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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:
August - October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP comnpliant

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:
from April 2002
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 16 - 18 g
- Housing: singly (dose range only) or pairs in suspended cages (dimensions 36.5 x 20.7 x 14 cm) with stainless steel grid tops and solid bottoms, nesting material (Nestlets, supplied by Datesand Limited, UK) was provided, wood shavings were used as bedding (a certificate of the wood shavings used in the study is available in the study report)
- Diet: Rat and Mouse No. 1 Maintenance Diet, supplied by Special Diets Services Limited, UK, available ad libitum (each batch of diet was analysed for major nutritive constituents and significant contaminants, the certificate for the batch used is available in the study report)
- Water: Water from domestic mains supply available ad libitum (the water used is analysed by the local water authority for dissolved items, heavy metals, pesticide residues, pH, nitrates and nitrites, certificate for the analysis conducted most recently before commencement of the study is available in the study report)
- Acclimation period: at least 8 days
- Indication of any skin lesions: nothing mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 50 (mean value)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Prescreen test: 50, 100%
Main test: 25, 50 and 100%
No. of animals per dose:
Prescreen test: 1
Main test: 4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: soluble in vehicle
- Irritation: no findings at concentrations of 50% in acetone/olive oil (4:1 v/v) and in 100% (undiluted test substance)
- Systemic toxicity: no abnormalities detected
- Ear thickness measurements: not performed
- Erythema scores: no findings, no scores mentioned

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation index (SI) ≥ 3, together with consideration of dose-response and, where appropriate statistical significance

TREATMENT PREPARATION AND ADMINISTRATION:
Prescreen test: Dose levels were selected based on existing data from similar products from the sponsor previously tested at the test laboratory (Inveresk project Nos. 505909 and 505956). 4 animals were allocated and treated as follows: one animal each receiving 50% and 100% test item (new), and 50% and 100% test item (old). The animals received an open application for above 3 consecutive days (25 µl each ear), reactions were then recorded using the following scoring scheme:
No eythema: 0
Very slight erythema: 1
Well defined erythema: 2
Moderate to severe erythema: 3
severe erythema (beet redness) to slight eschar formation (injuries in depth): 4

Main test: 4 test animals per dose group received the following concentrations: 0 (actone/olive oil (4:1 v/v) only as vehicle control), 25% , 50%, 100% test items (old and new) each. All animals received open applications of 25 µl of the appropiate formulation for 3 consecutive days onto the dorsum of each ear. There were no treatment on days 4 and 5. On day 6 each animal received an intravenous injection of 250 µl of phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine into the lateral tail vein. Approxymately 5 h after intravenous administration the animals were killed by exposure to carbon dioxide and exsanguinated. Each pair of draining auricular lymph nodes was collected from each animal. A single cell suspension of lymph node cells from each group was prepared by gentle mechanical desegration through 200 µm mesh stainless steel gauze. The lymph node cells were washed twice in an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at approximately 4°C for 18 h. After removal of excess TCA the pellets were resuspended and solubilised in 200 µl soluene. Scintillation fluid (10 ml) was added and incorporation of tritiated thymidine measured by ß-scintillation counting as disintegrations per minute (DPM).
- Observations: All animals were checked for viability early in the morning and again as late as possible on each day.
- Clinical observations: All main study animals were examined for reaction to treatment (at least 3 times) on each day of dosing, and once daily thereafter. The onset, intensity and duration of any signs were recorded. Assessments of the application sites during the main test were not considered to be necessary.
- Body weights: The body weight of each individual animal was recorded immediately before dosing (day 1) and on day 4 (prescreen) or day 6 (main study).
- Calculation of results: Reported DPM were corrected for background radiation. Results were expressed as the Stimulation Index (SI), this was obtained by dividing the DPM within each test item group by the DPM of the vehicle control group. The SI for the vehicle control group is 1.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Hexyl cinnamic aldehyde (HCA) was tested May and August 2003 in the test laboratory as validation of the Local Lymph Node Assay (Project Nos. 996459 and 996621). The studies were designed to be compliant with OECD Guideline No. 429, Skin Sensitisation: Local Lymph Node Assay, adopted April 2002. Four groups of 5 animals were used, 3 test groups and 1 control group in each study. During the induction exposure the test groups were exposed to 10, 20 or 40% HCA, and the control group was exposed only to the vehicle, acetone/olive oil (4:1 v/v). Stimulation Indices in the study completed in May 2003 were 1.9, 4.3, and 11.7 and in August 2003 were 1.7, 1.9, and 6.3. Under the conditions of these studies, HCA is considered to be a sensitiser in CBA/Ca mice, thus providing evidence that the procedure employed at the test laboratory were valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.1
Test group / Remarks:
25 % test substance (new)
Parameter:
SI
Value:
2.4
Test group / Remarks:
50 % test substance (new)
Key result
Parameter:
SI
Value:
6.4
Test group / Remarks:
100% test substance (new)
Key result
Parameter:
SI
Value:
7
Test group / Remarks:
100% test substance (old)
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION:
The Stimulation Indices, when compared with the vehicle control were calculated to be 1.1, 2.4, and 6.4 for 25%, 50% or 100% test item (new), respectively.
The Stimulation Indices, when compared with the vehicle control were calculated to be 1.8, 1.8, and 7.0 for 25%, 50% or 100% test item (old), respectively.

EC3 CALCULATION :
no EC3 value calculated

CLINICAL OBSERVATIONS:
no adverse clinical signs were noted during the observation period.

BODY WEIGHTS:
Body weight gain was considered satisfactory.

Any other information on results incl. tables

 Treatment  Dose level (%)  Disintegrations per minute (DPM)  Stimulation Indix (SI)
 Acetone/Olive oil (4:1 v/v)  0  2724  1
 Test item (new)  25  2902  1.1
   50  6403  2.4
   100  17515  6.4
 Test item (old)  25  4840  1.8
   50  4928  1.8
   100  19137  7.0

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the study, the test item produced by either the old or new manufacturing process is considered to be a weak skin sensitiser in mice.
Executive summary:

Using CBA/Ca mice this study investigated the delayed contact hypersensitivity potential of the test item, made using 2 separate production processes. The study was conducted based on OECD Guideline No. 429, Skin Sensitisation, Local Lymph Node Assay, April 2002 and in compliance with the principles of Good Laboratory Practice (GLP).

After dose ranging, 6 groups of 4 female mice received open applications of either test item (new) or test item (old) at concentrations of 25, 50 or 100%, another group of 4 females received only the vehicle, acetone/olive oil (4:1 v/v). Treatment was for 3 consecutive days. Three days later each animal received an intravenous injection of 3H methyl thymidine, and 5 h later the draining lymph nodes were collected and the incorporation of tritiated thymidine assessed by scintillation counting. The Stimulation Indices, when compared with the control group, were calculated to be 1.1, 2.4, and 6.4 for 25%, 50% , and 100% test item (new), respectively. The Stimulation Indices, when compared with the control group, were calculated to be 1.8, 1.8, and 7.0 for 25%, 50% , and 100% test item (old), respectively.

Under the conditions of the study, the test item produced by either the old or new manufacturing process is considered to be a weak skin sensitiser in mice.