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Description of key information

An in vitro skin irritation study showed no irritation to skin.
In an eye irritation study in rabbit with Sodium thiocyanate, performed according to OECD 405, ocular corrosion was observed.
No data on respiratory irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 June 2010 to 07 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Draft Proposal for a New Guideline: In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method
Deviations:
no
GLP compliance:
yes (incl. certificate)
Details on test animals and environmental conditions:
Test system - EPISKIN Standard Model (EPISKIN-SM(TM), 0.38 cm2, Lot no.: 10-EKIN-020), SkinEthic Laboratories, Nice, France.
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C. The level of Maintenance Medium was just beneath the tissue. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Nice, France.

Environmental conditions
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 76 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.7- 36.0°C) and humidity (with a maximum of 4%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg, moistened with 5 µl water. Sodium thiocyanate was spread to match the size of the tissue.

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 10 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 10 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
Exposure:5 minutes
Post incubation period: 42 hours
Details on study design:
STUDY DESIGN
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. At least 10 mg solid (with a small glass weight boat) with 5 μl Milli-Q water was added into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μl PBS (negative control) and 3 tissues with 10 μl 5% aq. SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully. The skin tissues were kept in new 12-well plates on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Irritation / corrosion parameter:
other: other: percentage viability
Value:
106
Remarks on result:
other:
Remarks:
Basis: other: percentage of control. Time point: 15 minutes. (migrated information)

Preliminary test for reduction of MTT by the test substance

Sodium thiocyanate was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that Sodium thiocyanate did not interact with MTT.

Acceptability of assay:

The positive control had a mean cell viability after 15 minutes exposure of 7%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Results:

The mean absorption at 570 nm measured after treatment with Sodium thiocyanate and controls are presented in Table 1.

Table 2 shows the mean tissue viability obtained after 15 minutes treatment with Sodium thiocyanate compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Sodium thiocyanate compared to the negative control tissues was 106%.

Table 1 Mean absorption in the in vitro skin irritation test with Sodium thiocyanate

  A (OD570) B (OD570) C (OD570) Mean (OD570)   SD
 Negative control   0.889   0.879   0.883   0.884  ±  0.005 
 Sodium thiocyanate   0.942   0.939   0.927   0.936  ±  0.008 
 Positive control   0.053   0.062   0.068   0.061  ±  0.008 

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption. Isopropanol was used to measure the background absorption.

Table 2 Mean tissue viability in the in vitro skin irritation test with Sodium thiocyanate

  Mean tissue viability
(percentage of control)
Negative control 100
Sodium thiocyanate 106
Positive control 7
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Sodium thiocyanate is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

In vitro skin irritation test with Sodium thiocyanate using a human skin model.

This report describes the ability of Sodium thiocyanate to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SMTM)). The possible skin irritation potential of Sodium thiocyanate was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Sodium thiocyanate was a white crystalline powder with a purity of 99.9% (dried material). Skin tissue was moistened with 5 μl of Milli-Q water and at least 10 mg of Sodium thiocyanate was applied directly on top of the skin tissue (0.38 cm2). After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Sodium thiocyanate compared to the negative control tissues was 106%. Since the mean relative tissue viability for Sodium thiocyanate was above 50% after 15 minutes treatment Sodium thiocyanate is considered to be non-irritant.

