1-hydroxybenzotriazole

Endpoint summary

Description of key information

Skin Irritation:

Slight erythema was noted in 1 of 6 New Zealand White rabbits and no edema was observed. Based on the overall observed effects, the test chemical was considered to be not irritating to the skin of rabbits.

Eye Irritation:

Both treated eyes initially showed mild conjunctival redness and chemosis with one instance of blood-tinged discharge; these effects resolved by 2 days in the rinsed eye and by 7 days in the unrinsed eye. Mild generalized opacity of the cornea was observed on the day of treatment in the unrinsed eye.

Hence, under the experimental test conditions it was concluded that the test chemical was considered to be mildly irritating to the eyes of rabbits and being classified as “Irritating to eyes in Category 2” as per CLP Regulation.

The mean % tissue viability of test substance was determined to be 2.2%. Hence, under the experimental test conditions it was concluded that test substance was considered to be irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

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: as mentioned below

Principles of method if other than guideline:

The skin irritation study was conducted on male New Zealand White rabbits to assess the irritation potential of the test compound.

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

0.5g (500 mg)

Duration of treatment / exposure:

24 hrs

Observation period:

At 24.5 and 48 hours

Number of animals:

6 male rabbits

Details on study design:

TEST SITE
- Area of exposure: Skin of the back
- % coverage: no data
- Type of wrap if used: Semi-occlusive wrap

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No data available
- Time after start of exposure: No data available

SCORING SYSTEM: Erythema and edema (according to the method described by Draize)

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48 h

Score:

0

Reversibility:

not specified

Remarks on result:

probability of weak irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48 h

Score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Slight erythema was noted in 1 of 6 New Zealand White rabbits.

Table for irritation response

Response	Erythema		Edema
	24 hours	48 hours	24 hours	48 hours
No response	6/6	5/6	6/6	6/6
Slight	0/6	1/6	0/6	0/6

Interpretation of results:

other: not irritating

Conclusions:

Slight erythema was noted in 1 of 6 New Zealand White rabbits and no edema was observed. Based on the overall observed effects, the test chemical was considered to be not irritating to the skin of rabbits.

Executive summary:

The skin irritation study was conducted on 6 male New Zealand White rabbits to assess the irritation potential of the test chemical. 0.5 g of the undiluted test chemical was applied to the clipped, intact skin of the back of rabbits under a semi-occlusive wrap. Exposure was terminated 24 hrs after contact and observations were made 24.5 and 48 hrs after start of the exposure. Slight erythema was noted in 1 of 6 New Zealand White rabbits and no edema was observed. Based on the overall observed effects, the test chemical was considered to be not irritating to the skin of rabbits.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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:

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: as mentioned below

Principles of method if other than guideline:

Primary eye irritation study was performed on male New Zealand White rabbits to assess the irritation potential of the test chemical

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

No data available

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.01g (10 mg) undiluted test sample.

Duration of treatment / exposure:

Duration of exposure: 20 secs (rinsed eyes)
Duration of treatment: 7 days (unrinsed eyes)

Observation period (in vivo):

2 and 4 hours, and at 1, 2, 3, and 7 days

Number of animals or in vitro replicates:

2 male rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, Approximately 20 seconds after the test material was administered, both eyes of 1 rabbit were rinsed for 1 minute with water.
- Time after start of exposure: 20 seconds after the test material was administered.

SCORING SYSTEM: According to the method of Draize

TOOL USED TO ASSESS SCORE: A biomicroscope and fluorescein stain were used at examinations beginning the day after treatment.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

7 d

Reversibility:

fully reversible within: 7 days

Remarks:

in the unrinsed eyes

Remarks on result:

probability of mild irritation

Irritant / corrosive response data:

Both treated eyes initially showed mild conjunctival redness and chemosis with one instance of blood-tinged discharge; these effects resolved by 2 days in the rinsed eye and by 7 days in the unrinsed eye. Mild generalized opacity of the cornea was observed on the day of treatment in the unrinsed eye.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Both treated eyes initially showed mild conjunctival redness and chemosis with one instance of blood-tinged discharge; these effects resolved by 2 days in the rinsed eye and by 7 days in the unrinsed eye. Mild generalized opacity of the cornea was observed on the day of treatment in the unrinsed eye.
Hence, under the experimental test conditions it was concluded that the test chemical was considered to be mildly irritating to the eyes of rabbits and being classified as “Irritating to eyes in Category 2” as per CLP Regulation.

Executive summary:

Primary eye irritation study was conducted on 2 male New Zealand White rabbits to assess the irritation potential of the test chemical. 0.01 g (10mg) of the undiluted test chemical was instilled into the lower conjunctival sac of the right eyes of 2 male New Zealand White rabbits. The left eye served as control. Approximately after 20 seconds of test material administration, both eyes of 1 rabbit were rinsed for 1 minute with water; the eyes of the other rabbit were not rinsed. Irritation was scored at 2 and 4 hours, and at 1, 2, 3, and 7 days. Biomicroscope and fluorescein stains were used at examinations beginning the day after treatment.

