Octan-2-one

Endpoint summary

Description of key information

Skin irritation:

No signs of erythema and edema were observed at 1.0% ICC and 10% ACC concentrations. Hence, the test chemical was considered to be non-irritating on guinea pigs in the preliminary irritation tests performed for the sensitization study.

Eye Irrritation:

The mean of OD for test chemical was determined to be 0.766.The mean % tissue viability of test chemical was determined to be 35.0%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the human eyes and and can thus be classified as “ Irritating to eyes in Category 2’’ as per CLP Regulation.

Rats were exposed to calculated vapor concentrations of the test chemical, 8.9 mg/L (1673 ppm) for 6 hr exhibited signs of mild eye irritation.

Hence, the test chemical was mildly irritating to rat eyes

 

 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: as per mentioned below

Principles of method if other than guideline:

Preliminary irritation tests were performed in guinea pigs to determine the concentration for the sensitization study

GLP compliance:

not specified

Species:

guinea pig

Strain:

Hartley

Details on test animals or test system and environmental conditions:

- Sex: Male/female
- Weight at study initiation: 450 g
- Housing: Housed in wire mesh cages in pairs of the same sex
- Diet (e.g. ad libitum): Pelleted guinea pig diet, cabbage and hay ad libitum
- Water (e.g. ad libitum): water ad libitum

Type of coverage:

open

Preparation of test site:

shaved

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

0.1 ml (Intradermal Concentration

Duration of treatment / exposure:

24 hrs

Observation period:

24 hours

Number of animals:

4 guinea pigs(same sex)

Details on study design:

TEST SITE
- Area of exposure:
Intradermal – shaved flanks
Topical - small circular areas on the clipped, shaved flanks

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24 h

Score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No irritation observed in the [reliminary tests

SENSITIZATION POTENTIAL OF SYNTHETIC PERFUME INGREDIENTS

Non-sensitizer = no evidence of sensitization

Test chemical	ICC(%)	ACC(%)	Results
111 -13 -71.0	1.0	10	Not sensitizer

 

 

Interpretation of results:

other: not irritating

Conclusions:

No signs of erythema and edema were observed at 1.0% ICC and 10% ACC concentrations. Hence, the test chemical was considered to be non-irritating on guinea pigs in the preliminary irritation tests performed for the sensitization study.

Executive summary:

Preliminary irritation tests were performed for the test chemical in guinea pigs to determine the concentration suitableforthe sensitization study[injection challenge concentration (ICC) and application challenge concentration(ACC)].

                                       

In the preliminary irritation study, 4 Albino Hartley guinea pigs of the same sexwereinjected intradermally on the shaved flanks with 0.1 ml aliquots of a range of concentrations of tests material in a suitable solvent. The reactions were examined for size (two largest diameters), erythema and oedema 24 h later and the concentration giving slight but perceptible irritationwith no oedema was selected as the injection challenge concentration (ICC).0.1 ml of the test material in arange of concentrations in a suitable solvent were applied in small circular areas to the shaved flanks of 4 guinea pigs of the same sex.The reactions were examined for erythema 24 hours later and the highest concentration which caused no irritation was selected as the application challenge concentration (ACC).

No signs of erythema and edema were observed at 1.0% ICC and 10% ACC concentrations. Hence, the test chemical was considered to be non-irritating on guinea pigs in the preliminary irritation tests performed for the sensitization study.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 05, 2017 to July 17, 2017

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

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:

no

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 30 µL of the test article was applied topically to the tissue 3. Post-exposure treatmentAfter 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): 30 µL
- 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: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): neat

POSITIVE 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.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1

Value:

2.1

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The MTT data show the assay quality controls were met.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study. The mean of OD for test chemical was determined to be 0.044.The standard deviation of viabilities for test chemical were calculated to be 0.14.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 2.1%. Thus, test chemical was considered to be irritating to the human skin.

Executive summary:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. 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. Tissues were exposed to the 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 show the assay quality controls were met and passed the acceptance of criteria. The mean of OD  for test chemical was determined to be 0.044.

The standard deviation of viabilities for test chemical were calculated to be 0.14.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 2.1%.Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritating to human skin and can thus be classified as “ Irritating to SKIN in Category 2'' as per CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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 inthe 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 Identification
All 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 applicability
EpiOcularTM Eye Irritation (OCL) by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakien.
The 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 Laboratories, Bratislava, Slovakien.
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 In Vitro Life Science Laboratories 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 μL of liquid 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, 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 liquid 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 30 minutes for liquid 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 liquid test articles and controls. 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 liquid test 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 assessment
MTT Pre-test
The 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 Test
Approximately 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:
After the recovery period, the MTT assay was performed on run 1 tissues by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 3 hours of 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 for liquid exposed tissues was overnight incubation. 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 was 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 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: 50 µL of liquid test article 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.
3. Post exposure treatment:
After the exposure, the tissues were rinsed 20 times with sterile 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 liquid 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 2 hours at approximately 37 degC, 5% CO2 in a humidified incubator.
- Doses of test chemical and control substances used
Test Article:
50 µL of liquid test article 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.
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 30 minute exposure time.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Tissues were exposed for approximately 30 minutes for liquid 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 formazan
The 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 model
Calculations and Statistical Methods
MTT Assay
Blanks:
· 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 Compounds
True 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 Compounds
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 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 VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean 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:

35

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The MTT data show the assay quality controls were met.

