Registration Dossier

Administrative data

Description of key information

Skin Irritation

The dermal irritation potential of test article was determined according to the OECD 439. In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”.

The Mean % tissue viability compared to negative control (n=3) of the test substance was determined to be 106.2 %. Thus, the test substance was considered to be not irritating to the human skin.

An in vivo dermal irritation test was performed in rabbits following the 16 CFR.1500.41 Guidelines to assess the dermal irritation potential of the test chemical.

Erythema was absent to barely perceptible at 24 hours postdose and absent at 72 hours.

Edema was absent to well defined at 24 post dose and absent at 72 hours.

Eye Irritation

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study.

The mean % tissue viability of test substance was determined to be 103.8 %. Hence, under the experimental test conditions it was concluded that test substance was considered to irritating to the human eyes.

An in vivo study was performed on rabbits according to FHSA 16 CFR 1500.42 Guidelines to assess the ocular irritation potential of the test chemical.

All six eyes appeared normal at each observation period. No irritant/corrosive effects were observed. Hence, the test chemical can be considered to be not irritating to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be dermal irritants. The dermal irritation potential of test article may be predicted by measurement of their cytotoxic effect, as reflected in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiDerm™ model.
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-phenylethyl benzoate
- Molecular formula :C15H14O2
- Molecular weight :226.274 g/mole
- Substance type:organic
- Physical state:Liquid
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: as provided by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Source strain:
other: Not applicable
Details on animal used as source of test system:
- Description of the cell system used:The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.Test System IdentificationAll of the EpiDerm™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues are included in this report. Tissue plates were appropriately labeled with study information.
Justification for test system used:
The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, In Vitro Life Science Laboratories, Bratislava, Slovakia) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
The tissues were exposed to the test article neat (undiluted) on April 25, 2018 (Run 1 of 1). EpiDerm™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.MTT and Color Pre-testsPretesting has actually been conducted for all chemicals, although the first intitial 8 test chemicals a pretesting was not conducted (for skin).MTT AssayFollowing the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 3 hours MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was approximately 3 hours with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean negative control tissues.Evaluation of Test Article in the Cell Models:1. Cell system: Upon receipt, the MatTek EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight (18 ± 3 hrs) at ~37°C, 5% CO2 in a humidified incubator
.2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test article are applied topically to tissues by pipette(liquid) Tissues were exposed to controls and the test article for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.a) Controls30 µL of negative control DPBS and 30 μL of the positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.b) Test Articles 25 mg of the test article was applied topically to the tissue 3. Post-exposure treatment: After the 1 hour exposure, the tissues were rinsed 15 times with sterile DPBS. After the 15th rinse from washing bottle, each insert wasw completely submerge 3 times in 150 ml DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for 24 ± 2 hours. After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 18 ± 3 hours, for a total of an approximately 42 hour post-exposure incubation.RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: The EpiDerm™ 3 dimensional human tissue model- Tissue Lot number(s): 26459- Date of initiation of testing: 6/08/2017TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: The test substance was rinsed from the tissues with sterile DPBS by filling and emptying the tissue insert 15 times to remove any residual test material. This was followed by completely submerge the insert 3 times in 150 ml DPBS.MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 300 µL MTT medium (1.0 mg/mL).- Incubation time: After 3 hours- Spectrophotometer: Synergy H4 spectrophotometer - Wavelength: 570 nm- Filter: No data- Filter bandwidth: No data- Linear OD range of spectrophotometer: No dataNUMBER OF REPLICATE TISSUES: 3CALCULATIONS and STATISTICAL METHODSAll data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT AssayBlanks:·        The optical density (OD) mean from all replicates for each plate (ODblank). Negative Controls (NC):·        The blank corrected value was calculated: ODNC= ODNCraw– ODblank. ·        The OD mean per NC tissue was calculated. ·        The mean OD for all tissues corresponds to 100% viability. ·        The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Positive Control (PC):·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank. ·        The OD mean per PC tissue was calculated. ·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Tested compound :·        Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·        The OD mean per tissue was calculated. ·        The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Data Correction Procedure for MTT Interfering Compounds (if applicable)True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).ODtvt= optical density of treated viable tissueODkt= optical density of killed tissuesODtkt= optical density of treated killed tissueODukt= optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored Compounds (if applicable)True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt– ODvt.ODtvt= optical density of treated viable tissue incubated in MTT mediaODvt= optical density of viable tissues incubated in media alone - Evaluation of data The results of the assay was evaluated and compared to negative control. Table: Criteria for in vitro Interpretation: In VitroResults In VivoPredictionMean tissue viability ≤50% Irritant (I), R38Mean tissue viability >50% Non-irritant (NI)- Assay quality controls- Negative Controls (NC)The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.  - Positive Controls (PC)5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.   - Standard Deviation (SD)The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 25 mg- Concentration (if solution): neat VEHICLE (Not used)- Amount(s) applied (volume or weight with unit): none- Concentration (if solution): none- Lot/batch no. (if required): none- Purity: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 30 µL sterile DPBS- Concentration (if solution): neatPOSITIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): 5% solution of sodium dodecyl sulfate
Duration of treatment / exposure:
The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
For a total of an approximately 42 hour post-exposure incubation.
Number of replicates:
3 tissues were used for test compound and control.
Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 ul
- Concentration (if solution): Neat

