N-tert-butylacrylamide

Endpoint summary

Description of key information

Skin Irritation:

The dermal irritation potential of target chemical was assessed in various in- vitro and in-vivo experimental studies.Based on the available studies,it can be concluded that the test chemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified''.

Eye Irritation:

The ocular irritation potential of target chemical was assessedin various in- vitro and in-vivo experimental studies which were conducted for test chemical.Based on the available key data and supporting study,it can be concluded that the testchemical is unable to cause eye irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified ".

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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Principles of method if other than guideline:

The objective of the study was to assess the irritant and/or corrosive effects of test chemical after dermal application on the intact skin in Rabbits.

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Procured from RABBI ROOF, Hyderabad
- Age at study initiation:3.5 to 4.5 Months (Approximately)
- Weight (Prior to Treatment):Minimum: 2.049 kg and Maximum: 2.336 kg
- Health Status : Healthy young adults rabbits were used for the study.
- Housing:The animals were housed individually in stainless steel cages.
- Room Sanitation:The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
- Cages and water bottle:All the cages and water bottles were changed minimum twice a week.
- Diet (e.g. ad libitum):All animals were provided conventional laboratory rabbit diet (Nutrivet Life Sciences, Pune) ad libitum.
- Water (e.g. ad libitum):Aqua guard filtered tap water was provided ad libitum.
- Acclimation period:Rabbits were acclimatised to the test conditions for a period of 5 days (Animal No.-1) and 8 days (Animal No.-2 and 3) prior to the application of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 20.00 °C and Maximum: 22.50 °C
- Humidity (%):Minimum: 47.20 % and Maximum: 68.40 %
- Air changes (per hr):More than 12 changes per hour
- Photoperiod (hrs dark / hrs light):12:12

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

other: distilled water

Controls:

yes, concurrent vehicle

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):0.5 gm

VEHICLE (distilled water)
- Amount(s) applied (volume or weight with unit):0.5 ml of distilled water
- Concentration (if solution): N/A
- Lot/batch no. (if required):N/A
- Purity:N/A

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3 male rabbits

Details on study design:

TEST SITE
- Area of exposure:The dorsal lumbar region at contralateral sites.
- % coverage:Approximately 6 X 6 cm.
- Type of wrap if used:A porous gauze dressing and non-irritating tape (Micropore 3”).

REMOVAL OF TEST SUBSTANCE
- Washing (if done):The residual test item was removed by using cotton soaked in distilled water.
- Time after start of exposure:4 hour

SCORING SYSTEM:Draize Method

Irritation parameter:

erythema score

Basis:

animal: #1 ,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal: #1 ,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The patch was removed after 4 hours and rabbits were observed for erythema and oedema at 1, 24, 48 and 72 hours after patch removal, evaluated and graded as per draize method.

In Animals No.1, 2 and 3 after post patch removal, revealed no erythema and oedema at 24 and 48 and 72 hour during the observation period.

The individual mean score at 24, 48 and 72 hours for Animal Nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively.

Other effects:

Clinical Observation
No systemic toxicity was observed at treated rabbits during the experimental period.

Mortality
No mortality was observed during the observation period.

Body weight
Body weight of Animal No. 1 was marginally decreased and in Animal No. 2 and 3 it was increase as compared with pre-treatment body weight.

Table 1

Skin Reaction

In Treated area Dose:0.5 gm of test item                                                                                          Sex:Male

Animal No.	Test	Treated  area*	Erythema score				Oedema score
			1h	24h	48h	72h	1h	24h	48h	72h
1	Initial	Left	0	0	0	0	0	0	0	0
2	Confirmatory	Left	0	0	0	0	0	0	0	0
3		Left	0	0	0	0	0	0	0	0

 

In Control area                

 Dose:0.5 ml of distilled water                              Sex:Male

Animal No.	Test	Treated area*	Erythema score				Oedema score
			1h	24h	48h	72h	1h	24h	48h	72h
1	Initial	Right	0	0	0	0	0	0	0	0
2	Confirmatory	Right	0	0	0	0	0	0	0	0
3		Right	0	0	0	0	0	0	0	0

Key: h = Hour.