Both treated eyes initially showed mild conjunctival redness and chemosis with one instance of blood-tinged discharge; these effects resolved by 2 days in the rinsed eye and by 7 days in the unrinsed eye. Mild generalized opacity of the cornea was observed on the day of treatment in the unrinsed eye.

Hence, under the experimental test conditions it was concluded that the test chemical was considered to be mildly irritating to the eyes of rabbits and being classified as “Irritating to eyes in Category 2” as per CLP Regulation.

Reference 2

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:

2.2

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.

Results of EpiOcular EIT 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	1.9415	1.9422	1.906	1.907	1.906	101.2
	2	1.8795	1.9102	1.844	1.875	1.859	98.8
PC	1	0.6014	0.6142	0.566	0.579	0.572	30.4
	2	0.7066	0.7091	0.671	0.673	0.672	35.7

2592-95-2	1	0.077	0.0778	0.041	0.042	0.042	2.2
	2	0.0765	0.0775	0.041	0.042	0.041	2.2

	mean	Dif.	mean of	Dif.	Dif./2	Classification
	of OD	of OD	viabilities [%]	of viabilities
NC	1.883	0.047	100.0	2.50	1.25	NI	qualified
PC	0.622	0.100	33.0	5.31	2.66	I	qualified

2592-95-2

0.042

0.000

2.2

0.02

0.01

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 % tissue viability of test substance was determined to be 2.2%. Thus, the 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. issues were exposed to solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours recovery after the post-soak.

The MTT data show the assay quality controls were met, passing the acceptance criteria.

The mean % tissue viability of test substance was determined to be 2.2%. Hence, under the experimental test conditions it was concluded that test substance was considered to be irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation

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/corrosion caused by the test chemical in living organisms. These results include in vivo experimental studies performed on rats and rabbits for the test chemical.

The skin irritation study was conducted on 6 male New Zealand White rabbits to assess the irritation potential of the test chemical. 0.5 g of the undiluted test chemical was applied to the clipped, intact skin of the back of rabbits under a semi-occlusive wrap. Exposure was terminated 24 hrs after contact and observations were made 24.5 and 48 hrs after start of the exposure. Slight erythema was noted in 1 of 6 New Zealand White rabbits and no edema was observed. Based on the overall observed effects, the test chemical was considered to be not irritating to the skin of rabbits.

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. Ten rats (5 male and 5 female) were used for conducting dermal irritation /corrosion 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 to shorn skin of 5 male and 5 female animals at 2000 mg/ kg body weight to 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. 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.

Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Also, the erythema and edema score of rats was calculated as 0. Administration of the test item did not result in any signs of toxicity and mortality during the 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 Sprague Dawley rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.

Based on the results from the available in vivo studies, the test chemical has strong possibility of not causing any dermal irritation or corrosion to the skin. Hence, the test chemical can be considered to be not irritating to skin. Comparing the annotations with the criteria of CLP regulation, the test chemical can be classified under the Category “Not Classified”.

Eye irritation

Various studies have been summarized to determine the extent of ocular damage caused by the test chemical in living organisms. These results include in vivo and in vitro experimental studies performed for the test chemical.

Primary eye irritation study was conducted on 2 male New Zealand White rabbits to assess the irritation potential of the test chemical. 0.01 g (10mg) of the undiluted test chemical was instilled into the lower conjunctival sac of the right eyes of 2 male New Zealand White rabbits. The left eye served as control. Approximately after 20 seconds of test material administration, both eyes of 1 rabbit were rinsed for 1 minute with water; the eyes of the other rabbit were not rinsed. Irritation was scored at 2 and 4 hours, and at 1, 2, 3, and 7 days. Biomicroscope and fluorescein stains were used at examinations beginning the day after treatment.

Both treated eyes initially showed mild conjunctival redness and chemosis with one instance of blood-tinged discharge; these effects resolved by 2 days in the rinsed eye and by 7 days in the unrinsed eye. Mild generalized opacity of the cornea was observed on the day of treatment in the unrinsed eye.

Hence, under the experimental test conditions it was concluded that the test chemical was considered to be mildly irritating to the eyes of rabbits and being classified as “Irritating to eyes in Category 2” as per CLP Regulation.

This in vivo result is supported by an in vitro study performed according to the OECD 492 test guideline for this study to determine the irritation potential of the test chemical. 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. issues were exposed to solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours recovery after the post-soak.

The MTT data show the assay quality controls were met, passing the acceptance criteria.

The mean % tissue viability of test substance was determined to be 2.2%. Hence, under the experimental test conditions it was concluded that test substance was considered to be irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation.

The in vivo and in vitro experimental results are in agreement with each other, indicating a very strong possibility that the test chemical can indeed 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

Based on the results from the available in vivo studies, the test chemical has strong possibility of not causing any dermal irritation or corrosion to the skin. Hence, the test chemical can be considered to be not irritating to skin. Comparing the annotations with the criteria of CLP regulation, the test chemical can be classified under the Category “Not Classified”.

 

The in vivo and in vitro experimental results are in agreement with each other, indicating a very strong possibility that the test chemical can indeed 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”.