VEHICLE: No data available
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 ul sterile DPBS
- Concentration (if solution): Neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% solution of sodium dodecyl sulfate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1
Value:
106.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.
Interpretation of results:
other: not irritating
Conclusions:
The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study. The Mean % tissue viability compared to negative control (n=3) of the test substancewas determined to be 106.2 %. Thus, the test substance was considered to be not irritating to the human skin.
Executive summary:

The dermal irritation potential of test article was determined according to the OECD 439. In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay.  The MTT data shows that the assay quality controls were met. The mean tissue viabilities for the Positive control, Methyl acetate were 6.5%, 10.7% respectively in the first and second run, whereas the tissue viabilities of the negative control, Tissue culture water remained at 100% in the both the runs.

The Mean % tissue viability compared to negative control (n=3) of the test substance was determined to be 106.2 %. Thus, the test substance was considered to be not irritating to the human skin.

Endpoint:
skin irritation: in vivo
Remarks:
Study performed in 2002/2003, before the changes in legislation concerning animal testing entered into force.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2002 - October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
data is from experimental reports
Qualifier:
according to
Guideline:
other: 16 CFR 1500.41
Deviations:
not specified
Principles of method if other than guideline:
To assess the dermal irritation potential of the test chemical in rabbits.
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Animals were received from Millbrook Breeding Labs, Amherst, MA, USA. Following an equilibration period of at least one week, six healthy New Zealand White rabbits were selected for this test from a larger group without conscious bias.
Pretest body weight range was 2.3- 2.7 kg.
The animals were identified by cage notation and a uniquely numbered metal eartag. The animals were housed 1/cage in suspended cages. Bedding, placed beneath the cages, was changed at least three times/week. Fresh PMI Rabbit Chow was provided daily. Water was available ad libitum.
The animal room, reserved exclusively for rabbits on acute tests, was temperature controlled, had a 12 hour light/dark cycle and was kept clean and vermin free.
Type of coverage:
semiocclusive
Preparation of test site:
other: The back and sides of each animal were clipped free of hair. The left side of each animal was abraded. The right side of each animal remained intact.
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
0.5 mL/site, 1 mL/rabbit
Duration of treatment / exposure:
24 hrs
Observation period:
72 hrs
Number of animals:
2 males and 4 females
Details on study design:
The test article was applied to two areas, 1 intact and 1 abraded, on the prepared site, on the back of each of six rabbits.
The test article was placed under a 2.5 x 2.5 em, 4 ply, surgical gauze patches which were secured with non-irritating adhesive tape. The torso was wrapped with plastic in an occlusive manner which was secured with non-irritating adhesive tape. The sites were occluded for 24 hours at which time the patches
were removed. Residual test article was gently wiped from the test site at the end of the exposure period, prior to scoring for dermal reactions.