The SD for the positive control (PC) is also high (D>20), however, the high value probably depends on a missspread of the PC liquid in this tissue (tissue 1 out of 2) during exposure and should thus be regarded as OK. If one of the values are deleted (1,5376 or 1, 4424), the SD error bar decreases under the cut-off-limit.

Results of the eye irritation in-vitro assay

Code N°	Tissue	Raw data		Blank corrected data		mean of OD	% of viability
	n	Aliq. 1	Aliq. 2	Aliq. 1	Aliq. 2
NC	1	2.5312	2.0912	2.496	2.056	2.276	104.0
	2	2.1457	2.1237	2.111	2.089	2.100	96.0
PC	1	1.5376	1.4424	1.502	1.407	1.455	66.5
	2	0.8738	0.8	0.839	0.765	0.802	36.6

111-13-7	1	0.6348	0.6092	0.600	0.574	0.587	26.8
	2	1.0045	0.9559	0.969	0.921	0.945	43.2

	mean	Dif.	mean of	Dif.	Dif./2	Classification
	of OD	of OD	viabilities [%]	of viabilities
NC	2.188	0.177	100.0	8.07	4.03	NI	qualified
PC	1.128	0.653	51.6	29.85	14.93	I	D>20

111-13-7

0.766

0.358

35.0

16.37

8.19

I

qualified

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study.The mean of OD for test chemical was determined to be 0.766.The mean % tissue viability of test chemical was determined to be 35.0%. Thus, test chemical was considered to be 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, passing the acceptance criteria.

The mean of OD for test chemical was determined to be 0.766.The mean % tissue viability of test chemical was determined to be 35.0%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the human eyes and and can thus be classified as “ Irritating to eyes in Category 2’’ as per CLP Regulation.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data from reliable databases

Qualifier:

equivalent or similar to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

To assess the ocular irritation potential of the test chemical

GLP compliance:

not specified

Species:

rat

Strain:

not specified

Details on test animals or tissues and environmental conditions:

no data available

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

8.9 mg/l (1673ppm)

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

6 hours

Duration of post- treatment incubation (in vitro):

no data available

Number of animals or in vitro replicates:

no data available

Details on study design:

no data

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 6 hours

Reversibility:

not specified

Remarks on result:

probability of mild irritation

Irritant / corrosive response data:

Mild irritation observed

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The test chemical was mildly irritating to rat eyes.

Executive summary:

Rats were exposed to calculated vapor concentrations of the test chemical, 8.9 mg/L (1673 ppm) for 6 hr exhibited signs of mild eye irritation.

Hence, the test chemical was mildly irritating to rat eyes

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

In various studies, the potential of the test chemical to cause dermal irritation or corrosion to living organisms has been reviewed. These include invivo experimental studies performed on guinea pigs, rats, rabbits and humans as well in vitro experimental study for the test chemical. The results are summarized as follows:

Preliminary irritation tests were performed for the test chemical in guinea pigs to determine the concentration suitable for the sensitization study[injection challenge concentration (ICC) and application challenge concentration(ACC)].                                      

In the preliminary irritation study, 4 Albino Hartley guinea pigs of the same sex were injected intradermally on the shaved flanks with 0.1 ml aliquots of a range of concentrations of tests material in a suitable solvent. The reactions were examined for size (two largest diameters), erythema and oedema 24 h later and the concentration giving slight but perceptible irritationwith no oedema was selected as the injection challenge concentration (ICC).0.1 ml of the test material in arange of concentrations in a suitable solvent were applied in small circular areas to the shaved flanks of 4 guinea pigs of the same sex.The reactions were examined for erythema 24 hours later and the highest concentration which caused no irritation was selected as the application challenge concentration (ACC).

No signs of erythema and edema were observed at 1.0% ICC and 10% ACC concentrations. Hence, the test chemical was considered to be non-irritating on guinea pigs in the preliminary irritation tests performed for the sensitization study.

This is supported by a study designed and conducted to determine the dermal reaction profile of the test chemical in Sprague Dawley rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures.

5 female young adult, non-pregnant rats were used for the study. The animals were kept in their cages for at least 5 days prior to administration for acclimatization to the laboratory condition and after acclimatization period, animals were randomly selected.

