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Administrative data

Description of key information

An in vitro skin irritation study showed no irritation to skin.
For eye irritation a BCOP (IVIS score of 121) and an OECD 405 study were performed under GLP. These studies show irreversible effects to the eyes.
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:
31-May-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.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: 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)
Test material information:
Composition 1
Details on test animals and environmental conditions:
Test system - EPISKIN Standard Model (TM) (EPISKIN-SM, 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

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:
Value:
79
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

Ammonium 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 Ammonium 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 Ammonium thiocyanate and controls are presented in Table 1.

Table 2 shows the mean tissue viability obtained after 15 minutes treatment with Ammonium 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 Ammonium thiocyanate compared to the negative control tissues was 79%.

 

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

 

A (OD570)

B (OD570)

C (OD570)

Mean (OD570)

SD

Negative control

0.889

0.879

0.883

0.884

±

0.005

Sodium thiocyanate

0.682

0.676

0.725

0.694

±

0.027

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 Ammonium thiocyanate

Mean tissue viability
(percentage of control)

Negative control

100

Sodium thiocyanate

79

Positive control

7

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
Ammonium thiocyanate is non-irritant in the in vitro skin irritation test under the experimental conditions described in the report.
Executive summary:

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

This report describes the ability of Ammonium thiocyanate to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SMTM)). The possible skin irritation potential of Ammonium 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.

Ammonium thiocyanate consisted of white crystals with a purity of 99.8% (dried material). Skin tissue was moistened with 5 μl of Milli-Q water and at least 10 mg of Ammonium thiocyanate was applied directly on top of the skin tissue. 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 Ammonium thiocyanate compared to the negative control tissues was 79%. Since the mean relative tissue viability for Ammonium thiocyanate was above 50% after 15 minutes treatment Ammonium thiocyanate is considered to be non-irritant.

Ammonium 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 Ammonium 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 vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-April-2010 to 14-April-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.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD guideline 437 “Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants”
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Details on test animals or tissues and environmental conditions:
Bovine eyes were used as soon as possible after slaughter on the same day. Bovine eyes from young cattle were obtained from the slaughterhouse
(Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container.

Preparation of corneas
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.
The isolated corneas were stored at 32 +/- 1°C in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32+/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

Cornea selection and Opacity reading
with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 3 were not used. Three corneas were selected at random for each treatment group.
Vehicle:
physiological saline
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl per cornea
- Concentration (if solution): 20% (w/w)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 20% (w/w) Imidazole (According to the OECD guideline (437) a positive control should result in an appropriate response. Whether this is 20% (w/v) or 20% (w/w) has no influence on the study integrity. Since the response was within the historical control data range, this deviation has no effect on the study integrity.)
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Three corneas were selected at random for each treatment group.
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 240 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed by immediate opacity measurement and permeability evaluation of the cornea.
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant
Irritation parameter:
other: In vitro irritancy score (IVIS)
Basis:
mean
Time point:
other: 240 minutes
Score:
121
Reversibility:
not reversible

Table 1 summarizes the opacity, permeability and in vitro irritancy scores of Sodium thiocyanate and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Tables 2-5.

 

The individual in vitro irritancy scores for the negative controls ranged from -0.9 to 0.0. The individual positive control in vitro irritancy scores ranged from 102 to 110 (Table 5).

 

The corneas treated with the positive control were turbid after the 240 minutes of treatment.

 

The corneas treated with Ammonium thiocyanate showed opacity values ranging from 60 to 83 and permeability values ranging from 3.036 to 3.690. The corneas were turbid after the 240 minutes of treatment with Ammonium thiocyanate. Hence, the in vitro irritancy scores ranged from 106 to 132 after 240 minutes of treatment with Ammonium thiocyanate.

