Tripotassium hexacyanoferrate

Endpoint summary

Description of key information

An in vitro skin irritation study was performed according to OECD test guideline, which showed no irritation to the skin.

An in vitro eye irritation study, the EpiOcular™ Cornea Epithelial Model, was performed according to OECD test guideline, which showed that potassium ferricyanide is potentially irritant or corrosive to the eye. An in vitro eye irritation study, BCOP, was performed according to the OECD test guideline but no prediction on the classification of potassium ferricyanide could be made as the test item induced an IVIS > 3 ≤ 55. An in vivo eye irritation study was therefore performed according to the OECD test guideline, which showed potassium ferricyanide is irritating to eyes.

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:

05 March 2018 - 12 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

To protect the test item from light glassware was wrapped in tin-foil.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

water

Remarks:

5 µL

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model(TM)
- Tissue batch number(s): 18-EKIN-010
- Surface: 0.38 cm^2
- Expiration date: 12 March 2018
- The skin was moistened with 5 µL Milli-Q water

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure to the test item: room temperature
- Temperature of incubations(°C): 36.4 - 37.4°C
- Humidity(%): 64 - 85

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: tissues were washed with phosphate buffered saline (1 washing step)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE: the test substance was checked for possible direct MTT reduction and color interference in a previous skin corrosion test using EpiDerm as a skin model. It was concluded that the test substance did not interfere with the MTT endpoint.
- Incubation time: 3 hours
- Measurement method: TECAN Infinite M200 Pro Plate Reader (570 nm)
- MTT concentration: 0.3 mg/mL

NUMBER OF REPLICATE TISSUES: 3 for the test substance, the negative and the positive control each.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 single run

CELL VIABILITY MEASUREMENT: After incubation, tissues were dried and incubated with 2 mL MTT-solution for 3 hours. After incubation, epidermis was separated from the collagen matrix and both parts were extracted with 500 μL isopropanol. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

PREDICTION MODEL / DECISION CRITERIA (see Table 1)
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

ACCEPTABILITY CRITERIA
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20.5 to 28.5 mg

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability(%): 3.3

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range and the SD of the % viability was 4.6.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability was 3.3% and the SD of the % viability was 0.3
- Acceptance criteria met for variability between replicate measurements: yes, the variability between tissue treated with the test item was 4.4%.

- Since the mean relative tissue viability for potassium ferricyanide was above 50% is considered to be non-irritant.

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation test, performed according to OECD guideline 439 and under GLP principles, potassium ferricyanide showed to be not irritant to the skin (mean tissue viability of 89%). Therefore, the substance does not need to be classified according to GHS and CLP criteria.

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:

07 August 2018 - 18 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Based on two previous performed in vitro eye irritation tests with Potassium Ferricyanide, no conclusion could be drawn on the eye irritating potential of the substance. In order to clarify classification it was decided to perform an in vivo study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008, part B: "Acute Toxicity: Eye Irritation/Corrosion"

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Appendix to Director General Notification, No. 12-Nousan-8147. Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan (JMAFF)

Version / remarks:

November 2000, including the most recent revisions.

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: approximately 13-14 weeks old
- Weight at study initiation: 2976 to 3221g
- Housing: individually, in cages with perforated floors. For psychological/environmental enrichment, animals were provided with shelters and wooden sticks.
- Diet: Pelleted diet for rabbits (Global Diet 2030 Teklad®, Mucedola, Milanese, Italy), once daily throughout the study period. Hay was fed in addition.
- Water: municipal tap-water, ad libitum
- Acclimation period: at least 5 days

It is considered that there were no known contaminants in the feed and water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 48 to 90
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09 August 2018 to 18 September 2018

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

- Amount applied: 99.9mg - 110.2mg (0.1 mL) to the conjunctival sac of the eye.

Duration of treatment / exposure:

single treatment

Observation period (in vivo):

14 days and/or 21 days

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: according to Draize scale

TOOL USED TO ASSESS SCORE: 2% fluorescein in water (pH adjusted to 7.0).

OBSERVATIONS:
- Mortality/moribundity checks: Twice daily, in the morning and at the end of the working day.
- Clinical observations: Once daily
- Body weights: Animals were weighed individually on day 1 (pre instillation) and on the day of the final observation.
- Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and/or 21 days after instillation of the test item. The irritation scores and a description of all other (local) effects were recorded.