Sodium thiocyanate did not cause direct MTT reduction. The positive control had a mean cell viability after 15 minutes exposure of 7%. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that Sodium thiocyanate is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 March, 2016 - 10 March, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
(2012)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF Guidelines (2000), including the most recent revisions.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France.
- Age at study initiation: 20 weeks
- Weight at study initiation: 4.315 kg
- Housing: Animals were individually housed in labeled cages with perforated floors
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 Harlan Teklad®, Italy). Hay and wooden sticks were available during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): set to maintain 18 – 24
- Humidity (%): set to maintain 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: One eye of each animal remained untreated and served as the reference control.
Amount / concentration applied:
TEST MATERIAL
- ground to a powder using a mortar and pestle prior to weighing.
- Amount applied: 60.7 mg (volume of approximately 0.1 mL)
Duration of treatment / exposure:
Single instillation on Day 1.
Observation period (in vivo):
The eyes of each animal were examined approximately 1 and 24 hours after instillation of the test substance.
Number of animals or in vitro replicates:
1 female
Details on study design:
STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). Based on the severity of the ocular lesions observed during the study, the animal was sacrificed for ethical reasons immediately after the 24 hours observation and the two further rabbits assigned to the study were not treated.

PREEMPTIVE PAIN MANAGEMENT
One hour prior to instillation of the test substance, buprenorphine (Buprenodale®, Dechra Ltd., Stokeon-Trent, United Kingdom) 0.01 mg/kg bw was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
Five minutes prior to instillation of the test substance, two drops of the topical anesthetic alcaine 0.5% (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.
TREATMENT
The animal was treated by instillation of, 60.7 mg of the test item (a volume of approximately 0.1 mL), in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test item. The other eye remained untreated and served as the reference control.
Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.
Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection.
Additional injections were supplied during the observation period to reduce pain and distress After the final observation, the animal was sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and on day of the final observation.
- Necropsy: No necropsy was performed according to protocol.
- The eyes of the animal were examined approximately 1 and 24 after instillation of the test item. The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to OECD 405.
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
animal #1
Time point:
other: 24 hrs.
Score:
2
Max. score:
4
Remarks on result:
other: Animal sacrificed after 24 hrs
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: 24 hrs.
Score:
1
Max. score:
2
Remarks on result:
other: Animal sacrificed after 24 hrs
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #1
Time point:
other: 24 hrs.
Score:
3
Max. score:
3
Remarks on result:
other: Animal sacrificed after 24 hrs
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: 24 hrs.
Score:
4
Max. score:
4
Remarks on result:
other: Animal sacrificed after 24 hrs
Irritant / corrosive response data:
Instillation of 61 mg of Sodium thiocyanate (a volume of approximately 0.1 mL) into an eye of one rabbit resulted in severe effects ocular effects and signs of discomfort of the animal (tilted head) necessitating the sacrifice the animal for humane reasons at 24 hours after instillation. The signs consisted of grey/white discouloration (sign of necrosis) of the eyelids and nictitating membrane. Moreover, injuries of the cornea and iris were noted. Corneal injury consisted of opacity and epithelial damage. Further, at 24 hours after dosing red discolouration of the lachrymal fluid was noted.
Other effects:
No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen.
No effects on Body weight was shown by the animal.
No further signs of systemic toxicity were observed in the animal during the test period and no mortality occurred.
Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an eye irritation study with a male rabbit with Sodium thiocyanate, performed according to OECD 405 test guideline, occular corrosion was observed.
Executive summary:

The acute eye irritation/corrosion of Sodium thiocyanate was determined in accordance with OECD 405 (2012) and according to GLP principles. A single sample of 61 mg (a volume of approximately 0.1 mL) of Sodium thiocyanate was instilled into an eye of one rabbit. Observations were made 1 and 24 hours after instillation.

Instillation of 61 mg of Sodium thiocyanate (a volume of approximately 0.1 mL) into an eye of one rabbit resulted in severe effects ocular effects and signs of discomfort of the animal (tilted head) necessitating the sacrifice the animal for humane reasons at 24 hours after instillation. The signs consisted of grey/white discouloration (sign of necrosis) of the eyelids and nictitating membrane. Moreover, injuries of the cornea and iris were noted. Corneal injury consisted of opacity and epithelial damage. Further, at 24 hours after dosing red discolouration of the lachrymal fluid was noted.

