cis-N-methyl-N-(1H-pyrrolo[2,3-d]pyrimidin4-yl)cyclobutane-1,3-diamine phosphate (1:1)

Endpoint summary

Description of key information

PF-03817968 was not considered to be corrosive or irritating in in-vitro studies. There was no effects seen in the BCOP eye irritiation study

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:

From 03 Dec 2018 to 07 Dec 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: not specified

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL Milli-Q water (negative control)
50 µL 8N KOH (positive control)

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

42 hr +/- 1 hours at 37°C.

Number of replicates:

Triplicate

Details on study design:

The test is based on the experience that corrosive chemicals show cytotoxic effects following short-term exposure to the stratum corneum of the epidermis. The test is designed to predict
and classify the skin corrosion potential of a test item by assessment of its effect on a threedimensional human epidermis model (1-4).
The test consists of topical application ofPF-03817968-09 on the skin tissue for 3-minute and 1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 4.2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

the mean tissue viability of the positive control

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was <_ 16%, indicating that the test system functioned properly

Interpretation of results:

other: Non-corrosive

Conclusions:

In conclusion,PF-03817968-09 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 Jan 2019 to 04 Feb 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test ". Official Journal of the European Union No. L142; Amended by EC No.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: not specified

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 µL PBS (negative control)
25 µL 5% SDS (positive control)

Duration of treatment / exposure:

15 +/- 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 f 1 hours at 37°C.

Number of replicates:

Triplicate

Details on study design:

The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin irritation potential of a test item by assessment of its effect on a three dimensional human epidermis model.
The test consists of topical application ofPF-03817968-09 on the skin tissue for 15 minutes.
After exposure the skin tissue is thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hours incubation period determination of the cytotoxic (irritancy) effect is performed.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (ITT) at the end of the treatment.

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 98

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

the mean tissue viability of the positive control

PF-03817968-09 was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20171443).
Because no color changes were observed it was concluded thatPF-03817968-09 did not interact with the MTT endpoint.

Interpretation of results:

other: Non-irritant

Conclusions:

In conclusion,PF-03817968-09 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations

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:

12 Nov 2018 to 13 Nov 2018

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)

GLP compliance:

yes

Specific details on test material used for the study:

Purity/Composition: 100%

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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:

Since no workable suspension of PF-03817968-09 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:

PF-03817968-09 was weighed in a bottle and applied directly on the corneas in such a way
that the cornea was completely covered (302.7 to 363.5 mg)

Duration of treatment / exposure:

The test consists of topical application of PF-03817968-09 on the epithelium of the bovine cornea. The non-surfactant solid test item is applied neat by direct application to the surface of the cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After exposure the corneas were thoroughly rinsed. The opacity of the corneas was determined directly after treatment and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Details on study design:

Treatment of Corneas and Opacity Measurements
The medium from the anterior compartment was removed and 750 µl of either the negative
control, positive control (20% (w/v) Imidazole solution) onto the epithelium of the cornea.
PF-03817968-09 was weighed in a bottle and applied directly on the corneas in such a way
that the cornea was completely covered (302.7 to 363.5 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
(Earle’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.


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 opacity value (measured with the device OP-KIT) was calculated according to:
((𝐼0 / 𝐼 )- 0.9894 )/ 0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows
and medium, and I the measured illuminance through a holder with cornea.
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.


Application of Sodium Fluorescein
Following the final opacity measurement, permeability of the cornea toNa-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-AldrichChemie 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.

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
490nm(OD490) of each sampling tube was measured in triplicate using a microplate reader
(TECAN Infinite® M200 Pro Plate Reader).
Opacity =
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. 0.2

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. -1.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: Permeability

Run / experiment:

Mean

Value:

ca. 0.14

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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 140 and within two standard deviations of the current historical positive control mean.
It was therefore concluded that the test conditions were adequate and
that the test system functioned properly.
PF-03817968-09 did not induce ocular irritation through both endpoints, resulting in a mean
in vitro irritancy score of 0.2 after 240 minutes of treatment.

PF-03817968-09 was tested as it is. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 116 to 168. The corneas treated with the positive control were turbid after the 240 minutes of treatment. The corneas treated with PF-03817968-09 showed opacity values ranging from -3.2 to -0.6 and permeability values ranging from 0.052 to 0.264. The corneas were clear after the 240 minutes of treatment with PF-03817968-09. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Therefore, the in vitro irritancy scores ranged from -1.2 to 1.0 after 240 minutes of treatment with PF-03817968-09.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since PF-03817968-09 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage

Executive summary:

The objective of this study was to evaluate the eye hazard potential of PF-03817968-09 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage ofPF-03817968-09 was tested through topical application for approximately 240 minutes. The study procedures described in this report were based on the most recent OECD guideline. PF-03817968-09 was a white to off-white solid with a purity of 100%. Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas. 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 140 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. PF-03817968-09 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.2 after 4 hours of treatment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available