Animals were observed for skin reactions at 24 and 72 hours following application of the test article.
Erythema and edema were scored according to the numerical Draize technique.
Body weights were recorded pretest.
The general health of the animals was monitored at each observation time.
All animals were humanely sacrificed using C02 following study termination.

The Primary Irritation Index was calculated by adding the mean values (6 rabbits) for erythema/eschar and · edema on intact and abraded skin at 24 and 72 hours (a total of 8 values) and dividing the sum by 4.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
72 h
Score:
ca. 0.75
Max. score:
0.83
Reversibility:
fully reversible within: 72 hrs
Irritant / corrosive response data:
Intact:
Erythema was absent to barely perceptible at 24 hours postdose and absent at 72 hours.
Edema was absent to well defined at 24 postdose and absent at 72 hours.

Abraded:
Erythema was absent to barely perceptible at 24 hours postdose and absent at 72 hours.
Edema was absent to well defined at 24 hours postdose, and absent at 72 hours.
Other effects:
There were no abnormal physical signs noted during the observaiton period.

SEX

F

M

M

F

F

F

 

PretestBodyWeight

•kg

2.5

 

2.6

2.6

2.3

2.7

2.4

Time:PostDose                                              erythema and eschar Formation                      Mean Scores         

Intactskin

24hours

1

0

1

0

1

1

0.67

72hours

0

0

0

0

0

0

0.0

Abradedskin

24hours

1

0

1

0

1

1

0.67

72 hours

0

0

0

0

0

0

0.00

EDEMA

Intactskin

24hours

1

0

2

0

1

1

0.83

72hours

0

0

0

0

0

0

0

Abradedskin

24hours

1

0

2

0

1

1

0.83

72 hours

0

0

0

0

0

0

0

Sum of the mean scores =

3.00

Primary Dermal Irritation Index[PDII]= Sum of the mean scores/4=

0.75

 

Systemic observations

 

24hours

A

A

A

A

A

A

72hours

A

A

A

A

A

A

 

A=Normal           *-.recllppe<l

Interpretation of results:
other: not irritating
Conclusions:
Erythema was absent to barely perceptible at 24 hours post dose and absent at 72 hours and Edema was absent to well defined at 24 hours post dose and absent at 72 hours under intact and abraded skin conditions.
The Primary Irritation Index was 0.75. Therefore,the test chemical not considered a dermal irritant.
Executive summary:

The dermal irritation potential of the test chemical was assessed in rabbits following the 16 CFR.1500.41 Guidelines. 2 males and 4 female young adult New Zealamd White rats were used for the study.

The test article was applied to two areas, 1 intact and 1 abraded, on the prepared site, on the back of each of six rabbits.

The test article was placed under a 2.5 x 2.5 em, 4 ply, surgical gauze patches which were secured with non-irritating adhesive tape. The torso was wrapped with plastic in an occlusive manner which was secured with non-irritating adhesive tape. The sites were occluded for 24 hours at which time the patches

were removed. Residual test article was gently wiped from the test site at the end of the exposure period, prior to scoring for dermal reactions.Animals were observed for skin reactions at 24 and 72 hours following application of the test article.

/Erythema and edema were scored according to the numerical Draize technique. Body weights were recorded pretest. The general health of the animals was monitored at each observation time. All animals were humanely sacrificed using C02 following study termination.

The Primary Irritation Index was calculated by adding the mean values (6 rabbits) for erythema/eschar and · edema on intact and abraded skin at 24 and 72 hours (a total of 8 values) and dividing the sum by 4.

Erythema was absent to barely perceptible at 24 hours postdose and absent at 72 hours.

