Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion

Two studies are available:

1) A skin irritation study in rabbit (Beerens-Heijnen, 2010) is available which is key study. This study showed that the test substance is not irritating.

2) An in vitro skin corrosion study using a human skin model (Buskens, 2010) is available which is supporting study. This study showed that the test substance is not corrosive.

Eye irritation

An eye irritation study with an isolated bovine (Verspeek-Rip, 2010) is available which is key study. It is concluded that the test substance is corrosive or severe irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 Aug to 02 Sep 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Purity: 100.0 % area (HPLC)
- Batch No.: E010010678 (PFI-09-01-097)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: between 10-20 weeks old
- Weight at study initiation: 2-4 kg
- Housing: housed in labeled cages with perforated floors and shelters
- Diet (e.g. ad libitum): Pelleted diet approximately 100 grams per day, Hay was provided at least three times a week.
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: At least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0ºC (actual range: 19.0-19.9ºC)
- Humidity (%): 40-70% (actual range: 49 - 87%)
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

IN-LIFE DATES: From: 2010-08-24 To: 2010-09-02
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
water
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 grams
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
3 males
Details on study design:
TEST SITE
- Area of exposure: approximately 150 square centimeters (10x15 cm)
- % coverage:
- Type of wrap if used: Micropore tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): tap water
- Time after start of exposure: 4 hours

SCORING SYSTEM: numerical scoring system
Irritation parameter:
erythema score
Basis:
animal: 475
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
other: The exposure period is 4 hours.
Irritation parameter:
erythema score
Basis:
animal: 486
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
other: The exposure period is 4 hours.
Irritation parameter:
erythema score
Basis:
animal: 489
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
other: The exposure period is 4 hours.
Irritation parameter:
edema score
Basis:
animal: 475
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
other: The exposure period is 4 hours.
Irritation parameter:
edema score
Basis:
animal: 489
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
other: The exposure period is 4 hours.
Irritation parameter:
edema score
Basis:
animal: 486
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 24 hours
Remarks on result:
other: The exposure period is 4 hours.
Irritant / corrosive response data:
Four hours exposure to 0.5 g of test substance resulted in very slight erythema in the treated skin areas of the three rabbits. The skin irritation resolved within 24 hours after exposure in all animals.
There was no evidence of a corrosive effect on the skin.
Other effects:
No staining of the treated skin by the test substance was observed and no test substance remnants were seen.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual skin irritation scores

Animal

475

489

486

Time after exposure

Erythema

(0-4)

Oedema

(0-4)

Erythema

(0-4)

Oedema

(0-4)

Erythema

(0-4)

Oedema

(0-4)

1 hour

1

0

1

0

1

0

24 hours

0

0

0

0

0

0

48 hours

0

0

0

0

0

0

72 hours

0

0

0

0

0

0

Interpretation of results:
not irritating
Conclusions:
No skin irritation was caused by 4 hours exposure to the test substance.
Executive summary:

Primary skin irritation/corrosion study with PF-00968603 in the rabbit (4-hour semi-occlusive application).

The study was carried out based on the guidelines described in:  OECD No.404, "Acute Dermal Irritation/Corrosion" (2002)  EC, No 440/2008; B4: "Acute Toxicity: Dermal Irritation/Corrosion" US EPA, OPPTS 870.2500 (1998), Acute Dermal Irritation   JMAFF Guidelines (2000) including the most recent partial revisions.

Three rabbits were exposed to 0.5 grams of PF-00968603, moistened with water, by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure.

Exposure to PF-00968603 resulted in very slight erythema in the treated skin areas of the three rabbits. The skin irritation resolved within 24 hours after exposure in all animals.

No staining of the treated skin by the test substance was observed and no test substance remnants were seen.