 

 

Table 1 Summary of opacity, permeability and in vitro scores

 

Treatment

Mean
Opacity

Mean
Permeability

Mean In vitro
Irritation Score1,2

Negative control

0

0.000

0

Positive control

76

1.998

106

Ammonium thiocyanate

71

3.328

121

1 Calculated using the negative control mean opacity and mean permeability values.

2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

 

 

INDIVIDUAL OPACITY, PERMEABILITY AND IN VITRO SCORES

 

Table 2 Opacity score

 

Eye

Opacity
before
treatment

Opacity
after
treatment

Final
Opacity1

Negative control
corrected Final
Opacity

Mean
Opacity

Negative control

1

0

0

0

-1

0

2

0

1

1

0

3

0

1

1

0

Positive control

4

1

82

81

80

76

5

0

74

74

73

6

0

76

76

75

Ammonium thiocyanate

10

0

84

84

83

71

11

1

61

61

60

12

0

72

72

71

1 Final Opacity = Opacity after treatment – Opacity before treatment.

 

 

Table 3 Permeability score individual values (uncorrected)

 

Eye

Dilution
factor

OD490
1

OD490
2

OD490
3

Average
OD

Final
OD

Mean final
negative
control

Negative control

1

1

0.018

0.020

0.019

0.019

0.019

0.014

2

1

0.008

0.010

0.007

0.008

0.008

3

1

0.013

0.012

0.016

0.014

0.014

Positive control

 

4

6

0.310

0.305

0.304

0.306

1.836

 

5

6

0.422

0.422

0.418

0.421

2.526

 

6

6

0.314

0.312

0.317

0.314

1.884

 

Ammonium thiocyanate

 

10

6

0.565

0.561

0.545

0.557

3.342

 

11

6

0.524

0.519

0.516

0.520

3.120

 

12

6

0.628

0.633

0.626

0.629

3.774

 

 

 

Table 4 Permeability score individual values (corrected)

 

Eye

Dilution
factor

OD490
1(1)

OD490
2(1)

OD490
3(1)

Average
OD

Final
OD

Mean final
negative
control

Negative control

1

1

0.004

0.006

0.005

0.005

0.005

0.000

2

1

-0.006

-0.004

-0.007

-0.006

-0.006

3

1

-0.001

-0.002

0.002

0.000

0.000

Positive control

4

6

0.296

0.291

0.290

0.292

1752

1.998

5

6

0.408

0.408

0.404

0.407

2442

6

6

0.300

0.298

0.303

0.300

1800

Ammonium thiocyanate

10

6

0.551

0.547

0.531

0.543

3.258

3.328

11

6

0.510

0.505

0.502

0.506

3.036

12

6

0.614

0.619

0.612

0.615

3.690

1 OD490 values corrected for the mean final negative control permeability (0.014).

 

 

Table 5 In Vitro irritancy score

 

Eye

Negative control
corrected Final
Opacity

Negative control
corrected Final
OD490

In vitro Irritancy
Score1

Negative control

1

-1

0.005

-0.9

2

0

-0.006

-0.1

3

0

0.000

0.0

Positive control

4

80

1.752

106

5

73

2.442

110

6

75

1.800

102

Ammonium thiocyanate

10

83

3.258

132

11

60

3.036

106

12

71

3.690

126

1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

 

 

Table 6 Historical control data for the BCOP studies

 

Negative control

Positive control

Opacity

Permeability

In vitro Irritancy
Score

In vitro Irritancy
Score

Range

0 - 1

-0.007 - 0.009

-0.1 - 1.1

96 - 146

Mean

0.24

0

0.24

120

SD

0.44

0

0.45

14

n

21

21

21

21

SD = Standard deviation

n = Number of observations

 

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of July 2008 to October 2009.

Interpretation of results:
corrosive
Remarks:
Migrated information and severe irritant Criteria used for interpretation of results: OECD GHS
Conclusions:
The mean IVIS was 121 after 240 minutes of treatment with Ammonium thiocyanate.
Executive summary:

Screening for the eye irritancy potential of Ammonium thiocyanate using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the ocular irritation properties of Ammonium thiocyanate on an isolated bovine cornea. The possible ocular irritancy of Ammonium thiocyanate was tested through topical application for 240 ± 10 minutes. The study procedures described in this report were based on the most recent OECD 437 guideline.