Approximately 24 hours before application, the hair of each rat was closely clipped from the trunk (dorsal surface and sides from scapular to pelvic area) with an electric clipper, so as to expose at least 10% of the body surface area. The test item was applied directly onto the exposed skin of the animal, taking care to spread the test item evenly over the entire area of approximately 10% of the total body surface area or as much of the area as can reasonably be covered. The test item was held in contact with the skin using a porous gauze dressing and non irritating tape around the animal to cover the exposure site for first 24 hours exposure period. Elizabethan collar was placed on each animal for first 24 hours after application of the test item. These collars prevent ingestion of test item. Following 24 hours of exposure, the wrapping was removed and the test site wiped free of excess test item. Distilled water was used to remove residual test item.

A single dose of 200 mg/kg, 1000 mg/kg and 2000 mg/kg body weight of the test item were applied to 1 female animal respectively in the dose range finding study. Since no signs of irritation were observed at the maximum dose of 2000 mg/kg in the test animals, 2 additional animals were tested in the main study with 2000 mg/kg. The animals were applied with a dose of 2000 mg/kg body weight in sequential manner at 48 hours intervals. Dermal reaction was observed daily for study period of 14 days.

The overall irritation score of the substance was determined to be 0 and no erythema and edema (skin irritation) were found at the end of 14 days observation period after patch removal.

Hence, it was concluded that the test chemical was Non-Irritating to the skin of female Sprague Dawley rats under the experimental conditions tested and classified as “Category- Not Classified” as per CLP Classification.

In an in vitro study performed according to OECD 439 test guideline the dermal irritation potential of test article was determined. 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. Tissues were exposed to the 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 show the assay quality controls were met and passed the acceptance of criteria. The mean of OD  for test chemical was determined to be 0.044.

The standard deviation of viabilities for test chemical were calculated to be 0.14.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 2.1%.Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritating to human skin and can thus be classified as “Irritating to SKIN in Category 2'' as per CLP Regulation.

The in vitro results are supported by an in vivo skin irritation performed on rabbits. The test chemical was applied full strength to the intact and abraded skin of rabbits under occlusion for 24 hours. The test chemical was slightly irritating to rabbit skin after 24 hours exposure.

Skin irritation effects were also estimated by four different models i.e, Battery, Leadscope, SciQSAR and CASE Ultra used within Danish QSAR database for the test chemical. Based on estimation, No severe skin irritation effects were known when the test chemical was exposed to rabbit skin.

The above estimated result is further supported by another skin irritation study performed on humans to assess the irritation potential of the test chemical. The test chemical was tested 5% in petrolatum in human volunteers in 48 hours closed patch test. The test chemical was not irritating to human skin after 48 hours exposure.

Even though results of some in vivo and in-vitro studies claim that the test chemical causes mild to moderate irritation to skin, but estimated and experimental studies indicate a strong possibility that the test chemical can be not irritating to skin.Also, when tested at the maximum dose of 2000mg/kg in rats; no signs of irritation were observed till end of observation than the concentrations tested on rabbits and guinea pigs.

Taking all these factors into consideration, the test chemical can be considered to be not irritating to skin.  Hence, the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Eye irritation

In several studies, the potential of the test chemical to cause ocular irritation was studied to a greater or lesser extent. The studies include in vivo experimental data on rabbits, rats, guinea pigs as well in vitro data for the test chemical. The results are mentioned as follows:

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, passing the acceptance criteria.

The mean of OD for test chemical was determined to be 0.766.The mean % tissue viability of test chemical was determined to be 35.0%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the human eyes and and can thus be classified as “ Irritating to eyes in Category 2’’ as per CLP Regulation.

The in vitro result is supported by a study performed in rats to assess the eye irritation potential. Rats were exposed to calculated vapor concentrations of the test chemical, 8.9 mg/L (1673 ppm) for 6 hr exhibited signs of mild eye irritation.

Hence, the test chemical was mildly irritating to rat eyes.

The above results are supported by another irritation study performed on rabbit eyes.

Undiluted test chemical was instilled into rabbit eyes and effects were observed (duration and dose not mentioned).

Undiluted test chemical was slightly irritating to rabbit eyes.

These results are further supported by accidental exposure of the test chemical in an acute inhalation study, guinea pigs were exposed to an essentially saturated atmosphere (1300 ppm) of the test chemical. Symptoms of eye irritation developed immediately after exposure. Hence, the test chemical can be considered as irritating to eyes.

The in vitro and in vivo results are in mutual agreement with each other indicating a very strong possibility of the test chemical to cause irritation to eyes. Hence, the test chemical can be considered to be irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Category 2”.

Justification for classification or non-classification

Even though results of some in vivo and in-vitro studies claim that the test chemical causes mild to moderate irritation to skin, but estimated and experimental studies indicate a strong possibility that the test chemical can be not irritating to skin.Also, when tested at the maximum dose of 2000mg/kg in rats; no signs of irritation were observed till end of observation than the concentrations tested on rabbits and guinea pigs.Taking all these factors into consideration, the test chemical can be considered to be not irritating to skin.  Hence, the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

The in vitro and in vivo results are in mutual agreement with each other indicating a very strong possibility of the test chemical to cause irritation to eyes. Hence, the test chemical can be considered to be irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Category 2”.