Edema was absent to well defined at 24 hours post dose and absent at 72 hours.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiOcular™ model.
GLP compliance:
no
Species:
human
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
Description of the cell system used:The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.- Test System IdentificationAll of the EpiOcular™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues is included in this report. Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.- Justification of the test method and considerations regarding applicabilityEpiOcularTM Eye Irritation (OCL) by MatTek In Vitro Life Science Laboratories, Bratislava, SlovakienThe test articles and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek In Vitro Life Science Lab. (Bratislava, Slovakia). The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL - Amount(s) applied (volume or weight with unit): 50 mg of solid test article
- Concentration (if solution): neat (undiluted)

VEHICLE (no vehicle)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat
Duration of treatment / exposure:
Tissues were exposed for approximately 30 minutes for liquid test article and controls 6 hrs ± 15 min for solid test articles, at approximately 37°C, 5% CO2 in a humidified incubator.
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Following the washing step and the post-soak, the tissues were incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of ~2 hours for 18 hrs for solid test articles, or 18 hrs for solid test articles, and controls.
Number of animals or in vitro replicates:
2 tissues were used for test compound and control.
Details on study design:
- Details of the test procedure used:
The tissues were exposed to the test article neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 6 hrs ± 15 min for solid test articles, and controls, at approximately 37°C, 5% CO2 in a humidified incubator. After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~12 minutes for 25 min for solid test articles articles and controls. Following the washing step and the, the tissues were incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~2 hours for 18 hrs for solid articles and controls. Tissue viability was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. - MTT Auto reduction and colouring assessmentMTT Pre-testThe test article was assessed for the potential to interfere with the assay. Approximately 50 µL of liquid test article was added to 1 mL of MTT media (~1 mg/mL) and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 3 hours. 50 µL of ultrapure water was used as a negative control. - Test Article Color TestApproximately 50 µL of liquid test article was added to 1.0 mL of ultrapure water and 2.0 mL isopropanol and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 04 minutes and 35 seconds. Samples were then added to the wells of a clear 96-well plate and the plate was read on a Thermo Scientific Multiskan FC Microplate Photometer to 570 nm. Test articles that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions).
- MTT Assay Solids: Inserts are removed from the 24-well plate after 3 hrs of incubation and the bottom of the insert is blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 1 ml isopropanol in each well so that no isopropanol is flowing into the insert. At the end of the non-submerged extraction inserts and tissues are discarded without piercing and 1 ml of isopropanol is added into each well. The extract solution is mixed and the optical density of the extracted formazan (200 μL/well of a 96-well plate) was determined using aThermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control - Evaluation of Test Article in the cell Models1. Cell System: Upon receipt, the MatTek EpiOcular™ tissue cultures were placed in 1.0 mL of fresh Maintenance medium (in a 6-well plate) for 60 minutes. After the 60 minutes incubation, the Maintenance medium was exchanged with fresh medium and the tissues were incubated overnight (16-24 hrs) at approximately 37°C, approximately 5% CO2 in a humidified incubator,2. Control and Test Article Exposures:20 µL of calcium and magnesium free DPBS was added to each tissue and the tissues placed back into the incubator for 30 minutes. The controls and the test article will be applied topically to tissues by pipette. Three tissues will be used per test compound and control.a)Controls: 50 µL of negative control sterile ultrapure water and positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time. b)Test Article: When a solid was tested, 50 mg of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min. 3. Post exposure treatment:After the exposure, the tissues were rinsed 20 times with sterile of DPBS to remove test material. The apical surface was gently blotted with a cotton swab and cultures were immediately transferred to a 12-well plate containing 5 mL of media per well. Tissues exposed to solid test articles (and the respective control) were incubated, submerged in the media for ~12 minutes at room temperature.For liquid test articles, tissues, Tissuses were then transferred to 6-well plates containing 1.0 mL fresh Maintenance medium per well and incubated for a post-exposure recovery period for 18 hrs overnight at approximately 37 degC, 5% CO2 in a humidified incubator.