Salicylonitrile

Endpoint summary

Description of key information

Skin sensitisation: Sensitising, EC3=21%, mouse LLNA, OECD 429, Weber 2008

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

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

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

Positive control results:

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

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

Parameter:

SI

Value:

9.1

Test group / Remarks:

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

Parameter:

SI

Value:

12.5

Test group / Remarks:

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

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

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).

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A single skin sensitisation study (Local Lymph Node Assay) conducted according to guideline OECD 429 and under GLP was available (Weber, 2008). The study is considered to be relevant, reliable (Klimisch 1) and adequate for the purposes of risk assessment, classification and labelling. The LLNA resulted in stimulation indices above 3 at all concentrations tested; the test substance is therefore considered to be a sensitiser.

 

The EC3 value was not reported in the original study report, and has been calculated by the registrant for the purposes of classification and labelling in accordance with Commission Regulation (EU) No. 286/2011. The following dose response values were reported by Weber (2008):

 

	Concentration	SI
Low dose (c,d)	c=25%	d=4.1
Mid dose (a,b)	a=50%	b=9.1
High dose	64.8%	12.5

 

Because no Stimulation Index was reported below 3, a linear interpolation could not be employed, and a log-linear extrapolation was used to determine the EC3 value (Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency, Ryan et al, Cutan Ocul Toxicol, 2007, 26(2), 135-145).

EC3_{(extrapolated)}= 2^(log₂(c)+(3-d)/(b-d)*(log₂(a)-log₂(c))) = 21%

where logarithms are base 2, and variable pairs (a,b) and (c,d) correspond to the mid and low doses respectively, on a SI vs concentration plot (see reference for further details).

 

The value EC3=21% was carried forward for potency classification purposes.

Justification for selection of skin sensitisation endpoint:
GLP study according to OECD testing guideline

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance meets the criteria for a sensitising substance according to the test method criteria in Regulation (EU) 440/2008, B.42. The derived EC3 value is >2%. As a result and in accordance with Regulation (EU) No. 1272/2008 as amended by Regulation (EU) No. 286/2011, 3.4.2.2.3.3, the substance is classified as a skin sensitiser Category 1B (H317: May cause an allergic skin reaction).