Imidodicarbonic acid, 2-[5-bromo-3-[[(4-methylphenyl)sulfonyl]oxy]-2-pyridinyl]-, 1,3-bis(1,1-dimethylethyl) ester

Endpoint summary

Description of key information

Skin irritation:

Two studies are available:

1) An in vitro skin corrosion study (Eurlings I.M.J., PhD., 2016) is availible which is a key study. This study demonstrated that GHS criteria were not meant for the classification of the test substance as corrosive to the skin.

2) A Primary Skin Irritation/Corrosion Study in Rabbit (Latour J.E.H.M., MSc., 2016) is available which is a key study. This study demonstrated that GHS criteria were not meant for the classification of the test substance as a skin irritant.

Eye irritation:

Two studies are available:

1)       An in vitro study is available (Eurlings I.M.J., PhD., 2016) is available which is a key study. This study demonstrated that GHS criteria were not meant for the test substance to be classifiied as an eye irritant.

2)       An in vivo study is available (Latour J.E.H.M., MSc., 2016) is available which is a key study. This study demonstrated that GHS criteria were not meant for the test substance to be classifiied as an eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 January 2016 to 29 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method (adopted 28 July 2015).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Remarks:

Exception: The characterisation of the test item was conducted in a pre-GIP environment.

Specific details on test material used for the study:

Purity by HPLC, % a/a: 99.7

Test system:

human skin model

Remarks:

EpiDerm Skin Model (EPI-200, Lot no.: 23297 kit M and N)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Details on animal used as source of test system:

Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

MTT medium
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by
MatTek Corporation.

Environmental conditions
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 79 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 36.7°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, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

water

Remarks:

The skin was moistened with 25 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue

Details on test system:

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm2) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

30.5 to 35.0 mg of the solid test item was added into the 6-well plates on top of the skin tissues

Duration of treatment / exposure:

The test was performed on a total of 4 tissues per test item. Two tissues were used for a 3-minute exposure to PF-06841215 and two for a 1-hour exposure.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application

Value:

84

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application

Value:

81

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Table 3 Mean tissue viability in the in vitro skin corrosion test with PF-06841215

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
PF-06841215	84	81
Positive control	11	9

Interpretation of results:

GHS criteria not met

Conclusions:

Because the mean relative tissue viability for PF-06841215 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment PF-06841215 is considered to be not corrosive.

Finally, it is concluded that this test is valid and that PF-06841215 is not corrosive in the in vitro skin
corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 2016 to 09 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No.404: "Acute Dermal Irritation / Corrosion", Paris, 2015.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B4: "Acute Toxicity: Dermal Irritation/Corrosion". Official Journal of the European Union No. L142, May 2008, including most recent amendments.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.2500, Acute Dermal Irritation. Office of Prevention, Pesticides and Toxic Items (7101), EPA 712-C-98-196, August 1998.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF

Version / remarks:

Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000; including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity by HРLС, % a/a: 99.7

Species:

rabbit

Strain:

New Zealand White

Remarks:

Albino, SPF-Quality

Details on test animals or test system and environmental conditions:

Age and body weight:
At start of dosing, the animals were between 12 and 24 weeks old and body weights were at least 1.5 kg.

Identification:
Earmark.

Health inspection:
At least prior to dosing. It was ensured that the animals were healthy and that the skin to be treated was intact and free from any abnormality

Conditions:
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation:
Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
Acclimatization period was at least 5 days before start of treatment under laboratory conditions.

Diet:
Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period.

Water:
Free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5 grams of the test item moistened with 1 mL of the vehicle

Duration of treatment / exposure:

Four hours

Observation period:

72 hours

Number of animals:

1 Male, 2 Females.

Details on study design:

6.5. Study Design
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner one week later, after considering the degree of skin irritation observed in the first animal.

6.6. Treatment
Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimetres (10x15 cm). To facilitate scoring, treated skin areas were re-clipped.

Each animal was treated by dermal application of 0.5 grams of the test item. The test item was moistened with 1 mL of the vehicle and applied to the skin of one flank, using a metalline patch# of 2x3 cm. The patch was mounted on Micropore tape#, which was wrapped around the abdomen and secured with Coban elastic bandage#.
Four hours after the application, the dressing was removed and the skin cleaned of residual test item using tap water.

After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

6.7. Observations
Mortality/Viability Twice daily.
Toxicity At least once daily.
Body Weight Day of treatment (prior to application) and on the day of the final observation.
Irritation The skin reactions were assessed at approximately 1, 24, 48 and 72 hours after the removal of the dressings and test item. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of the untreated skinof each animal served as controls.

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

8.1. Irritation
No skin irritation was caused by 4 hours exposure to PF-06841215.
8.2. Corrosion
There was no evidence of a corrosive effect on the skin.

Other effects:

8.3. Coloration / Remnants
No staining of the treated skin by the test item was observed and no test item remnants were seen.
8.4. Toxicity / Mortality
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Table 2: Mean Value Irritation Scores

Animal Mean 24, 48 and 72 hrs

		Erythema	Oedema
38		0.0	0.0
49		0.0	0.0
50		0.0	0.0

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results PF-06841215 does not have to be classified and has no obligatory
labelling requirement for skin irritation according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments),
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Justification for type of information:

6.4. Weight of Evidence Analysis
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed, prior to the start of this in vivo skin irritation study in the rabbit. As recommended in the test guidelines, all available information was evaluated (e.g. existing human and animal data, literature, item data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity) and in vitro, ex-vivo and in vivo tests) to determine the need for in vivo skin testing. It was concluded that there is need to perform this in vivo skin irritation study in rabbit in order to establish the possible skin irritating properties of the test item.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 April 2016 to 21 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No.405, "Acute Eye Irritation / Corrosion", Paris, 2012.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B5: "Acute Toxicity: Eye Irritation/Corrosion". Official Journal of the European Union No. L142, May 2008, including most recent amendments.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.2400, Acute Eye Irritation. Office of Prevention, Pesticides and Toxic Items (7101), EPA 712-C-98-195, August 1998.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF

Version / remarks:

Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000; including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes

Remarks:

Exception: The characterisation of the test item was conducted in a pre-GIP environment.

