Registration Dossier

Administrative data

Description of key information

Skin Irritation:

The skin of all 3 animals did not show any reactions. Signs of systemic intoxication were not observed. The Primary Irritation Index (PII) of the test chemical was 0.0

Based on these results and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as Skin Irritant.

Eye Irritation:

Instillation of the test substance in one of the three animals resulted in slight to moderate redness (persisting for 72 hours) and slight swelling (persisting for 48 to 72 hours) of the conjunctivae. Seven days after dosing the conjunctival effects had disappeared. The iris of one of the rabbits was affected one hour after dosing, but this effect disappeared during the next 24 hours. Iris effects were not observed in the other two animals. Adverse effects on the cornea were not observed in any of the rabbits during the entire observation period. Treatment of the eyes with sodium fluorescien 24 hours post instillation didnot reveal any signs of epithelial damage. Signs of systemic intoxication were not observed.

Based on these observations and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as an Eye Irritant.

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 reports
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Principles of method if other than guideline:
The purpose of the study was to evaluate the local irritating/corrosive effects on the rabbit skin following a single application of the test chemical
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tetrylammonium bromide- Molecular formula: C8H20N.Br- Molecular weight: 210.157 g/mol- Smiles notation: [N+](CC)(CC)(CC)CC.[BrH-]- InChl : 1S/C8H20N.BrH/c1-5-9(6-2,7-3)8-4;/h5-8H2,1-4H3;1H/q+1;/p-1- Substance type: Organic- Physical state: solid- Purity: 100.1 -100.2% based on Br-- Storage: At ambient temperature in the dark- Batch No: HH 85-83A
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS- Sex: Female - Source: The Broekmen Institute, Someren, The Netherlands - Age at study initiation: 2- 3 months old - Weight at study initiation: 2617- 2751 g - Housing: individually housed in metal cages with perforated floors - Diet (e.g. ad libitum): standard lab diet (100g/ day); obtained from Hope Farms - Water (e.g. ad libitum): tap water, ad libitum - Acclimation period: A quarantine period of 12 days followed by another 3 weeks of acclimation period ENVIRONMENTAL CONDITIONS - Temperature (°C): 20 - 21 degC - Humidity (%): 60 -70% (relative humidity) - Air changes (per hr): no data available - Photoperiod (hrs dark / hrs light): 12 hours ligth, 12 hours dark
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
yes
Amount / concentration applied:
0.5 gm of test substance moistened with 0.5 ml of milli RO water
Duration of treatment / exposure:
4 hours
Observation period:
responses were scored at 60 minutes then 24, 48, 72 hours after patch removal
Number of animals:
3
Details on study design:
TEST SITE - Area of exposure: left flank, central flank - % coverage: 6cm square patch of Metalline, mounted on a permeable tape - Type of wrap if used: flexible bandage (COBAN, 3M, USA) REMOVAL OF TEST SUBSTANCE - Washing (if done): yes - Time after start of exposure: 4 hours after exposure of the test chemicalOBSERVATION TIME POINTS(indicate if minutes, hours or days) : The exposed areas were examined for signs of erythema and edema and the responses were scored at 60 minutes then 24, 48, 72 hours after patch removalSCORING SYSTEM: - Method of calculation:The test reaults were evaluated according to the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC)
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The skin of all 3 animals did not show any reactions.Signs of systemic intoxication were not observed.

Table 1: Primary Skin Irritation Scores for TEAB in the rabbit

Rabbit No. and Sex

Body Weight (gm)

Observation time after exposure period (hours)

Erythema

Edema

269, Female

2617

1

0

0

24

0

0

48

0

0

72

0

0

Subtotala)/ Mean valuec)

 

 

 

 

0/0

0/0

271, Female

2751

1

0

0

24

0

0

48

0

0

72

0

0

Subtotala)/ Mean valuec)

 

 

 

 

0/0

0/0

276, Female

2646

1

0

0

24

0

0

48

0

0

72

0

0

Subtotala)/ Mean valuec)

 

 

 

 

0/0

0/0

Totalb)/ Mean value

 

 

0/0

0/0

Where,

a)Subtotal = sum of 24,48,72 hours scores for each animals individually

b)Total = sum of 24, 48, 72 hours scores calculated over all animals

c)Mean value = mean score of 24,48 and 72 hours reading time

   

Primary Irritation Index (PII)*= 0

*- Total of 24 and 72 hours scores for erythema and edema, divided by 6

Interpretation of results:
other: not irritating
Conclusions:
The skin of all 3 animals did not show any reactions.Signs of systemic intoxication were not observed.The Primary Irritation Index (PII) of the test chemical was 0.0
Based on these results and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as Skin Irritant.
Executive summary:

The test chemical was applied in a single dose to the skin of the experimental animals, each animal serving as its own control.The degree of irritation was evaluated and scored according to OECD 404 Guidelines at specific intervals.

