1-dodecylpyridinium chloride

Endpoint summary

Description of key information

Skin Irritation

A case study of 28-year old male has been summarized for evaluating the irritation and dermal sensitization potential of the test chemical. Of 23 controls tested with 0.1% aq. test chemical , 16 were negative, 6 had a ? +, and 1 a+ reaction.

A positive reaction was observed to 0.1% aq. test chemical (48 h+, 72 h + +). Therefore, the test chemical was considered to be irritating to the human skin.

An in vitro study was performed according to OECD 439 Guidelines to determine the dermal irritation potential of test article. The mean of OD for test chemical was determined to be 0.068.

Eye Irritation

The standard deviation of viabilities for test chemical were calculated to be 0.60.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 3.2%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritating to human skin.

An in vivo study was performed according to Draize test on guinea pigs to determine the ocular irritation potential. % w/v solution of the test chemical causes irritation to guinea pig eyes. Hence, the test chemical can be considered to be irritating to 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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: Patch tests with the European standard series (ICDRG)

Principles of method if other than guideline:

A patch was conducted on a 28-year-old male for test chemical according to the European standard series (ICDRG) after he had recurrent itchy dermatitis on the left side of the chest and adjacent area of the arm.

GLP compliance:

not specified

Species:

other: human

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

- Age at study initiation: 28-year-old

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

other: Aqueous solution

Controls:

yes

Amount / concentration applied:

0.1%

Duration of treatment / exposure:

72 hr

Observation period:

48 and 72 hr

Number of animals:

1 (male)

Details on study design:

No data available

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 48/ 72 hr

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Of 23 controls tested with 0.1% aq. test chemical , 16 were negative, 6 had a ? +, and 1 a+ reaction.

Skin irritation scores

Time (hours)	Rating
48	+
72	++

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

A positive reaction was observed to 0.1% aq. test chemical (48 h+, 72 h + +). Therefore, the test chemical was considered to be irritating to the human skin.

Executive summary:

A case study of 28-year old male has been summarized to for test chemical. In this study, a patch test was conducted on a 28-year-old male according to the European standard series (ICDRG) after he had recurrent itchy dermatitis on the left side of the chest and adjacent area of the arm at concentration of 0.1% aq for 48 and 72-hrs.

Of 23 controls tested with 0.1% aq. test chemical , 16 were negative, 6 had a ? +, and 1 a+ reaction.

A positive reaction was observed to 0.1% aq. test chemical (48 h+, 72 h + +). Therefore, the test chemical was considered to be irritating to the human skin.

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 (MatTek Corp., Ashland, MA).

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ 3-dimensional human tissues used in this study

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, Ashland, MA) 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 June 28, 2017 (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 for MTT auto-reduction and coloring was not performed for this study but was based on the results obtained from another study (CYP1690_R1b).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 2 hours, 57 minute and 25 second 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 2 hours 04 minutes and 11 seconds with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer 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 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. Tissues were exposed to controls and the test articles 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, 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 ArticleFor solid test article, the tissues were moistened with 25 μL of ultrapure water to improve contact of the tissue surface with the test article. Approximately 25 mg of each test article was evenly applied to the apical surface of each tissue (n=3). All the tissues were placed into the ~37°C incubator with 5% CO2. The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature. 3.Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 20 to 25 times with 1 mL of 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 either 25 hours, 38 minutes and 23 seconds or for 24 hours, 10 minutes and 09 seconds (as there were numerous tissues, they had to be broken down into 2 sets to complete dosing in a timely manner). 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 17 hours, 03 minutes and 34 seconds prior to performing the MTT assay, 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: 6/08/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Twice
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL MTT medium (1.0 mg/mL)
- Incubation time: After 2 hours, 57 minute and 25 second MTT incubation
- Spectrophotometer: Synergy H4 spectrophotometer
- Wavelength: 570 nm
- Filter: No data- Filter bandwidth: No data
- Linear OD range of spectrophotometer: No data

NUMBER OF REPLICATE TISSUES: 3

CALCULATIONS and STATISTICAL METHODS: All 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 (undiluted)
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 will be used per test compound and control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1

Value:

3.2

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, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was SD>18 passing the acceptance criteria.