Erythema                                                                                                                                           Oedema

0 =No erythema                                                                                                                            0 =No oedema

 

Table 1 Continued…

Mean Individual Animal Score at 24, 48 and 72 hours

 

Animal Number                   Observations	1	2	3
Erythema	0.00	0.00	0.00
Oedema	0.00	0.00	0.00

 

Table 2

Individual Animal BodyWeight

Sex:Male

Animal No.	Body Weight (kg)
	Prior to Dosing	At termination
1	2.049	1.954
2	2.178	2.378
3	2.336	2.396

 

Table 3

Individual AnimalClinicalSigns

Sex:Male

Animal No.	Days (Post dosing Observation)
	0	1	2	3
1	1	1	1	1
2	1	1	1	1
3	1	1	1	1

Key: 1 = Normal.

Interpretation of results:

other: not irritating

Conclusions:

No erythema and oedema (skin irritation) were found at the end of 72 hour observation period after patch removal.The individual mean score at 24, 48 and 72 hours for Animal Nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively. 
Hence, under the experimental test conditions it was concluded that the test chemical was Non-Irritating to the skin of Male New Zealand White rabbits.

Executive summary:

Acute Dermal Irritation/corrosion Study of test chemical was performed as per OECD guideline No. 404 using three healthy young adult male New Zealand White rabbits.

Rabbits with good intact skin were selected for the study. The hairs of all the rabbits were clipped at contralateral sites, approximately 24 hours prior to treatment. A dose of 0.5 gm of test item moistened with 0.5 ml of distilled water was applied to the skin, over an area of approximately 6 x 6 cm clipped of hair on one side of rabbits. The other untreated side was kept as control area and0.5 ml of distilled water was applied at this site. At the end of 4 hours, the gauze patch was removed and test item application site was wiped with water without altering the integrity of the epidermis. 

 

Initially, the test item was applied to the clipped area of skin of one rabbit. The test site was covered with gauze patch. After 4 hours of exposure in Animal No. 1, there was no erythema and oedema observed at 1, 24 and 48 hours observation. Hence the confirmatory test was conducted on additional two rabbits (No. 2 and 3)to confirm the non irritant nature of the test item.

 

In Animals No. 2 and 3 after post patch removal, revealed no erythema and oedema at 1, 24, 48 and 72 hours observation.

The patch was removed after 4 hours and rabbits were observed for erythema and oedema at 1, 24, 48 and 72 hours after patch removal, evaluated and graded as per draize method.

 

The individual mean score at 24, 48 and 72 hours for Animal Nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively.  

 

Hence, under the experimental test condition it was concluded that the test chemical was Non-Irritating to the skin of Male New Zealand White rabbits and Classified as “Category- Not Classified” 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 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:

91.7

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Mean O.D.:1.849 ; Non-irritant

Other effects / acceptance of results:

The MTT data show the assay quality controls were met.

Interpretation of results:

other: not irritating

Conclusions:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance was determined to be 91.7 %. Thus, the test chemical was considered to be not irritating to the human eyes.

Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to solid test articles (50mg) and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours 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 % tissue viability of test substance was determined to be 91.7 %. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human eyes and can thus be classified as "Not Classified" as per CLP Regulation.

Reference 2

Endpoint:

eye irritation: in 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 reports

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Principles of method if other than guideline:

The objective of the study was to assess the irritant and/or corrosive effects of test chemical on eye, when exposed by the ocular route in rabbits.

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:Procured from RABBI ROOF, Hyderabad.
- Age at study initiation:4.5 to 5.5 Months (Approximately)
- Weight at study initiation:Minimum: 2.036 kg and Maximum: 2.408 kg (Prior to Treatment)
- Health Status :Healthy young adult, Females were nulliparous and non pregnant.
- Housing:The animals were housed individually in stainless steel cages.
- Room Sanitation:The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
- Cages and water bottle:All the cages and water bottles were changed minimum twice a week.
- Diet (e.g. ad libitum):All animals were provided conventional laboratory rabbit diet (Nutrivet Life Sciences, Pune) ad libitum.
- Water (e.g. ad libitum):Aqua guard filtered tap water was provided ad libitum.
- Acclimation period:Rabbits were acclimatised to the test conditions for a period of 6 days (Animal No.-1) and 9 days (Animal No. 2 and 3) prior to the application of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Minimum: 19.50 °C and Maximum: 22.20 °C
- Humidity (%)::Minimum: 41.20 % and Maximum: 59.20 %
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light):: 12:12

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):100 mg
- Concentration (if solution):N/A

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

Duration of treatment / exposure:

24 hrs

Observation period (in vivo):

All the animals were observed at 1, 24, 48 and 72 hours after instillation of test item.