 

Based on the results, Sodium thiocyanate should be classified as Irreversible effects on the eye (Category 1) and labeled as H318: Causes serious eye damage, according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation:

There is no existing human data available indicating to corrosive or skin irritant properties of Sodium thiocyanate. Cross-reading to an available in vivo study (OECD 404) on NH4SCN also indicates no skin irritating properties. Profiling (Toolbox v.3.2) indicates that BfR inclusion rules for irritation (skin and eye) are not met, and all of the BfR exclusion criteria for irritation are met for which data is available. QSARs based on molecular structure are not suitable for salts. The pH was 6 at 1000 g/L.

 

Dermal irritancy of Sodium thiocyanate was tested on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SMTM)) through topical application for 15 minutes. The study was performed according to EU guideline B.46, which has recently been validated and adopted in EU as a full replacement method. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Sodium thiocyanate compared to the negative control tissues was 106% indicating a result not different from control. Since the mean relative tissue viability for Sodium thiocyanate was above 50% after 15 minutes treatment Sodium thiocyanate is considered to be non-irritant.

Sodium thiocyanate did not cause direct MTT reduction. The positive control had a mean cell viability after 15 minutes exposure of 7%. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Following these results, further (in vivo) studies for the evaluation of dermal irritation are not considered necessary.

 

The non-irritating properties to skin are confirmed in a sensitization study available for Sodium thiocyanate. The highest applicable concentration of 50% in a LLNA study showed no signs of irritation.

 

Eye irritation:

There is no data related to human exposures.

 

The eye irritancy potential of Sodium thiocyanate was evaluated using the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD 437 (Sep. 2009) under GLP.

Sodium thiocyanate was a white crystalline powder with a purity of 99.9% (dried material). The test substance was applied as a 20% (w/w) solution (750 μl) directly on top of the corneas for 240 ± 10 minutes. The mean in vitro irritancy score (IVIS) was 49, which is just below the threshold for severe irritancy/corrosion of 55, indicating eye irritation.

 

As there were still uncertainties as to the appropriate classification for eye irritation of sodium thiocyanate, a subsequentin vivoeye irritation study in rabbits was performed according to OECD 405 guidelines.Instillation of 61 mg of Sodium thiocyanate (a volume of approximately 0.1 mL) into an eye of one rabbit resulted in severe effects ocular effects and signs of discomfort of the animal (tilted head) necessitating the sacrifice the animal for humane reasons at 24 hours after instillation. The signs consisted of grey/white discouloration (sign of necrosis) of the eyelids and nictitating membrane. Moreover, injuries of the cornea and iris were noted. Corneal injury consisted of opacity and epithelial damage. Further, at 24 hours after dosing red discolouration of the lachrymal fluid was noted.

The study concluded that instillation of Sodium thiocyanate into the rabbit eye resulted to ocular corrosion.

 

Respiratory irritation:

No data available. Besides, the likelihood for exposure via inhalation is low.The vapour pressure is extremely low (< 1.33 x 10-8 Pa) and thus does not present any potential for inhalation exposure due to volatilization of the salt. Furthermore thiocyanates are very hygroscopic (see granulometry). Inhalable particles are not available and will also not be formed during handling and use of the substance.


Justification for selection of skin irritation / corrosion endpoint:
Only available study, sufficient to decide upon classification.

Justification for selection of eye irritation endpoint:
Only available in vivo study, decisive for classification purposes.

Effects on eye irritation: corrosive

Justification for classification or non-classification

Based on the negative result from in vitro skin irritation study it can be concluded that classification is not warranted. All further available data support this conclusion.

 

An eye irritation study in rabbit with Sodium thiocyanate resulted to ocular corrosion. Sodium thiocyanate therefore needs to be classified under GHS as Eye Damage 1 and according to the Regulation (EC) 1272/2008 labeled as H318: Causes serious eye damage.

 

There is no information available regarding possible respiratory irritation. However, considering the low irritation potential to skin and eyes, and the low likelihood for exposure via inhalation, there is no need for classification for respiratory irritation.