Registration Dossier

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:
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
lot L--005457795-001 G001
Specific details on test material used for the study:
L-005225800-000B008 White powder Used as received analysis 98% w/w

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:
foreskin from a single donor
Source strain:
not specified
Details on animal used as source of test system:
Type: Normal human epidermal keratinocytes (NHEK)
Genetic make-up: Single donor
Derived from: Neonatal-foreskin tissue (NHEK)
Alternatives: NHEK from adult breast skin
Screened for: HIV, Hepatitis-B, Hepatitis-C, mycoplasma
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
- Production date:
- Shipping date:
- 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
- Temperature of post-treatment incubation (if applicable): N/A

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one rinse, volume not specified
- Observable damage in the tissue due to washing: not disclosed
- Modifications to validated SOP: none dislcosed

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

Resultsopen allclose all
Irritation / corrosion parameter:
other: ET50 (hrs)
Run / experiment:
L-005225800-000B008
Value:
> 24
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
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-005225800-000B008 results

    dose 100 mg     concentration : neat      
 Time (hrs)  OD1  OD2  Mean (OD)  SD  Viability %  Error %
 1  1.767  1.726  1.747  0.029  94.3  1.6
 4.0  2.15  1.942 2.046   0.147  110.4  7.9
 24.0  1.268  1.976  1.622  0.501  87.6  27.0
 neg control  1.903  1.802  1.853  0.071  100.0  3.9
    ET50 (hrs) >24     Irritancy classification: Non-irritating      

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 articles

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 540

nm 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-005225800-000B008                                   1, 4, 24                     >24.0              Non-Irritating

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)