Ammonium thiocyanate consisted of white crystals with a purity of 99.8% (dried material). The test substance was applied as a 20% (w/w) solution (750 μl) directly on top of the corneas.

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% w/w Imidazole) was 106 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Ammonium thiocyanate induced severe ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 121 after 240 minutes of treatment.

Since Ammonium thiocyanate induced an IVIS ≥ 55.1, it is concluded that Ammonium thiocyanate is a corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

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 Ammonium thiocyanate. There is no adequate in vivo data available from Cross-reading to other thiocyanate salts. 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 (Toolbox v.3.2). QSARs based on molecular structure are not suitable for salts. The pH was 4.8 at 1000 g/L.

 

An in vivo skin irritation study according to OECD 404 showed no irritation to skin. However there are some deviations from the current guideline, such as: intact and abraded skin was exposed and the body weights of the animals were not recorded. More importantly, it was not indicated whether the solid test item was moistened to ensure good skin contact. Due to these deviations the study report was considered to have limited validity.

 

Consequently, the dermal irritancy of Ammonium 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 Ammonium thiocyanate compared to the negative control tissues was 79%. Since the mean relative tissue viability for Ammonium thiocyanate was above 50% after 15 minutes treatment Ammonium thiocyanate is considered to be non-irritant.

Ammonium 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 studies available for Ammonium thiocyanate. Concentration of 100% in a maximization study only resulted to slight erythema (0.5 ml, 48 hrs, closed patch).

 

Eye irritation:

An in vivo study is performed according to OECD 405 and under GLP. For animals 4-6 the eyes were washed out 4 sec. p.a.. Effects on the eyes were scored 1, 24, 48 and 72 hours after instillation. Moderate to severe eye irritation was observed in all rabbits and flushing the eyes after 4 seconds did not seem to make a difference (if anything those animals even showed slightly higher effects on Conjunctivae). These effects were not reversible in 72 hours. No further observations were made.

The study was performed according to US GLP. The authors of this study conclude that the test substance is not an eye irritant. The applicant does not agree with this conclusion. At least 2 out of 3 animals show >= 1 for iritis and >= 2 for redness. Furthermore the reversibility of the effects cannot be judged from this study. Interpretation of the results of this study varies between non-irritant (report) and severe irritant R41 (by authorities in DAR KSCN, 2008).

 

In order to come to a conclusion on eye irritating properties, a new study was initiated in which the ocular irritation of Ammonium thiocyanate was evaluatedusing the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD 437 (Sep. 2009) under GLP.

Ammonium thiocyanate consisted of white crystals with a purity of 99.8% (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) was121. Since Ammonium thiocyanate induced an IVIS >= 55.1, it is concluded that Ammonium thiocyanate is corrosive or severe irritant to the eyes.

 

Respiratory irritation:

No data available. Besides, the likelihood for exposure via inhalation is low. The vapour pressure is low (0.0152 Pa at 20°C) and does not indicate a 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:
Selected key study representing the most reliable data.

Justification for selection of eye irritation endpoint:
Study with highest reliability.

Effects on eye irritation: corrosive

Justification for classification or non-classification

No signs of irritation were observed in an available in vivo (OECD 404) and in vitro (OECD 439) skin irritation study. The non-irritating properties to skin are confirmed in sensitization studies available for Ammonium thiocyanate. Concentration of 100% in a maximization study only resulted to slight erythema (0.5 ml, 48 hrs, closed patch). Consequently, the test substance is not classified as a skin irritant.

 

Based on the available test results the test substance meets the criteria under GHS to be classified "Irreversible effects to the eyes" Category 1.

There is no information available regarding possible respiratory irritation. Also there are no existing human data available that would point at possible respiratory irritation. Considering the low irritation potential to skin and the low likelihood for exposure via inhalation, there is no need for classification for respiratory irritation.