N-cyclohexylcyclohexanamine 2-[1-[[[(1R)-1-[3-[(1E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanyl]methyl]cyclopropyl]acetate

Endpoint summary

Description of key information

Montelukast dicyclohexylamine salt did not elicit a biological response in either the in vitro skin corrosion test or the in vitro skin irritation test

under the conditions of the studies. The substance did not provoke a response in the Bovine Corneal Opacity & Permeability test.

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:

January 20th to January 26th 2015

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not defined

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN Small Model TM (EPISKIN-SM TM, 0.38 cm2, Lot no.: 14-EKIN-003, See APPENDIX 4).
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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

No correction was made for the purity/composition of the test compound.

The solid test substance (14.2 to 15.8 mg) was applied directly on top of the skin tissue.
Montelukast dicyclohexylamine salt was spread to match the size of the tissue.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 per test substance

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean absorption at 570 nm measured after treatment with Montelukast dicyclohexylamine salt
and controls are presented in APPENDIX 1,Table 1. The individual OD
measurements are
presented in APPENDIX 2.

Table 2 shows the mean tissue viability obtained after 15 minutes treatment with Montelukast
dicyclohexylamine salt compared to the negative control tissues. Skin irritation is expressed as the
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remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained
after 15 minutes treatment with Montelukast dicyclohexylamine salt compared to the negative control
tissues was 104%. Since the mean relative tissue viability for Montelukast dicyclohexylamine salt was
above 50% Montelukast dicyclohexylamine salt is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 9%. The absolute mean
OD
of the negative control tissues was within the laboratory historical control data range
(See APPENDIX 3). The standard deviation value of the percentage viability of three tissues treated
identically was less than 13%, indicating that the test system functioned properly.

Interpretation of results:

not classified

Conclusions:

It is concluded that this test is valid and that Montelukast dicyclohexylamine salt 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:

22 December to 23 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes

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 on the same day.
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)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

371.4 to 392.0 mg per cornea

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

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:

Corneas were treated in triplicate with either the test substance, positive control compound or negative control (0.9% sodium chloride solution).

Details on study design:

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
(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-Ferrand, 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

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
(OP-KIT, MC2, Clermont-Ferrand, 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 7 were not used. Three corneas were selected at random for each
treatment group.

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.
Montelukast dicyclohexylamine salt was weighed in a bottle and applied directly on the corneas in
such a way that the cornea was completely covered (371.4 to 392.0 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, Invitrogen Corporation). Possible pH effects of the test substance 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 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 post-treatment 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°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 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
was performed, the OD490 of each reading was corrected for the mean negative control OD
490 beforethe dilution factor was applied to the readings.

Electronic data capture

Observations/measurements in the study were recorded electronically using the following programme:
Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

Irritation parameter:

in vitro irritation score

Value:

ca. -0.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Value:

ca. -1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Value:

ca. 0.012

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Positive control results: opacity value 78; Permeability 1.786; and in vitro irritancy 104.8

Interpretation
In vitro irritancy score
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)

Additionally the opacity and permeability values were evaluated independently to determine whether
the test substance induced irritation through only one of the two endpoints.

The IVIS cut-off values for identifying the test substances as inducing eye damage (UN GHS
Category 1) and test substances not requiring classification for eye irritation or serious eye damage
(UN GHS No Category) are given hereafter:

≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1


Acceptability of the assay

The assay is considered acceptable if:

- The positive control gives an in vitro irritancy score that falls within the laboratory historical mean
value.
- The negative control responses should result in opacity and permeability values that are less than
the upper limits of the laboratory historical range.

Interpretation of results:

GHS criteria not met

Conclusions:

TThe 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 105 and within the
historical positive control data range (APPENDIX 3, Table 6). It was therefore concluded that the test
conditions were adequate and that the test system functioned properly.

Montelukast dicyclohexylamine salt did not induce ocular irritation through both endpoints, resulting in
a mean in vitro irritancy score of -0.5 after 240 minutes of treatment.

Finally, it is concluded that this test is valid and that Montelukast dicyclohexylamine salt is not irritant in
the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this
report.

Since Montelukast dicyclohexylamine salt 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

Additional information

Justification for selection of skin irritation / corrosion endpoint:

The in vitro skin irritancy test was selected as the most sensitive assay for signs of irritancy.

Justification for selection of eye irritation endpoint:

A single in vitro Eye Irritancy study was performed, which indicated that there was no irritant potential. Therefore further testing was not required.

Justification for classification or non-classification

Montelukast dicyclohexylamine salt did not elicit a biological response in either the in vitro skin corrosion test or the in vitro skin irritation test

under the conditions of the studies. Furthermore, the substance did not provoke a response in the Bovine Corneal Opacity & Permeability test, with an IVIS of less than 3. Therefore no classification or labeling is required.