PAIN MANAGEMENT:
- One hour prior to instillation of the test item, buprenorphine 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.
- Five minutes prior to instillation of the test item, two drops of the topical anaesthetic 0.5% proparacaine hydrochloric ophthalmic solution were applied to both eyes.
- Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg were administered by subcutaneous injection. Additional injections of buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg were administered immediately after the 48-hour observation to reduce pain and distress.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Instillation of the test item caused corneal injury, iridial irritation and irritation to the conjunctivae.

The corneal injury consisted of opacity and epithelial damage. As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent 7 and/or 14 days after instillation for two animals. The corneal injury resolved within 7 days in one animal and within 14 days in the other two animals.
Iridial irritation was observed and resolved within 24 hours, 48 hours or 7 days.
The irritation of the conjunctivae consisted of redness, chemosis and discharge and was completely resolved within 14 days in all animals.

Other effects:

- Coloration/remnants: Yellow staining of the fur on the head and paws caused by the test item was observed. 72 hours after instillation the staining had dissappeared in all animals.
- Toxicity/mortality: No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

other: Irritating to eyes (Category 2)

Remarks:

according to EC No 1272/2008

Conclusions:

In an in vivo eye irritation test performed according to OECD test guideline 405 and GLP principles, Potassium Ferricyanide caused effects on the cornea, iris and conjunctivae and is classified as irritating to eyes (Category 2).

Executive summary:

An in vivo eye irritation study was performed in new zealand white rabbits with Potassium Ferricyanide according to OECD guideline 405 and GLP principles. A single application of 99.9mg - 110.2mg (approximately 0.1 mL) Potassium Ferricyanide was applied to the conjunctival sac of the eye under systemic and local anesthetic. Observations period were at 1, 24, 48 and 72 hours and 7, 14 and/or 21 days after instillation of the test item. Corneal epithelial damage was assessed with 2% fluorescein in water. Observations indicated corneal injury that consisted of opacity and epithelial damage. As a results of the corneal injury, pannus (neovascularization of the cornea) was apparent 7 and/or 14 days after instillation for two animals.  The corneal injury resolved within 7 days in one animal and within 14 days in the other two animals. Iridial irritation was observed and resolved within 24 hours, 48 hours or 7 days. Irritation of the conjunctivae was observed after instillation and consisted of redness, chemosis and discharge and was completely resolved within 14 days in all animals. No signs of systemic toxicity were observed and no mortality occurred. Based on these results, Potassium Ferricyanide is classified as: mildly irritating to eyes (Category 2B) according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and classified as irritating to eyes (Category 2), labeled as H319: Causes serious eye irritation, according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an in vitro skin irritation test, performed according to OECD guideline 439 and under GLP principles, potassium ferricyanide showed to be not irritant to the skin (mean tissue viability of 89%). As a consequence, the substance does not need to be classified according to GHS and CLP criteria.

Based on the results of an in vitro eye irritation test EpiOcular, Potassium ferricyanide is potentially irritant or corrosive to the eyes with a mean cell viability of 1% compared to the negative control.

Based on an in vitro eye irritation study (BCOP) no prediction on the classification of Potassium Ferricyanide can be made as the test item induced an IVIS > 3 ≤ 55.

An in vivo eye irritation study was performed in new zealand white rabbits with Potassium Ferricyanide according to OECD guideline 405 and GLP principles. A single application of 99.9mg - 110.2mg (approximately 0.1 mL)Potassium Ferricyanide was applied to the conjunctival sac of the eye under systemic and local anesthetic. Observations period were at 1, 24, 48 and 72 hours and 7, 14 and/or 21 days after instillation of the test item. Corneal epithelial damage was assessed with 2% fluorescein in water. Observations indicated corneal injury that consisted of opacity and epithelial damage. As a results of the corneal injury, pannus (neovascularization of the cornea) was apparent 7 and/or 14 days after instillation for two animals.  The corneal injury resolved within 7 days in one animal and within 14 days in the other two animals. Iridial irritation was observed and resolved within 24 hours, 48 hours or 7 days. Irritation of the conjunctivae was observed after instillation and consisted of redness, chemosis and discharge and was completely resolved within 14 days in all animals.No signs of systemic toxicity were observed and no mortality occurred. Based on these results, Potassium Ferricyanide is considered irritating to eyes.

Justification for classification or non-classification

According to the available data, potassium ferricyanide does not have to be classified for skin irritation according to CLP Regulation (EC) No. 1272/2008.

According to the available data for eye irritation, potassium ferricyanide is classified as Irritating to eye (Category 2) and labeled as H319: Causes serious eye irritation according to CLP Regulation (EC) No. 1272/2008.