Number of animals or in vitro replicates:

3 female rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done):The treated eye of rabbit was washed with normal saline.
- Time after start of exposure:24 hours

SCORING SYSTEM:Draize Method

TOOL USED TO ASSESS SCORE: Ophthalmoscope and fluorescein strips.

Irritation parameter:

cornea opacity score

Basis:

animal: #1,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Remarks:

For treated eye

Irritation parameter:

iris score

Basis:

animal: #1,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

not specified

Remarks on result:

no indication of irritation

Remarks:

For treated eye

Irritation parameter:

conjunctivae score

Basis:

animal: #1 and #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Remarks:

For treated eye

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Remarks:

treated group

Irritation parameter:

chemosis score

Basis:

animal: #1,#2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Remarks:

For treated eye

Irritant / corrosive response data:

The following were observed in treated rabbits.
Observation at 1 hour after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals. Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible in animal no. 1 and some blood vessels definitely hyperaemic (injected) animal no. 2 whereas in animal no. 3 blood vessels were normal; Chemosis: Some swelling above normal (includes nictitating membranes) in animal no. 1 whereas no swelling was observed in animal no. 2 and 3.

Observation at 24 hours after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals. Conjunctivae - Some blood vessels definitely hyperaemic (injected) was observed in animal no.3 and normal in Animal Numbers 1 and 2; Chemosis: No swelling was observed in all the 3 animals.

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 0 % damage in animal no. 1, 2 and 3.

Observation at 48 and 72 hours after instillation of test item revealed: Cornea- No ulceration or opacity in all the animals; Area of Opacity- Zero in all the animals; Iris: Normal in all the animals. Conjunctivae – Blood vessels were normal in all the animals; Chemosis: No swelling was observed in all the animals.
The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for Corneal opacity, iris, conjunctiva and chemosis were found 0.00, 0.00, 0.00, 0.00; 0.00, 0.00, 0.00, 0.00; and 0.00, 0.00, 0.33, 0.00, respectively.

Other effects:

Clinical Observation
No systemic toxicity was observed in treated rabbits during the experimental period.

Mortality
No mortality was observed during the observation period.

Body weight
All rabbits were weighed on test day 0 (prior to application) and at termination.

Table 1 : Individual Animal Eye Irritation Scores

 

In Treated area Dose:100 mg (0.1 gm) of test item                                          Sex:Female

Animal Numbers	1					2					3
Application Side	Right					Right					Right
Eye Reactions	At hour					At hour					At hour
	*	1	24	48	72	*	1	24	48	72	*	1	24	48	72
Corneal Opacity	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Area of Opacity	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Iris	0	2	0	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctiva	0	1	0	0	0	0	1	0	0	0	0	0	1	0	0
Chemosis	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Corneal Damage%	0					0					0

 

 

Dose:Untreated (Control Eye)                                                                       Sex:Female

Animal Numbers	1					2					3
Application Side	Left					Left					Left
Eye Reactions	At hour					At hour					At hour
	*	1	24	48	72	*	1	24	48	72	*	1	24	48	72
Corneal Opacity	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Area of Opacity	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Iris	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Conjunctiva	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Chemosis	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Corneal Damage%	0					0					0

Key:*= Pre-exposure eye examination.

