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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
28 April 2010 - 25 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sodium thiocyanate
- Substance type: White crystalline powder (determined at NOTOX)
- Physical state: solid
- Analytical purity: 99.9% (dried material)
- Impurities (identity and concentrations): See attached document 'Certificate of analysis'.
- Composition of test material, percentage of components: See attached document 'Certificate of analysis'.
- Isomers composition: Not indicated.
- Purity test date: See attached document 'Certificate of analysis'.
- Lot/batch No.: 0600801070919-0 (date Jan. 09, 2008)
- Expiration date of the lot/batch: 19 September 2001 (taken from label)
- Stability under test conditions: Stable under storage conditions; Stability in vehicle is not indicated.
- Storage condition of test material: At room temperature in the dark in desiccator
- Other:
pH: 5.5 at concentration of 5% aqueous solution (CoA)
Hygroscopic: yes

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Horst, The Netherlands.
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 19-23 gram
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment.
The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period:The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).
- Other:
Species: Mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA)Identification: Tail mark with marker pen.
A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.
Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 – 23.3ºC
- Humidity (%): 37 - 84%. Temporary deviations from the minimum level of relative humidity (i.e. 40%) occurred, but historical data do not indicate an effect of the deviations. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

IN-LIFE DATES: From: 28 April 2010 To: 25 May 2010

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
from Merck, Darmstadt, Germany
Concentration:
1 0 % test substance (vehicle only)
2 10% test substance
3 25% test substance
4 50% test substance
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.
- Irritation: A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2) at the highest concentration. Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (50% for solids). The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (Source: Source: Charles River France, L’Arbresle Cedex, France; Age: 8-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were not determined on Day 3 (Evaluation: Sufficient data available for the selection of dose levels in the main study). The animals were sacrificed after the final observation and no necropsy was performed.
- Lymph node proliferation response: Not determined in the preliminary irritation study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM. The results were evaluated according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labeling and packaging of substances and mixtures. Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3).

TREATMENT PREPARATION AND ADMINISTRATION:
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.

Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that the vehicle was administered instead of the test substance.

Excision of the nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6:
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) stored in the refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On Days 1 (pre-treatment) and 6.
Necropsy: No necropsy was performed according to protocol.
Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the numerical scoring system described in 'Any other information on materials and methods incl. tables'. Furthermore descriptions of all other (local) effects were recorded.
Positive control substance(s):
other: The results of a reliability test with Alpha-hexylcinnamicaldehyde, performed not more than 6 months previously, are summarized in the attached document 'Reliability check'.
Statistics:
Not performed.

Results and discussion

Positive control results:
The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 10, 25 and 50% were 1.6, 2.7 and 1.5 respectively. See table 4 and figure 1 of attached document 'Figures and tables'.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 674, 1142 and 615 DPM respectively. The mean DPM/animal value for the vehicle control group was 420 DPM. See tables 3 and 4 in the attached doument 'Figures and tables'.

Any other information on results incl. tables

Preliminary irritation study:

The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in Table 1 of the attached document 'Figures and tables'. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

 

Main study(see Table 2 of attached document 'Figures and tables'):

No irritation of the ears was observed in any of the animals examined.

All auricular lymph nodes of animals at 25% and 50% were considered enlarged and all auricular lymph nodes of animals at 10% and control animals were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

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 one individual animal, was considered not toxicologically significant.

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

 

 

Table 1: Skin reactions after epidermal exposure and body weights

animal

number

test

substance1

(% w/w)

Day 1

 

Day 3

Body

weight (g)

 

skin reactions dorsal surface ear

 

left

right

 

erythema

oedema

erythema

oedema

 

 

 

 

 

 

 

 

1

252

22

 

      0

       0

      1

       0

2

503

24

 

      0

       0

      1

       0

 

 

 

 

 

 

 

 

1. Vehicle: Dimethyl formamide.

2. Applied using a pipette with the tip cut off.

3. Applied using a spatula, instead of using a pipette. The 50% formulations appeared dry, but stuck on the ears sufficiently.

