Vehicle Control (Dimethyl Formamide)

373 ± 48

1.0

2

25 % Test Substance

411 ± 188

1.1 ± 0.5

3

50 % Test Substance

385 ± 105

1.0 ± 0.3

4

100 % Test Substance

243 ± 137

0.7 ± 0.6

 

OBSERVATIONS (MAIN STUDY)

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study. The majority of the nodes of the experimental and control groups were considered normal in size (visual inspection). The nodes of two animals treated at 25 and 100 % were decreased in size. No other macroscopic abnormalities of the nodes were noted. 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.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100 % were 411, 385, and 243 respectively. The mean DPM/animal value for the vehicle control group was 373 (NOTOX Project 418534). The SI values calculated for the substance concentrations 25, 50, and 100 % were 1.1,1.0, and 0.7 respectively. There was no indication that the test substance could elicit an SI > 3.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met