 

Table 1 Continued…

 

Eye Irritation Scores - Mean Values after 24, 48, 72 Hours (Treated eye)

Animal No.  Eye Reaction	1	2	3
Corneal Opacity	0.00	0.00	0.00
Iris	0.00	0.00	0.00
Conjunctiva	0.00	0.00	0.33
Chemosis	0.00	0.00	0.00

 

 

Formula :

 

Mean Eye Irritation Score =  

Sum of the Individual Animal Score for eye reactionat24, 48 and 72 hours

Number of the Observations (3)

 

 

Table 2 : Individual AnimalClinicalSigns

 

Sex:Female

Animal No.	Days (Post application observation)
	0	1	2	3
1	1	1	1	1
2	1	1	1	1
3	1	1	1	1

Key:1 = Normal

  

Table 3: Individual Animal Body Weight

 

Sex :Female

Animal No.	Animal Body Weight (kg)
	Prior to application	At termination
1	2.340	2.432
2	2.408	2.508
3	2.036	2.048

Key:kg = Kilogram

Interpretation of results:

other: not irritating

Conclusions:

The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for Corneal opacity, iris, conjunctiva and chemosis were found 0.00, 0.00, 0.00, 0.00; 0.00, 0.00, 0.00, 0.00; and 0.00, 0.00, 0.33, 0.00, respectively.
Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbit no. 1 and 2 was observed at 24 hours and rabbit no. 3 at 48 hours

Hence, the test chemical was “Non Irritant” to New Zealand White Female rabbit eyes and is being classified as “Not Classified” as per the CLP Regulation.

Executive summary:

Acute eye irritation/corrosion study was conducted in rabbits to evaluate the eye irritant nature of the test chemical. The study was performed as per OECD 405 Guidelines using 3 female New Zealand White rabbits.

 

Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 100 mg of test item was instilled in the other (treated) eye of each rabbit.The eye was observed at 1, 24, 48 and 72 hour after test item instillation.Ophthalmoscope was used for scoring of eye lesions.

In the initial test,100 mg of test item (Pulverized Form) was applied into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. Ocular lesions were seen in animal no.1 at 1 hour observation which recovered at 24 hour hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mg of test item was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. Ocular lesions were seen in animal no. 2 at 1 hour observation which was recover at 24 hour and in animal no.3 at 24 hour which was recover at 48 hour observation.

 

Untreated eye of all the three rabbits was normal throughout the experimental period of 72 hours.

 

The following grading scores were observed in treated eye of tested rabbits.

Observation at 1 hour after instillation of test item revealed: Cornea-No ulceration or opacity in all 3 animals; Area of Opacity-Zero in all the animals;Iris:Normal in all the animals.Conjunctivae -Diffuse, crimson color; individual vessels not easily discerniblein animal no. 1 and some blood vessels definitely hyperaemic (injected) animal no. 2 whereas in animal no. 3 blood vessels were normal;Chemosis:Some swelling above normal (includes nictitating membranes) in animal no. 1 whereas no swelling was observed in animal no. 2 and 3.

 

Observation at 24 hours after instillation of test item revealed: Cornea-No ulceration or opacity in all 3 animals; Area of Opacity-Zero in all the animals;Iris:Normal in all the animals.Conjunctivae -Some blood vessels definitely hyperaemic (injected) was observed in animal no.3 and normal in Animal Numbers 1 and 2;Chemosis:No swelling was observed in all the 3 animals

 

At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 0 % damage in animal no. 1, 2 and 3.

 

Observation at 48 and 72 hours after instillation of test item revealed: Cornea-No ulceration or opacity in all the animals; Area of Opacity-Zero in all the animals;Iris:Normal in all the animals.Conjunctivae -Blood vessels were normal in all the animals;Chemosis:No swelling was observed in all the animals.

 

The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for Corneal opacity, iris, conjunctiva and chemosis were found 0.00, 0.00, 0.00, 0.00; 0.00, 0.00, 0.00, 0.00; and 0.00, 0.00, 0.33, 0.00, respectively.

Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbit no. 1 and 2 was observed at 24 hours and rabbit no. 3 at 48 hours. Hence, the test chemical was considered as Non-Irritating to the eyes of female New Zealand White rabbits and Classified as “Category- Not Classified” as per CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation :

In different studies, the test chemical has been investigated for potential for dermal irritation to a greater or lesser extent. The studies are based on in-vivo experimental conducted in different subjects which have been summarized as below:

Acute Dermal Irritation/corrosion Study of test chemical was performed as per OECD guideline No. 404 using three healthy young adult male New Zealand White rabbits.