 

 

Table 2: Skin reactions, body weights and relative size auricular lymph nodes

group

 

test substance1

(% w/w)

an2

 

Day 1

 

Day 3

 

Day 6

 

bw

(g)3

 

skin reactions dorsal surface ear

 

bw

(g)3

 

size nodes4

 

 

left

right

 

 

left

right

 

 

erythema

oedema

erythema

oedema

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

0%

1

 

 20

 

0

0

0

0

 

 21

 

n

n

 

(vehicle)

2

 

 20

 

0

0

0

0

 

 22

 

n

n

 

 

3

 

 21

 

0

0

0

0

 

 22

 

n

n

 

 

4

 

 21

 

0

0

0

0

 

 22

 

n

n

 

 

5

 

 19

 

0

0

0

0

 

 20

 

n

n

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2

10%

6

 

 23

 

0

0

0

0

 

 23

 

n

n

 

 

7

 

 20

 

0

0

0

0

 

 20

 

n

n

 

 

8

 

 22

 

0

0

0

0

 

 23

 

n

n

 

 

9

 

 19

 

0

0

0

0

 

 21

 

n

n

 

 

10

 

 19

 

0

0

0

0

 

 19

 

n

n

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3

25%

11

 

 21

 

0

0

0

0

 

 22

 

+

+

 

 

12

 

 19

 

0

0

0

0

 

 19

 

+

+

 

 

13

 

 22

 

0

0

0

0

 

 25

 

+

+

 

 

14

 

 20

 

0

0

0

0

 

 22

 

+

+

 

 

15

 

 23

 

0

0

0

0

 

 24

 

+

+

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4

50%5

16

 

 21

 

0

0

0

0

 

 22

 

+

+

 

 

17

 

 21

 

0

0

0

0

 

 22

 

+

+

 

 

18

 

 19

 

0

0

0

0

 

 19

 

+

+

 

 

19

 

 20

 

0

0

0

0

 

 21

 

+

+

 

 

20

 

 23

 

0

0

0

0

 

 22

 

+

+

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.Vehicle: Dimethyl formamide.

2. Animal number.

3.Body weight (grams).

4.Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be

   normal).

5.Applied using a spatula, instead of using a pipette. The 50% formulations appeared dry, but stuck on the ears sufficiently.

 

  

Table 3: Radioactivity measurements (individual animals)

group

 test substance1(% w/w)

animal

DPM / animal

 

 

 

 

1

0%

1

669

 

 

(Vehicle)

2

226

 

 

 

3

364

 

 

 

4

449

 

 

 

5

390

 

 

 

 

 

 

2

10%

6

281

 

 

 

7

924

 

 

 

8

467

 

 

 

9

536

 

 

 

10

1161

 

 

 

 

 

 

3

25%

11

764

 

 

 

12

829

 

 

 

13

950

 

 

 

14

1318

 

 

 

15

1850

 

 

 

 

 

 

4

50%

16

313

 

 

 

17

608

 

 

 

18

562

 

 

 

19

1092

 

 

 

20

501

 

 

 

 

 

1.    Vehicle: Dimethyl formamide.

 

 

Table 4: Disintegrations Per Minute (DPM) and Stimulation Index (SI).

 

group

test substance1

(% w/w)

mean

DPM ±

 

SI ±

 

 

 

 

 

 

 

 

2

10%

674

±

161

1.6

±

0.5

3

25%

1142

±

201

2.7

±

0.7

4

50%

615

±

129

1.5

±

0.4

 

 

 

 

 

 

 

 

1

0% (vehicle)

420

±

72

1.0

±

0.2

 

 

 

 

 

 

 

 

1.    Vehicle: Dimethyl formamide.

 

Figure1: Dose-response curve

An individual Stimulation Index (SI) is calculated for each animal. The individual SI

is the ratio of the DPM/animal compared to the DPM/vehicle control group.

A mean SI is calculated for each group using the individual SI values.

(See attached figure)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%, Sodium thiocyanate was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 50%.

Based on these results Sodium thiocyanate would not be regarded as skin sensitizer according to the recommendations made in the test guidelines and does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.
Executive summary:

The study was carried out based on the guidelines described in:

OECD, Section 4, Health Effects, No.429 (2002),

EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"

EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

 

Test substance concentrations selected for the main study were based on the results of a preliminary study.

 

In the main study, three experimental groups of five female/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Dimethyl formamide).

 

Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating theof the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

 

 

No irritation of the ears was observed in any of the animals examined.

 

All auricular lymph nodes of animals at 25% and 50% were considered enlarged and all auricular lymph nodes of animals at 10% and control animals were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 674, 1142 and 615 DPM respectively.

The mean DPM/animal value for the vehicle control group was 420 DPM.

 

 

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.6, 2.7 and 1.5 respectively.

 

Since there was no indication that the test substance elicits an SI3 when tested up to 50%, Sodium thiocyanate was considered not to be a skin sensitizer. It was established that the EC3 value(the estimated test substance concentration that will give a SI =3)(if any) exceeds 50%.

 

The six-month reliability check withAlpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

 

Based on these results Sodium thiocyanate would not be regarded as skin sensitizer according to the recommendations made in the test guidelines and does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and theRegulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.