Based on these results PF-00968603 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 (2007), - Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

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:
From 12 to 15 Jul 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Purity: 100.0 % area (HPLC)
- Batch No.: E010010678 (PFI-09-01-097)
Species:
other: cattle
Strain:
other: an isolated bovine cornea
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Age at study initiation:
- Weight at study initiation:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: minimium of 1 hour
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 301.5 to 302.0 mg
Duration of treatment / exposure:
240±10 minutes
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:
Negative control: Physiological saline
Positive control: 20% (w/v) Imidazole in physiological saline

Treatment of corneas and opacity measurements:
The medium from the anterior compartment was removed and 750 l of the negative control and 20% (w/w) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. Three corneas were covered with 301.5 to 302.0 mg the test substance. 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℃. 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. Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Opacity measurement:
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final posttreatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance 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.

Application of sodium fluorescein:
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) 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. 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℃.

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 l 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 (Multiskan spectrum, Thermo labsystems, Breda, The Netherlands). 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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.
Irritation parameter:
in vitro irritation score
Remarks:
Mean
Value:
ca. 143
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
other:
Remarks:
Permeabillity Mean
Value:
ca. 9.152
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Remarks:
Mean
Value:
ca. 6
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

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

79

3.122

126

Test substance

6

9.152

143

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).

Interpretation of results:
Category 1 (irreversible effects on the eye)
Conclusions:
It is concluded that the test substance is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.
Executive summary:

Screening for the eye irritancy potential of PF-00968603 using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the ocular irritation properties of PF-00968603 on an isolated bovine cornea. The possible ocular irritancy of PF-00968603 was tested through topical application for 240 ± 10 minutes.  

The study procedures described in this report were based on the most recent OECD guideline.

Batch E010010678 (PFI-09-01-097) of PF-00968603 was a white powder with lumps with a purity of 100.0 % area (HPLC). Since no workable suspension of PF-00968603 in physiological saline could be obtained, the test substance was added pure on top of the corneas (301.5 to 302.0 mg).

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/v Imidazole) was 126 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.  

PF-00968603 induced severe ocular irritation at one endpoint mainly (permeability), resulting in a mean in vitro irritancy score of 143 after 240 minutes of treatment.

Since PF-00968603 induced an IVIS ≥ 55.1, it is concluded that PF-00968603 is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.  

Based on these results: According to the test guideline, PF-00968603 should be regarded as severe eye irritant.  According to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), PF-00968603 would be classified as:  having irreversible effects on the eyes (Category 1). According to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures, PF-00968603 would be classified as: Irreversible effects on the eye (Category 1) and labeled as H318: Causes serious eye damage.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation/corrosion

Two studies are available:

1) A skin irritation study was conducted according to OECD 404 using rabbits (Beerens-Heijnen, 2010). Key study.

This study showed that the test substance is not irritating.

2) An in vitro skin corrosion study was conducted according to OECD 431 using a human skin model (Buskens, 2010). Supporting study.

In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a human skin model test was performed. This study showed that the test substance is not corrosive. Based on this, in vivo skin irritation study in rabbit was performed to establish the possible skin irritating properties of the test substance.

Eye irritation:

An eye irritation study with an isolated bovine cornea (Verspeek-Rip, 2010) was conducted according to OECD 437. This study is considered as reliable and key study. It is concluded that the test substance is corrosive or severe irritant.

Justification for selection of skin irritation / corrosion endpoint:

Study run to a method comparable with current guidelines and to GLP

Justification for selection of eye irritation endpoint:

Study run to a method comparable with current guidelines and to GLP

Effects on eye irritation: corrosive

Justification for classification or non-classification

Skin irritation/corrosion: Mean scores at 24, 48 & 72 hours for erythema were < 2.3 (actual value 0) for oedema were < 2.3 (actual value 0). In addition all reactions were fully reversible within the observation period (actual value within 24 hours).

Serious eye damage/eye irritation: An eye irritation study with an isolated bovine cornea showed that the test substance is corrosive or severe irritant. The test substance induced an IVIS ≥ 55.1.

Therefore in accordance with Regulation (EC) No. 1272/2008 Tables 3.2.2 & 3.3.1 the substance is not classified for the skin corrosion/irritation and classified for Category 1 (irreversible effects on the eye) for the serious eye damage/eye irritation endpoint.