Salicylonitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-01-29 to 2008-02-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxybenzonitrile

IUPAC Name:

2-hydroxybenzonitrile

Constituent 2

Reference substance name:

Salicylonitrile

EC Number:

210-259-3

EC Name:

Salicylonitrile

Cas Number:

611-20-1

Molecular formula:

C7H5NO

IUPAC Name:

2-hydroxybenzonitrile

Details on test material:

TEST MATERIAL
- Name of test material (as cited in study report): 2-hydroxybenzonitrile
- Substance type: pure test substance, recrystallised 2-cyanophenol
- Physical state: solid, yellowish (beige) crystalline powder
- Melting range: 95.4°C
- Stability under test conditions: not determined
- Storage condition of test material: room temperature, stable

RADIOLABEL
- 3H-methyl thymidine
- 80 microcurie / mL (3H, tritium)
- radiochemical purity: 96%
- thymine content: 0.9%
- specific activity of stock solution: 2.0 Ci/mmol
- radioactive concentration of stock solution: 1 mCi/mL
- amount of stock solution: 1.296 mL
- diluted with phosphate-buffered saline (PBS), 14.904 mL
- dose per animal: 20 microcurie

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 19.0 - 23.0 g
- Housing: optimal hygienic conditions (OHC), single caging in Makrolon type II cages
- Diet: ad libitum, Ssniff Maintenance diet for rats and mice R/M-H (item V1534-3)
- Water: communal drinking water from Makrolon bottles, ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 (continuous monitoring)
- Humidity (%): 30 - 70% (continuous monitoring)
- Air changes (per hr): not reported (OHC)
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2008-01-29 To: 2008-02-05

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

DMF allows highest test substance concentration

Concentration:

25%, 50%, 64.8% (w/w),
equal to 37.5, 75, 97.2 mg test substance / animal

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 64.8 % (highest solubility found in any of the guideline-recommended vehicles)
- Irritation: Range finding study with 2 animals / concentration (64.8% and 50% w/w, applied 3 times on consecutive days): no systemic toxicity, no excessive local skin irritation, nor important increase in ear thickness
- Lymph node proliferation response: Concurrent positive control with 25% hexyl cinnamic aldehyde in acetone/olive oil proves the sensitivity of the strain of animals (main study).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node assay LLNA
- Criteria used to consider a positive response: induction of a 3-fold or greater increase in 3H-methyl thymidine incorporation in lymph node cells, relative to control lymph nodes, together with the consideration of dose response.

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl of the test compound solution in DMF was administered epicutaneously to the dorsal surface of each ear of each mouse. The application was repeated on days 2 and 3. On day 6 an injection of 250 µl phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Approximately five hours later, the draining auricular lymph node of each ear was rapidly excised into PBS; the lymph nodes of each group were pooled. A single cell suspension of lymph node cells was prepared by gentle mechanical disaggregation through a 70 micrometer cell strainer. The cell suspension was centrifuged (4°C, max. 200 g, 10 min) and washed twice with PBS. Cell macromolecules were precipitated with 5% trichloroacetic acid (TCA) at 4 °C overnight. Each precipitate was pelleted by centrifugation (as above) and resuspended in 1 ml TCA.
Suspensions were transferred to 10 mL scintillation cocktail, and 3H-TdR incorporation was determined with a beta-scintillation counter.

BODY WEIGHT:
- recorded on days 1 and 6

SKIN REACTIONS:
- application sites were visually checked for local irritations once daily (days 1-6)

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Stimulation index (SI) = disintegrations per minute (dpm) in the test group (pooled) / dpm in the negative control group (pooled)
dpm corrected by subtraction of background

Results and discussion

Positive control results:

SI of positive control group (hexyl cinnamic aldehyde) = 16.2

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

4.1

Test group / Remarks:

Group A (low dose; 25% (w/w))

Remarks on result:

other: group K (negative control): 1 group P (positive control): 16.2

Results 2

Parameter:

SI

Value:

9.1

Test group / Remarks:

Group B (mid dose; 50% (w/w))

Results 3

Parameter:

SI

Value:

12.5

Test group / Remarks:

Group C (high dose; 64.8% (w/w))

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: group K (negative control): 2752 group A (low dose): 11186 group B (mid dose): 25022 group C (high dose): 34377 group P (positive control): 44455

Any other information on results incl. tables

BODY WEIGHT DEVELOPMENT:

- Weights and weight gains were within the expected range for this strain, sex, and age.

- Slight body weight losses were noted in animals of all groups, including negative and positive controls.

SYMPTOMS:

- No clinical signs in any animal.

SKIN REACTIONS:

- No local irritations in the negative control group and the three dose groups.

- Slight erythema of the application site in the positive control (day 3).

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The substance is considered to be sensitising. The study is considered to be relevant and reliable.