3 young adult female New Zealand White rabbits were used for the study. One day before dose administration, the fur was removed from the central back of the rabbits by clipping, exposing an area of skin of approximately 10*10 cm. 0.5 gm of test substance moistened with 0.5 ml of milli RO water  was applied on  6cm square patch of Metalline, mounted on a permeable tape.this was applied to the left flank of each animal.The right flank being covered with same dressing with the test chemical served as control.Finally the animals were wrapped in a flexible bandage. The exposure duration was 4 hours, after which the remaining test substance was gently removed using a tissue moistened with tap-water. The exposed areas were examined for signs of erythema and edema and the responses were scored at 60 minutes then 24, 48, 72 hours after patch removal. The test results were evaluated according to the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).

The skin of all 3 animals did not show any reactions.Signs of systemic intoxication were not observed.The Primary Irritation Index (PII) of the test chemical was 0.0

Based on these results and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as Skin Irritant.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

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 reports
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Principles of method if other than guideline:
The purpose of the study was to evaluate the ability of the test chemical to produce ocular irritation/corrosion in rabbits following a single application.
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
- Sex: Female - Source: The Broekmen Institute, Someren, The Netherlands
- Age at study initiation: 2- 3 months old
- Weight at study initiation: 2617- 2751 g
- Housing: individually housed in metal cages with perforated floors
- Diet (e.g. ad libitum): standard lab diet (100g/ day); obtained from Hope Farms
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: A quarantine period of 12 days followed by another 6 weeks of acclimation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21 degC
- Humidity (%): 60 -70% (relative humidity)
- Air changes (per hr): no data available
- Photoperiod (hrs dark / hrs light): 12 hours ligth, 12 hours dark
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
approximately 72 mg (equivalent to 0.1 ml)
Duration of treatment / exposure:
single exposure
Observation period (in vivo):
Immediately after instillation of the test substance, the animals were observed and abnormalities were recorded.The eyes were examined approximately 1,24,48, 72 hours and 7 days after instillation of the test chemical
Duration of post- treatment incubation (in vitro):
no data available
Number of animals or in vitro replicates:
3
Details on study design:
SCORING SYSTEM:The ocular lesions were scored according to OECD 405 Guidelines and the results were evaluated according to the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC)

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein: Fluorscein treatment
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
1.3
Reversibility:
fully reversible within: 7 days
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0.7
Reversibility:
fully reversible within: 7 days
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Instillation of the test substance in one of the three animals resulted in slight to moderate redness (persisting for 72 hours) and slight swelling (persisting for 48 to 72 hours) of the conjunctivae.Seven days after dosing the conjunctival effects had disappeared.The iris of one of the rabbits was affected one hour after dosing, but this effect disappeared during the next 24 hours.Iris effects were not observed in the other two animals.Adverse effects on the cornea were not observed in any of the rabbits during the entire observation period.Treatment of the eyes with sodium fluorescien 24 hours post instillation didnot reveal any signs of epithelial damage.Signs of systemic intoxication were not observed.

Table 1: Eye Irritation Scores in rabbits (EEC Scores table)

Rabbit No, Sex

Body Weight (gm)

Observation time after instillation (hours)

Cornea Opacity

Iris Lesion

Conjunctivae

 

Redness

Swelling (Chemosis)

266, female

2751

1

0

1

2

1

24

0

0

2

1

48

0

0

2

1

72

0

0

1

1

Day 7

0

0

0

0

Subtotala)/ Mean valuec)

 

 

0/0

0/0

5/1.7

3/1.0

267, female

2830

1

0

0

1

1

24

0

0

1

1

48

0

0

2

1

 

 

72

0

0

1

0

Day 7

0

0

0

0

Subtotala)/ Mean valuec)

 

 

0/0

0/0

4/1.3

2/0.7

269, female

2850

1

0

0

1

1

24

0

0

1

0

48

0

0

1

1

72

0

0

1

0

Day 7

0

0

0

0

Subtotala)/ Mean valuec)