- Doses of test chemical and control substances usedTest Article: 50 mg of solid test article were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs exposure time Controls: 50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs exposure time. - Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Tissues were exposed for approximately 30 minutes for 6 hrs for solid test articles and controls, at approximately 37°C, 5% CO2 in a humidified incubator. Following the washing step and the, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~2 hours for liquid test articles and controls.- Justification for the use of a different negative control than ultrapure H2O (Not applicable)- Justification for the use of a different positive control than neat methyl acetate (Not applicable)- Number of tissue replicates used per test chemical and controls: 2 tissues were used for test compound and control.- Description of the method used to quantify MTT formazanThe blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction for liquid exposed tissues was overnight incubation with a 20 minute 24 second shake the following morning. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. The blue formazan salt was extracted by placing the tissue insterts in 1 mL isopropanol in a 6-well plate. The extraction for solid exposed tissues was 3 hrs incubation. After an addition of 1 ml isopropanol and mixing, the optical density of the extracted formazan (200μL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm.- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction modelCalculations and Statistical MethodsMTT AssayBlanks: ·  The OD mean from all replicates for each plate (ODblank). Negative Controls (NC): ·  The blank corrected value was calculated: ODNC= ODNCraw– ODblank. ·  The OD mean per NC tissue was calculated. ·  The mean OD for all tissues corresponds to 100% viability. ·  The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. ODblank= optical density of blank samples (isopropanol alone). ODNCraw= optical density negative control samples. ODNC= optical density of negative control samples after background subtraction. Positive Control (PC): ·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank. ·        The OD mean per PC tissue was calculated. ·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. ODPCraw= optical density positive control samples. ODPC= optical density of positive control samples after background subtraction. Tested Articles: ·  Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·  The OD mean per tissue is calculated. ·  The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100. ·  The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues. ·  The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated. ODTTraw= optical density test article samples. ODPC= optical density of test article samples after background subtraction. Data Correction Procedure for MTT Interfering CompoundsTrue viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt). ODtvt = optical density of treated viable tissue ODkt = optical density of killed tissues ODtkt = optical density of treated killed tissue ODukt = optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored CompoundsTrue viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt – ODvt. ODtvt = optical density of treated viable tissue incubated in MTT media ODvt = optical density of viable tissues incubated in media alone. Proposed Statistical methods The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated. - Evaluation of data The results of the assay was evaluated and compared to negative control. Table: Irritancy Prediction In VitroResults In VivoPredictionMean tissue viability ≤60% Irritant (I) – Category 1 or 2Mean tissue viability >60% Non-irritant (NI) – No Category- Assay quality controls- Negative Controls (NC)The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density (OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.   - Positive Controls (PC)Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.   - Standard Deviation (SD)Each test of ocular irritancy potential is predicted from the mean viability determined on 3 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the replicates is <18% for three replicate tissues.  
Irritation parameter:
other: Mean % tissue viability
Run / experiment:
Run 1
Value:
103.8
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.
Interpretation of results:
other: not irritating
Conclusions:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance was determined to be 103.8 %. Thus, the test substance was considered to be not irritating to the human eyes.
Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak.  The viability of each tissue was determined by MTT assay.  The MTT data show the assay quality controls were met, and passing the acceptance criteria.

