Potassium cyanate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2009-02-17 to 2009-04-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

440/2008/EC

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24th April 2002

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:

Sodium cyanate

EC Number:

213-030-6

EC Name:

Sodium cyanate

Cas Number:

917-61-3

Molecular formula:

CNO.Na

IUPAC Name:

sodium cyanate

Details on test material:

- Chemical name: sodium cyanate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Hungarian Supplier, WOBE Kereskedelmi Kft., H-1164, Budapest, Garmada u. 10.
- Age at study initiation: Young adult, 11-12 weeks old, age-matched within one week
- Weight at study initiation: 20.3 – 21.5 grams (the weight variation in animals involved in the study did not exceed +/- 20 % of the mean weight)
- Housing: Individual caging / mice were provided with glass tunnel-tubes
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch Number: 266 0944, Expiry Date: January 2009 and Batch Number: 376 1603, Expiry Date: May 2009) produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum. The contents of the diet provided by the Supplier are listed in Appendix 6. The diet and drinking water are routinely analysed and are considered to not contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are retained in the Archive at LAB Research Ltd.
- Acclimation period: 36 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 24 - 70 %
- Air changes (per hr): 15-20 air exchange/hour by central air-condition system.
- Photoperiod (hrs dark / hrs light): 12 light hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 2009-02-26 To: 2009-03-04

Study design: in vivo (LLNA)

Vehicle:

other: 1 (w/v) % Pluronic PE9200 in water

Concentration:

2.5 %, 5 % and 10 %

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
The test item vehicle compatibility was verified in a Preliminary Compatibility Test with
1. Acetone: Olive oil 4:1 mixture (AOO),
2. N,N-dimethylformamide,
3. Propylene glycol,
4. Dimethyl sulphoxide and
5. 1 (w/v) % Pluronic.
The test item was insoluble in solvents 1-4. The suitable vehicle was 1 % Pluronic. The maximum soluble concentration was 10% in this vehicle. Pluronic PE9200 is a surfactant, which ensured the formulation remained on the ears of the animals when applied topically. Although this vehicle is not specifically listed in the OECD guideline, it has been validated with positive control materials in international laboratories and LAB Research Ltd and represents a worst case vehicle.

- Irritation:
No cutaneous reaction was observed in any of the treated groups.
- Lymph node proliferation response:
The observations recorded in this preliminary test suggest that the formulations, the application of the material and the local effects on the animal are acceptable for a valid LLNA.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
During the assay each mouse was topically dosed on the dorsal surface of each ear with 25 mL of the appropriate formulation applied using a pipette. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

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

Statistics:

mean

Results and discussion

Positive control results:

The positive control group animals were treated with 25 (w/v) % HCA solution in a relevant vehicle (1% Pluronic) concurrent to the test item groups. The stimulation index of the test item was determined to be 8.9.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control (1% Pluronic)

Results 2

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

10% sodium cyanate

Results 3

Key result

Parameter:

SI

Value:

2.4

Test group / Remarks:

5% sodium cyanate

Results 4

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

2.5% sodium cyanate

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The following disintegrations per minute (DPM) were determined:
Vehicle: 691.5 dpm
10% test item: 951.5 dpm
5.0% test item: 1635.5 dpm
2.5 % test item: 548.5 dpm
Positive control: 6169.5 dpm

The following DPM per group was determined:
Vehicle: 728.0 dpm/group
10% test item: 988.0 dpm/group
5.0% test item: 1672.0 dpm/group
2.5 % test item: 585.0 dpm/group
Positive control: 6206.0 dpm/group

The following DPN was determined:
Vehicle: 86.4 dpn
10% test item: 118.9 dpn
5.0% test item: 204.4 dpn
2.5 % test item: 68.6 dpn
Positive control: 771.2 dpm

DETAILS ON STIMULATION INDEX CALCULATION
SI = DPN value of a treated group divided by the DPN value of the negative control group

CLINICAL OBSERVATIONS: No mortality or signs of systemic toxicity were observed during the study. No irritation or other cutaneous effect was observed in any treated of the groups. Test item precipitate was observed on the ears of the animals in the 10 % treated group (Days 2 and 3).

BODY WEIGHTS: No significant effects were observed on animal body weights.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the test item analogue, sodium cyanate, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay under the test conditions used in this study.

Executive summary:

Local Lymph Node Assay according to OECD 429 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42 was performed with CBA/CaOlaHsd mice. The aim of this study was to determine the skin sensitisation potential of the test item analogue following dermal exposure in the Local Lymph Node Assay. The test item was a white solid; vehicle compatibility was verified in a Preliminary Compatibility Test with Acetone: Olive oil 4:1 mixture (AOO), N,N-dimethylformamide, Propylene glycol, Dimethyl sulphoxide and 1 (w/v) % Pluronic. The test item was insoluble in first – fourth solvents. The suitable vehicle was 1 (w/v) % Pluronic. The maximum available concentration was 10% in this vehicle. Pluronic PE9200 is a surfactant, which ensures that the formulation remained on the ears of the animals when applied topically. Although this vehicle is not specifically listed in the OECD guideline, it has been validated with positive control materials in international laboratories and LAB Research Ltd. A Preliminary Irritation/Toxicity test was performed with the test item at concentrations of 10 % and 5 % in the selected vehicle. The 10 % concentration was considered acceptable as the maximum concentration in the main assay. In the main assay twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received the appropriate formulation of test item analogue at concentrations of 10 %, 5 % and 2.5 %,

- the negative control group received the vehicle (1 % Pluronic),

- the positive control group received 25 (w/v) % HCA in relevant vehicle. The test item solutions were applied on the dorsal surface of ears of experimental animals (25 l/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or systemic clinical signs were observed during the study. No treatment related effect on the body weights was observed. No signs of irritation or other cutaneous effect were observed in any of the treated groups. No significant lymphoproliferative response (SI 3) of test item analogue was observed at the concentrations applied compared to the negative control. Stimulation index values of the test item were 1.4, 2.4 and 0.8 at treatment concentrations of 10 %, 5 % and 2.5 %, respectively. No dose-response to treatment was observed. α-Hexylcinnamaldehyde (25 (w/v) % dissolved in 1% Pluronic) was used as the positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 8.9) was noted for the positive control chemical, this result confirmed the validity of the assay.

The test item analogue, sodium cyanate, is not considered to be a skin sensitizer under the test conditions chosen.