Code N°	Tissue	Raw data		Blank corrected data		mean	% of viability
	n	Aliq. 1	Aliq. 2	Aliq. 1	Aliq. 2	of aliquotes
NC	1	2.2655	2.2455	2.229	2.209	2.219	104.9
	2	2.0693	2.0371	2.033	2.001	2.017	95.4
	3	2.1138	2.1778	2.078	2.142	2.110	99.7
PC	1	0.0737	0.0743	0.037	0.038	0.038	1.8
	2	0.0784	0.0804	0.042	0.044	0.043	2.0
	3	0.0781	0.0805	0.042	0.044	0.043	2.0
C1	1	0.1012	0.1026	0.065	0.066	0.066	3.1
	2	0.1175	0.1187	0.081	0.082	0.082	3.9
	3	0.093	0.0934	0.057	0.057	0.057	2.7

	mean	SD	mean of	SD	CV %
	of OD	of OD	viabilities [%]	of viabilities	[%]
NC	2.115	0.101	100.0	4.79	4.79
PC	0.041	0.003	2.0	0.15	7.48
C1	0.068	0.013	3.2	0.60	18.54

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 0.068.The standard deviation of viabilities for test chemical were calculated to be 0.60.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 3.2%. 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 0.068. The standard deviation of viabilities for test chemical were calculated to be 0.60.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 3.2%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritating to human skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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 in the (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 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 chemical
- 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 6 hrs ± 15 min for solid test articles, 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 and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals 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 ~25 minutes for solid test articles and controls.Following the washing and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals 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:
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 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.2 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 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:
When a solid was tested, 6 hours 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.
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 6 hours for solid test articles and controls, at approximately37°C, 5% CO2 in a humidified incubator.
Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling 18 hours for solid 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 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 meanthe 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 2 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:

1.3

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean of OD :0.025; Irritant

Other effects / acceptance of results:

The MTT data show the assay quality controls were met.

Tissue 2 (well C1 and D1) of the negative control did not look 100% okay after the treatment, which may be due to some kind of damage of the tissue. This gives us a SD of D>20, however, the negative control can still be used since the SD error bar is above the cut-off-limit of the assay (i.e. 60%).

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 0.025 .Tissue 2 (well C1 and D1) of the negative control did not look 100% okay after the treatment, which may be due to some kind of damage of the tissue. This gives us a SD of D>20, however, the negative control can still be used since the SD error bar is above the cut-off-limit of the assay (i.e. 60%). The mean % tissue viability of test chemical was determined to be 1.3%. 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 0.025.Tissue 2 (well C1 and D1) of the negative control did not look 100% okay after the treatment, which may be due to some kind of damage of the tissue. This gives us a SD of D>20, however, the negative control can still be used since the SD error bar is above the cut-off-limit of the assay (i.e. 60%).The mean % tissue viability of test chemical was determined to be 1.3%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the human eyes.

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 is from handbooks

Qualifier:

according to guideline

Guideline:

other: Draize test

Principles of method if other than guideline:

Draize test was performed on guinea pigs to determine the ocular irritation potential of the test chemicals

GLP compliance:

not specified

Species:

guinea pig

Strain:

not specified

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

5% w/v solution of the test chemical

Duration of treatment / exposure:

no data available

Observation period (in vivo):

no data available

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 available

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: no data available

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

5% w/v solution of the test chemical cause irritation to guinea pig eyes.

Other effects:

no data available

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

5% w/v solution of the test chemical causes irritation to guinea pig eyes. Hence, the test chemical can be considered to be irritating to eyes.

Executive summary:

Draize test was performed on guinea pigs to determine the ocular irritation potential of the test chemical. 5% w/v solution of the test chemical were instilled into the eyes of guinea pigs and observed for signs of irritation (duration, number of animals and observation period not mentioned).

5% w/v solution of the test chemical causes irritation to guinea pig eyes. Hence, the test chemical can be considered to be irritating to eyes.       

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

Various studies have been summarized to determine the level of dermal irritation caused by the test chemical in living organisms. These include in vivo as well as in vitro experimental results for the test chemicals. The results are summarized below:

A case study of 28-year old male has been summarized to for test chemical. In this study, a patch test was conducted on a 28-year-old male according to the European standard series (ICDRG) after he had recurrent itchy dermatitis on the left side of the chest and adjacent area of the arm at concentration of 0.1% aq for 48 and 72-hrs.

Of 23 controls tested with 0.1% aq. test chemical , 16 were negative, 6 had a ? +, and 1 a+ reaction.

A positive reaction was observed to 0.1% aq. test chemical (48 h+, 72 h + +). Therefore, the test chemical was considered to be irritating to the human skin.

This is supported by an in vitro study performed according to OECD 439 Guidelines to determine the dermal irritation potential of test article. 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 0.068. The standard deviation of viabilities for test chemical were calculated to be 0.60.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 3.2%. Hence, under the current experimental test conditions it was concluded that test chemical was considered to be irritating to human skin.

The in vitro result is supported by an in vivo 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.

In the dose range finding study a single dose of 200 mg/kg body weight of the test item was administered to 1 female animal. No death or clinical signs of toxicity was observed during first 48 hours, hence, additional 1 female animal was administered with the dose of 1000 mg/kg body weight. Administration of 1000 mg/kg body weight revealed no clinical signs of toxicity or mortality during first 48 hours, hence, additional 1 female animal was administered at the dose of 2000 mg/kg body weight.