The mean % tissue viability of test substance was determined to be 103.8 %. Hence, under the experimental test conditions it was concluded that test substance was considered to irritating to the human eyes.

Endpoint:
eye irritation: in vivo
Remarks:
The study was done in 2002/2003, before the changes in legislation concerning animal testing entered into force.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2002 - October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: FHSA 16 CFR 1500.42
Deviations:
not specified
Principles of method if other than guideline:
To assess the ocular irritation potential of the test chemical in rabbits
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
New Zealand While rabbits, received from Millbrook Breeding Labs, Amherst, MA, USA were equilibrated for at least one week. Only animals in apparent good health were made available for study assignment. Prior to being selected for this study, both eyes of each animal were examined according to the Draize technique for any evidence of irritation or abnormalities of the cornea, iris and/or conjunctiva. A Mini-Maglite® flashlight equipped with a high intensity bulb was used to aid in the examination. Based on this examination, six rabbits, free from evidence of ocular irritation or abnormalities, were assigned to this study without conscious bias.
The pretest body weight range was 2.6- 3.2 kg. The animals were identified by cage notation and a uniquely numbered metal eartag.
The animals were housed 1/cage in suspended cages. Bedding was placed beneath the cages and changed at least three times/week. Fresh PMI Rabbit Chow was provided daily. Water was available ad libitum. The animal room, reserved exclusively for rabbits on acute tests, was temperature controlled, had a 12 hour light/dark cycle and was kept clean and vermin free.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
After instillation, the lids were held together briefly to insure adequate distribution of the test article.
Observation period (in vivo):
3 days
Number of animals or in vitro replicates:
3 males and 3 females
Details on study design:
One eye of each rabbit was dosed. The contralateral eye served as a control. The test article (0.1 ml) was placed by syringe into the conjunctival sac, which was formed by gently pulling the lower eyelid away from the eye. After instillation, the lids were held together briefly to insure adequate distribution of the test article.

Using a Mini-Maglite® flashlight equipped with a high intensity bulb, the treated eye of each rabbit was examined for irritation of the cornea, iris and conjunctiva on days 1, 2 and 3 postdose. Ocular reactions were graded according to the numerical Draize technique.
Body weights were recorded pretest.
The general health of the animals was monitored at each observation time. All animals were humanely sacrificed using C02 following study termination.

The primary eye irritation score for each rabbit was calculated from the weighted Draize scale. The method of calculation is indicated on the attached scale.
The irritation potential was determined by counting the number of rabbits with positive irritation on days 1, 2 or 3. Positive irritation is defined as any score for opacity or iritis, or 2 or more for redness or chemosis:
Irritant: 4 or more rabbits with a positive score at any time period.
Non-irritant: 0 or 1 animal with a positive score at any time period.
Indeterminate: 2 to 3 animals with a positive score at any time period.
Irritation parameter:
other: primary eye irritation score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
0
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
All six eyes appeared normal at each observation period. No irritant/corrosive effects were observed.
Other effects:
There were no abnormal physical signs noted during the observation period.
Interpretation of results:
other: not irritating
Conclusions:
All six eyes appeared normal at each observation period. No irritant/corrosive effects were observed.
Hence, the test chemical can be considered to be not irritating to eyes.
Executive summary:

The ocular irritation potential of the test chemical was assessed in rabbits. The study was performed according to FHSA 16 CFR 1500.42 Guidelines. 3 males and 3 females young adult New Zealand White rabbits were used for the study. One eye of each rabbit was dosed with 0.1ml of the test chemical. The contralateral eye served as a control. The test article (0.1 ml) was placed by syringe into the conjunctival sac, which was formed by gently pulling the lower eyelid away from the eye. After instillation, the lids were held together briefly to insure adequate distribution of the test article. The treated eyes were observed for 3 days and scored for corneal opacity, iris, conjunctival redness, discharge according to the FHSA Guidelines.

All six eyes appeared normal at each observation period. No irritant/corrosive effects were observed. Hence, the test chemical can be considered to be not irritating to eyes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

In different studies, the test chemical has been investigated for potential to cause dermal irritation to a greater or lesser extent. The studies are based on in- vitro and in-vivo experiments conducted on human, mice, swine and rabbits which have been summarized below:

The dermal irritation potential of test article was determined according to the OECD 439. In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay.  The MTT data shows that the assay quality controls were met. The mean tissue viabilities for the Positive control, Methyl acetate were 6.5%, 10.7% respectively in the first and second run, whereas the tissue viabilities of the negative control, Tissue culture water remained at 100% in the both the runs.

