(2S,3S)-2,3-Bis[(4-methylbenzoyl)oxy]butanedioic acid — methyl {(4S)-8-fluoro-2-[4-(3methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydroquinazolin-4yl}acetate — ethyl acetate (1:1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-06-2104 to 20-08-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

The critieria for a positive outcome differ from the standard Test guideline- the MB model examines the tissue viability (EC50) at three set exposure timepoints 1,4 and 24 hours, whereas the validated OECD method has an exposure time of 60 minutes and a post exposure period of incubation period of 42 hours before the tissue viability is measured for Mattek EpiDerm. The MB protocol doses 100 mg/tissue whereas the OECD guideline for Epiderm is only 25 mg/tissue plus 25µL dulbeccos PBS for tissue wetting (39mg/cm2)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2S,3S)-2,3-Bis[(4-methylbenzoyl)oxy]butanedioic acid — methyl {(4S)-8-fluoro-2-[4-(3methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydroquinazolin-4yl}acetate — ethyl acetate (1:1:1)

EC Number:

947-432-4

Cas Number:

2241754-97-0

Molecular formula:

C30H30F4N4O4.C20H18O8.C4H8O2

IUPAC Name:

(2S,3S)-2,3-Bis[(4-methylbenzoyl)oxy]butanedioic acid — methyl {(4S)-8-fluoro-2-[4-(3methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydroquinazolin-4yl}acetate — ethyl acetate (1:1:1)

Test material form:

solid

Details on test material:

lot L--005457795-001 G001

Specific details on test material used for the study:

L-005457795-001 G001 Off-white powder Used as received analysis >90% w/w 6-9% ethyl acetate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Justification for test system used:

To provide an estimate of dermal irritation potential using an alternative to the Draize methodology. The
MatTek EpiDerm ™ MTT Viability Assay has been demonstrated to be a quantitative method for
assessing potential skin hazards.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Mattek Epiderm
- Tissue batch number(s): Lot 20521 Kit F
- Delivery date: 24 Jun 2014
- Date of initiation of testing: 24 Jun 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml in DMEM
- Incubation time: 3 hrs extraction time overnight
- Spectrophotometer:
IJQuant Plate Reader, Bio-Tek Instruments,Winooski, VT
- Wavelength:
540nm subtracting absorbance at 690nm

NUMBER OF REPLICATE TISSUES:
2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
For each test article, 100 mg of the test articie were mixed with 1 ml of MTT solution (1 mg/ml Methyl
thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium (DMEM)). A Negative Control, 100 IJI
of tissue culture water, was tested concurrently. The soiutions were incubated at room temperature in the
dark for 60 minutes. After incubation, the solutions were Visually inspected for purple coloration, which
indicates that the test articie reduced MTT. Since tissue viability is based on MTT reduction, direct
reduction by a test article can exaggerate viability, making a test article seem less irritating than it really is.
Neither of the test articles was found to have reduced MTT and the assay continued as per the protocol.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

As per MatTek, as a general guideline, the following groups can be used to assign expected in vivo
irritancy responses based on the ET50 resuits obtained using the EpiDerm™ MIT Viability Assay:

Expected In vivo Irritancy Example ET50 (hrs)

Severe, Probable Corrosive Concentrated Nitric acid < 0.5
Moderate 1% Sodium Dodecyl Sulfate 0.5 - 4
Moderate to Mild 1% Triton'" X-100 4 -12
Very Mild Baby shampoo 12 - 24
Non-irritating 10% Tween'" 20 > 24

Control samples:

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

100 mg/tissue

Duration of treatment / exposure:

1, 4 and 24 hours

Duration of post-treatment incubation (if applicable):

none

Number of replicates:

duplicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: ET50 (hrs)

Run / experiment:

L-005457795-001 G001

Value:

> 24

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Test Article L-005457795-001 G001 results

dose 100mg		test concentration : neat
time (Hrs)	OD1	OD2	OD mean	SD	viability %	error %
1	1.862	1.753	1.808	0.077	97.6	4.2
4.0	2.179	2.124	2.152	0.039	116.1	2.1
24.0	1.745	2.001	1.873	0.181	101.1	9.8
neg control	1.903	1.802	1.853	0.071	100.0	3.9
ET50 (hrs) >24		Irritancy classification Non-irritating

 

Positve control 1% Triton X-100

 

dose 100µl		concentration: neat
time (Hrs)	OD1	OD2	OD mean	SD	Viability %	error %
4	1.824	1.628	1.726	0.139	93.2	7.5
9	0.154	0.142	0.148	0.008	8.0	0.5
neg control	1.903	1.802	1.853	0.071	100.0	3.6
ET50 (hrs): 6.0		Irritancy classificaiton : within range (4.8 -8.7)

Applicant's summary and conclusion

Interpretation of results:

other: non guideline critieria- likely non-irritant

Conclusions:

The test article was not irritant according to the criteria set out by the Testing laboratory- the assay was not conducted in accordance with the OECD test guideline or EU test method, therefore no conclusion can be made regarding irritancy on the basis of the validated acceptance critieria. Expert judgement would suggest that the substance is non-irritant based on the tissue viabilities observed.

Executive summary:

 

OBJECTIVE: To provide an estimate of dermal irritation potential using an alternative to the Draize methodoiogy. The MatTek EpiDerm ™ MIT Viability Assay has been demonstrated to be a quantitative method for assessing potential skin hazards.

METHOD SYNOPSIS: MatTek EpiDerm™ tissue samples were treated in duplicate with the test article and Positive Control for various exposure times listed below. Negative Controls (undosed tissues) were tested at 4 hours only. Following treatment, the viability of the tissues was determined using Methyl thiazole tetrazolium (MTT) uptake and reduction. The absorbance of each sample was measured at 540nm using a reference wavelength of 690 nm. The viability was then expressed as a percent of Control values. The mean percent viability for each time point was used to calculate an ET50, which represents the time at which the EpiDerm™ tissue viability was reduced 50% compared to Negative Control tissues.

The ET 50 scores were converted to an irritancy classification.

SUMMARY/CONCLUSION:

 

Test and Control Article Identity              Exposure Times (hrs)       ET50 (hrs)       Irritancy Classification

L-005457795-001 G001                                   1,4,24                     >24.0                         Non-Irritating

1% Triton® X-100 (Positive Control)                     4,9                            6.0              Within Range (4.8 - 8.7)