 

 

0/0

0/0

3/1.0

1/0.3

Totalb)/ Mean value

 

 

0/0

0/0

12/1.3

6/0.7

Where,

a)Subtotal = sum of 24,48,72 hours scores for each animals individually

b)Total = sum of 24, 48, 72 hours scores calculated over all animals

c)Mean value = mean score of 24,48 and 72 hours reading time

Interpretation of results:
other: Not irritating
Conclusions:
Instillation of the test chemical in one of the three animals resulted in slight to moderate redness (persisting for 72 hours) and slight swelling (persisting for 48 to 72 hours) of the conjunctivae.Seven days after dosing the conjunctival effects had disappeared.Adverse effects on the cornea were not observed in any of the rabbits during the entire observation period.Treatment of the eyes with sodium fluorescien 24 hours post instillation didnot reveal any signs of epithelial damage.Signs of systemic intoxication were not observed.Based on these observations and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as an Eye Irritant.
Executive summary:

The purpose of the study was to evaluate the ability of the test chemical to produce ocular irritation/corrosion in rabbits following a single application. The study was performed according to OECD 405 Guidelines and evaluated according to EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).

3 young adult female New Zealand White rabbits were used for the study. Before dosing the test substance was ground to a fine powder using a mortar and pestle.The bulk density of the test substance was determined by NOTOX and amounted to 0.725 g/ml.On the day of dose administration, three portions of 72±1.5 mg of the powder were dispensed in glass containers with screw caps. Approximately 72 mg (equivalent to 0.1 ml) was instilled into the conjunctival sac of the right eye of the rabbit using a spatula.The lids were then gently held together for two seconds and then released Immediately after instillation of the test substance, the animals were observed and abnormalities were recorded.The eyes were examined approximately 1,24,48, 72 hours and 7 days after instillation of the test chemical.Immediately after treatment, the animals were transfered to metal cages.The left eye remained untreated and served as control. Immediately after instillation of the test substance, the animals were observed and abnormalities were recorded.The eyes were examined approximately 1,24,48, 72 hours and 7 days after instillation of the test chemical. Approximately 24 hours after instillation of the test substance(immediately after scoring the corneal opacity and the alterations of the iris and conjunctivae), a solution of 2% sodium fluorescien in water (pH adjusted to 7.0) was applied to both eyes of the test animals to examine quantitatively the potential for corneal injury.The brightly green staining area indicating epithelial damage was estimated as a percentage of total corneal area.Any observed local effects other than those indicated above were recorded, The ocular lesions were scored according to OECD 405 Guidelines and the results were evaluated according to the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).

Instillation of the test substance in one of the three animals resulted in slight to  moderate redness (persisting for 72 hours) and slight swelling (persisting for 48 to 72 hours) of the conjunctivae.Seven days after dosing the conjunctival effects had disappeared.The iris of one of the rabbits was affected one hour after dosing, but this effect disappeared during the next 24 hours.Iris effects were not observed in the other two animals.Adverse effects on the cornea were not observed in any of the rabbits during the entire observation period.Treatment of the eyes with sodium fluorescien 24 hours post instillation didnot reveal any signs of epithelial damage.Signs of systemic intoxication were not observed.

Based on these observations and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as an Eye Irritant.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected 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:
29.3
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.

Code N° Tissue  Raw data Blank corrected data mean of OD % of viability
  n Aliq. 1 Aliq. 2 Aliq. 1 Aliq. 2
NC 1 2.1392 2.1045 2.104 2.070 2.087 98.7
  2 2.1925 2.1633 2.158 2.128 2.143 101.3
PC 1 0.692 0.715 0.657 0.680 0.669 31.6
  2 0.7927 0.7855 0.758 0.751 0.754 35.7

71-91-0 1 0.7895 0.7876 0.755 0.753 0.754 35.6
  2 0.5314 0.512 0.496 0.477 0.487 23.0

  of OD of OD viabilities [%] of viabilities      
NC 2.115 0.056 100.0 2.65 1.33 NI qualified
PC 0.711 0.086 33.6 4.05 2.02 I qualified

71-91-0 0.620 0.267 29.3 12.62 6.31 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 29.3%. 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 chemical by determining the viability of the tissues following exposure to the test article via MTT. Tissues 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 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 29.3%. 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 ‘’Irritating to eyes in Category 2” 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

Various studies have been reviewed to determine the level of dermal irritation/corrosion caused by the test chemical in living organisms. The studies include experimental results on rabbits for the test chemical. The results are summarized as follows:

The test chemical was applied in a single dose to the skin of the experimental animals, each animal serving as its own control. The degree of irritation was evaluated and scored according to OECD 404 Guidelines at specific intervals.