The Mean % tissue viability compared to negative control (n=3) of the test substance was determined to be 106.2 %. Thus, the test substance was considered to be not irritating to the human skin.

This in vitro result is supported by an in vivo dermal irritation test performed in rabbits following the 16 CFR.1500.41 Guidelines to assess the dermal irritation potential of the test chemical. 2 males and 4 female young adult New Zealamd White rats were used for the study.

The test article was applied to two areas, 1 intact and 1 abraded, on the prepared site, on the back of each of six rabbits.

The test article was placed under a 2.5 x 2.5 em, 4 ply, surgical gauze patches which were secured with non-irritating adhesive tape. The torso was wrapped with plastic in an occlusive manner which was secured with non-irritating adhesive tape. The sites were occluded for 24 hours at which time the patches

were removed. Residual test article was gently wiped from the test site at the end of the exposure period, prior to scoring for dermal reactions. Animals were observed for skin reactions at 24 and 72 hours following application of the test article. Erythema and edema were scored according to the numerical Draize technique. Body weights were recorded pretest. The general health of the animals was monitored at each observation time. All animals were humanely sacrificed using C02 following study termination.

The Primary Irritation Index was calculated by adding the mean values (6 rabbits) for erythema/eschar and · edema on intact and abraded skin at 24 and 72 hours (a total of 8 values) and dividing the sum by 4.

Erythema was absent to barely perceptible at 24 hours postdose and absent at 72 hours.

Edema was absent to well defined at 24 post dose and absent at 72 hours.

Another skin irritation studies were performed on mice and swine to assess the irritation potential of test chemical. Undiluted test chemical was applied under occlusion to the backs of hairless mice and swine for 24 hours. The treated animals were observed for signs of irritation. No signs of skin irritation were observed in treated animals. Hence the test material was considered to be not irritating to hairless mice and swine after 24 hours exposure.

These results are further supported by a skin irritation study performed on humans to assess the irritation potential of test chemical.8% test chemical in petrolatum was applied to the skin of human volunteers in a closed patch skin for 48 hours. The test chemical failed to produce any dermal irritation on the skin of humans. Hence the test material was not irritating to humans after 48 hours exposure

All these studies lead to a conclusion that Test chemical was indeed not irritating to skin. Comparing the above annotations with the criteria of CLP regulation, Test chemical can be classified under the category “Not Classified”.

Eye Irritation

In different studies, the test chemical has been investigated for potential to cause ocular damage to a greater or lesser extent. The studies are based on in- vitro and in-vivo experiments carried out on rabbits which have been summarized below:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak.  The viability of each tissue was determined by MTT assay.  The MTT data show the assay quality controls were met, and passing the acceptance criteria.

The mean % tissue viability of test substance was determined to be 103.8 %. Hence, under the experimental test conditions it was concluded that test substance was considered to irritating to the human eyes.

The in vitro result is supported by an in vivo study performed on rabbits according to FHSA 16 CFR 1500.42 Guidelines to assess the ocular irritation potential of the test chemical. 3 males and 3 females young adult New Zealand White rabbits were used for the study. One eye of each rabbit was dosed with 0.1ml of the test chemical. The contralateral eye served as a control. The test article (0.1 ml) was placed by syringe into the conjunctival sac, which was formed by gently pulling the lower eyelid away from the eye. After instillation, the lids were held together briefly to insure adequate distribution of the test article. The treated eyes were observed for 3 days and scored for corneal opacity, iris, conjunctival redness, discharge according to the FHSA Guidelines.

All six eyes appeared normal at each observation period. No irritant/corrosive effects were observed. Hence, the test chemical can be considered to be not irritating to eyes.

The in vitro and in vivo experimental results are in mutual agreement with each other indicating a very strong possibility that the test chemical can be indeed not irritating to eyes. Hence, comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified’.

Justification for classification or non-classification

The in vitro and in vivo experimental results are in mutual agreement with each other indicating a very strong possibility that the test chemical can be indeed not irritating to eyes and skin. Hence, comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified’.