Specific details on test material used for the study:

Molecular Weight: 543.43
Puritty 99.7 %a/a (HPLC)

Species:

rabbit

Strain:

New Zealand White

Remarks:

SPF-Quality

Details on test animals or tissues and environmental conditions:

Age and body weight: At start of dosing, the animals were between 12 and 24 weeks old and body weights were at least 1.5 kg.

Identification: Earmark.

Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that eyes were free from any abnormality.

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation: Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm). Acclimatization period was at least 5 days before start of treatment under laboratory conditions.

Diet: Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period.

Water: Free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

93.2 mg (range 93.1 – 93.2 mg) of the test item (a volume of approximately 0.1 mL)

Duration of treatment / exposure:

The lids were gently held together for about one second to prevent loss of the test item

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

1 Male and 2 Females.

Details on study design:

6.5. Study Design
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner one week later, after considering the degree of eye irritation observed in the first animal.

6.6. Preemptive Pain Management
One hour prior to instillation of the test item, buprenorphine (Buprenodale®, Dechra Ltd., Stoke-on-Trent, United Kingdom) 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 anesthetic alcaine 0.5% (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.

6.7. Treatment
Animals were treated by instillation of, on average, 93.2 mg (range 93.1 – 93.2 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.

After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

6.8. Observations
Mortality/Viability Twice daily.
Toxicity At least once daily.
Body Weight Day of treatment (prior to instillation) and after the final observation.
Irritation The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test item. The irritation scores and a description of all other (local) effects were recorded.

Irritation parameter:

conjunctivae score

Basis:

animal: 63

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal: 70

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal: 71

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritant / corrosive response data:

8.1. Irritation
Instillation of approximately 93 mg of PF-06841215 (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 72 hours in all animals.

No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test item instillation revealed no corneal epithelial damage.

8.2. Corrosion
There was no evidence of ocular corrosion.

Other effects:

8.3. Coloration / Remnants
No staining of (peri) ocular tissues by the test item was observed and no test item remnants were seen.

8.4. Toxicity / Mortality
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Table 2: Mean Value Eye Irritation Scores
                                                Mean 24, 48 and 72 hours                                

	Animal	Corneal opacity	Iris	Conjunctivae Redness	Conjunctivae Chemosis
	63	0.0	0.0	0.7	0.0
	70	0.0	0.0	0.7	0.0
	71	0.0	0.0	0.7	0.0

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results, PF-06841215 does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).

Based on the maximum group mean of 8.0, PF-06841215 is classified as mild irritant to the rabbit eye according to the Kay and Calandra classification system.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 January 2016 to 19 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 437: " Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage”(adopted July 26, 2013).

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity by HPLC, %a/a 99.7

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

Source: 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.\

Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Remarks:

A solubility test in physiological saline was performed. Since no workable suspension of PF-06841215 in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

320.8 to 343.0 mg of the substance was applied.

Duration of treatment / exposure:

240 ± 10 minutes at 32 ± 1°C.

Observation period (in vivo):

Immediate opacity measurement and permeability evaluation of the cornea.

Number of animals or in vitro replicates:

Three corneas for each treatment group (total 9 corneas).

Details on study design:

6.5.1. Preparation of corneas
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light.
Those exhibiting defects were discarded.

The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) 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.

6.5.2. Cornea selection and Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

6.5.3. Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 μl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. PF-06841215 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (320.8 to 343.0 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

6.5.4. Opacity measurement
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.

The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.

The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

6.5.5. Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.

The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for
90 ± 5 minutes at 32 ± 1°C.

6.5.6. Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

ca. 2.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

ca. 2.5

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Perneability

Run / experiment:

Mean

Value:

ca. 0.005

Negative controls validity:

valid

Positive controls validity:

valid

Table 1 Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score 1' 2
Negative control	0.3	0.015	0.5
Positive control	112.3	1.286	131.6
PF-06841215	2.5	0.005	2.6

1. Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2. In vitro irritancy score (IVIS) = mean opacity value+ (15 x mean 0 D490 value).

Interpretation of results:

GHS criteria not met

Conclusions:

The negative control responses for opacity and permeability 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/v) Imidazole) was 132 and within two standard deviations of the current historical positive control mean (APPENDIX 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

PF-06841215 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.6 after 240 minutes of treatment.

Since PF-06841215 induced an IVIS £ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation: Mean scores at 24, 48 and 72 hours for erythema were < 2.3 (actual value 0) and for edema were < 2.3 (actual value 0).

Skin Corrosion: The mean relative tissue viability for PF-06841215 was not below 50% after 3 minutes treatment (actual value 84%) and not below 15% after 1 hour treatment (actual value 81%) PF-06841215 is considered to be not corrosive.

Eye Irritation:

An in vitro study is availible with a IVIS score <=3 (actual mean score of 2.6) after 240 minutes of treatement.

An in vivo study is availible with fully reversible conjunctivae score < 1 over 72 hours (actual values of 0.7, 0.7, and 0.7).

Neither results fufill any GHS classification for the substance as an eye irritant.