Administration of 2000 mg/kg body weight resulted in mortality on day 1.

As the dose range finding study revealed mortality at the maximum dose of  2000 mg/kg, the main study was initiated with two additional animals at a dose of 1000 mg/kg body weight. The animals were administered with a dose of 1000 mg/kg body weight in sequential manner at 48 hours intervals.

Administration of 1000 mg/kg body weight revealed minimal to moderate erythema at the site of application from day 1 to day 14 and exhibited normal body weight gain, no clinical signs of toxicity or mortality during the study period of 14 days from dose range finding study and main study.Gross pathological examination revealed moderate to severe erythema on site of application in animals from 1000 mg/kg dose group.

The mean erythema score on day 14 of observation was 3 in the dose range finding study and main study for the 1000 mg/kg dose group rats.

Based on the above findings the test chemical can be considered to be irritating to the skin of female Sprague Dawley rats. It can be further classified under the category "Category 2" as per CLP Regulation.

The in vitro and in vivo results are in mutual agreement with each other indicating very strong possibility that the test chemical can be irritating to skin. Comparing the above annotations with the criteria of the CLP Regulations, the test chemical can be classified under the category “Category 2”.

Eye Irritation

Various studies have been summarized to evaluate the ocular irritation potential caused by the test chemical in living organisms. These include in vivo as well as in vitro experimental results for the test chemicals. The results are 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, passing the acceptance criteria.

The mean of OD for test chemical was determined to be 0.025.Tissue 2 (well C1 and D1) of the negative control did not look 100% okay after the treatment, which may be due to some kind of damage of the tissue. This gives us a SD of D>20, however, the negative control can still be used since the SD error bar is above the cut-off-limit of the assay (i.e. 60%).The mean % tissue viability of test chemical was determined to be 1.3%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the human eyes.

This is supported by another in vitro study performed using the pollen tube growth test (PTG test) to assess the eye irritating potential of test chemical based on a quantitative photometric measurement of the Pollen tube mass produced in vitro.

Tobacco plants of the species, Nicotiana sylvestris were cultivated from seeds in a greenhouse and set out to flower in the Botanical Garden of the University of Hamburg. The anthers of flowers that were not yet fully open were Harvested and dried for two days at 20°Cin a Petri dish. The pollen was obtained by shaking the dried anthers and then freezing them in Eppendorf tubes (1.5 ml) at minus 18°C.

 Frozen pollen grains were resuspended in culture medium at a concentration of 1 mg/ml. Test substances were serially diluted by a factor of 2. Dilutions were transferred to the pollen suspension and incubated for 18 h at 25°C. Pollen tube cultures were washed twice with double distilled water and then stained with Alcian blue (0.05%) for 30 min. The dyed pollen tube walls were washed and finally destained with citric acid (40%). The optical density was measured photometrically at 607 nm. Each concentration was tested in quadruplicates. TheED5o (50% effective dose) was calculated.

 The ED50 of the test substance was observed to be 5.05 ppm. The results of the PTG test are compared with their in vivo classification according to the Draize eye irritation assay. This comparison clearly reveals that the compound tested that produced strong irritation in the rabbit eye are also highly toxic in the PTG test. Thus the chemical was considered as irritating to eyes.

These in vitro results are supported by the in vitro Cell Detachment Assay conducted for test chemical by using BHK-21/C13: commercially available fibroblastic cell line derived from Baby Hamster Kidney (BHK) to assess its eye irritation potential.

 

Cells were seeded in culture plates and incubated for 24 h prior to exposure to ensure the proper attachment of the cells to the substrate. Several concentrations of the test substance were applied.The total number of plated cells before treatment and the number of adherent cells after exposure was determined by trypsinating the cell layer and counting the cells with a coulter counter. The loss of cells after treatment was calculated, and the endpoint value (CD-25) was determined from a dose-response curve.

 The CD-25 of the test chemical was observed to be 9.0 μg/ml. The results of the test are compared with In vivo Draize eye irritation test performed on guinea pigs. According to the Draize eye irritation assay, the chemical was considered as severely irritating using a 5% (w/v) solution. Thus on the basis of overall observations, the test chemical can be considered as irritating to the eye.

These in vitro results are supported by an in vivo study performed according to Draize test on guinea pigs to determine the ocular irritation potential of the test chemical. 5% w/v solution of the test chemical were instilled into the eyes of guinea pigs and observed for signs of irritation (duration, number of animals and observation period not mentioned). 5% w/v solution of the test chemical causes irritation to guinea pig eyes. Hence, the test chemical can be considered to be irritating to eyes.       

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

Justification for classification or non-classification

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