Rabbits with good intact skin were selected for the study. The hairs of all the rabbits were clipped at contralateral sites, approximately 24 hours prior to treatment. A dose of 0.5 gm of test item moistened with 0.5 ml of distilled water was applied to the skin, over an area of approximately 6 x 6 cm clipped of hair on one side of rabbits. The other untreated side was kept as control area and0.5 ml of distilled water was applied at this site. At the end of 4 hours, the gauze patch was removed and test item application site was wiped with water without altering the integrity of the epidermis.Initially, the test item was applied to the clipped area of skin of one rabbit. The test site was covered with gauze patch. After 4 hours of exposure in Animal No. 1, there was no erythema and oedema observed at 1, 24 and 48 hours observation. Hence the confirmatory test was conducted on additional two rabbits (No. 2 and 3)to confirm the non irritant nature of the test item.In Animals No. 2 and 3 after post patch removal, revealed no erythema and oedema at 1, 24, 48 and 72 hours observation.The patch was removed after 4 hours and rabbits were observed for erythema and oedema at 1, 24, 48 and 72 hours after patch removal, evaluated and graded as per draize method. The individual mean score at 24, 48 and 72 hours for Animal Nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively.  

Hence, under the experimental test condition it was concluded that the test chemical was Non-Irritating to the skin of Male New Zealand White rabbits and Classified as “Category- Not Classified” as per CLP Regulation.

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 % tissue viability compared to negative control (n=3) of the test substance was determined to be 96.7%.Hence, under the current experimental test conditions it was concluded that test substance was considered to be not irritating to human skin and can thus be classified as “Not Classified'' as per CLP Regulation.

All these studies lead to a conclusion that Test chemical is indeed not irritating to skin. Hence, comparing the above annotations with the criteria of CLP regulation, Test chemical cannot be classified for skin irritation as per CLP.

Eye Irritation:

Various studies have been investigated for the test chemical to observe the potential for ocular irritation to a greater or lesser extent. The studies are based on in-vitro and in-vivo experiments conducted for target chemical which have beensummarized as below;

 

 

The in-vitro study was conducted on test chemical to determine the ocular irritation potential of test article was determined according to the OECD 492 test guideline. 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 solid test articles (50mg) and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours 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 % tissue viability of test substance was determined to be 91.7 %. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human eyes.

 

The above in-vitro study was supported by in-vivoAcute eye irritation/corrosion studyconducted in rabbits as per OECD 405 Guidelines using 3 female New Zealand White rabbits. Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 100 mg of test item was instilled in the other (treated) eye of each rabbit.The eye was observed at 1, 24, 48 and 72 hour after test item instillation. Ophthalmoscope was used for scoring of eye lesions. In the initial test,100 mg of test item (Pulverized Form) was applied into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. Ocular lesions were seen in animal no.1 at 1 hour observation which recovered at 24 hour hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 100 mg of test item was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. Ocular lesions were seen in animal no. 2 at 1 hour observation which was recover at 24 hour and in animal no.3 at 24 hour which was recover at 48 hour observation. Untreated eye of all the three rabbits was normal throughout the experimental period of 72 hours. The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for Corneal opacity, iris, conjunctiva and chemosis were found 0.00, 0.00, 0.00, 0.00; 0.00, 0.00, 0.00, 0.00; and 0.00, 0.00, 0.33, 0.00, respectively. Under the experimental conditions tested, eye irritation and reversibility of effects on eyes of rabbit no. 1 and 2 was observed at 24 hours and rabbit no. 3 at 48 hours. Hence, the test chemical was considered as Non-Irritating to the eyes of female New Zealand White rabbits and.

 

Based on the above summarized studies for target chemical,it can be concluded that the testchemical is unable to cause eye irritation and considered as not irritating.Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

Justification for classification or non-classification

The skin and eye irritation potential of test chemical was observed in experimental studies. The results obtained from these studies indicate that the chemical is not likely to cause skin and eye irritation. Hence the test chemical can be classified under the category “Not Classified” for skin and eye as per CLP.