3 young adult female New Zealand White rabbits were used for the study. One day before dose administration, the fur was removed from the central back of the rabbits by clipping, exposing an area of skin of approximately 10*10 cm. 0.5 gm of test substance moistened with 0.5 ml of milli RO water was applied on  6cm square patch of Metalline, mounted on a permeable tape.this was applied to the left flank of each animal. The right flank being covered with same dressing with the test chemical served as control. Finally the animals were wrapped in a flexible bandage. The exposure duration was 4 hours, after which the remaining test substance was gently removed using a tissue moistened with tap-water. The exposed areas were examined for signs of erythema and edema and the responses were scored at 60 minutes then 24, 48, 72 hours after patch removal. The test results were evaluated according to the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).

The skin of all 3 animals did not show any reactions. Signs of systemic intoxication were not observed. The Primary Irritation Index (PII) of the test chemical was 0.0

Based on these results and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as Skin Irritant.

This is supported by another study designed and conducted to determine the dermal Irritation/corrosion potential of the test chemical in Sprague Dawley rats. This study was performed as per OECD guideline No. 402. 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 moistened with distilled water. The test item was applied 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.   

The test chemical was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. 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. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.

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- Unclassified” as per CLP Classification.

Available results for the test chemical indicate a strong possibility that the test chemical was indeed not irritating to skin. Hence the test chemical can be classified under the category “Not Classified” as per CLP Classification.

Eye Irritation

Various studies have been reviewed to ascertain the level of ocular damage caused by the test chemical in living organisms. These include in vivo as well as in vitro experimental results for the test chemical. The results have been summarized below:

The purpose of the study was to evaluate the ability of the test chemical to produce ocular irritation/corrosion in rabbits following a single application. The study was performed according to OECD 405 Guidelines and evaluated according to EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).

3 young adult female New Zealand White rabbits were used for the study. Before dosing the test substance was ground to a fine powder using a mortar and pestle.The bulk density of the test substance was determined by NOTOX and amounted to 0.725 g/ml.On the day of dose administration, three portions of 72±1.5 mg of the powder were dispensed in glass containers with screw caps. Approximately 72 mg (equivalent to 0.1 ml) was instilled into the conjunctival sac of the right eye of the rabbit using a spatula. The lids were then gently held together for two seconds and then released immediately after instillation of the test substance, the animals were observed and abnormalities were recorded. The eyes were examined approximately 1,24,48, 72 hours and 7 days after instillation of the test chemical. Immediately after treatment, the animals were transferred to metal cages. The left eye remained untreated and served as control. Immediately after instillation of the test substance, the animals were observed and abnormalities were recorded. The eyes were examined approximately 1,24,48, 72 hours and 7 days after instillation of the test chemical. Approximately 24 hours after instillation of the test substance(immediately after scoring the corneal opacity and the alterations of the iris and conjunctivae), a solution of 2% sodium fluorescien in water (pH adjusted to 7.0) was applied to both eyes of the test animals to examine quantitatively the potential for corneal injury.The brightly green staining area indicating epithelial damage was estimated as a percentage of total corneal area.Any observed local effects other than those indicated above were recorded,

The ocular lesions were scored according to OECD 405 Guidelines and the results were evaluated according to the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).

Instillation of the test substance in one of the three animals resulted in slight to moderate redness (persisting for 72 hours) and slight swelling (persisting for 48 to 72 hours) of the conjunctivae. Seven days after dosing the conjunctival effects had disappeared. The iris of one of the rabbits was affected one hour after dosing, but this effect disappeared during the next 24 hours. Iris effects were not observed in the other two animals. Adverse effects on the cornea were not observed in any of the rabbits during the entire observation period. Treatment of the eyes with sodium fluorescien 24 hours post instillation didnot reveal any signs of epithelial damage. Signs of systemic intoxication were not observed.

Based on these observations and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as an Eye Irritant.

This is supported by the results of an in vitro study performed according to the OECD 492 test guideline for determining the ocular irritation potential of the test chemical. The MatTek

EpiOcular™ model was used to assess the potential ocular irritation of the test chemical by determining the viability of the tissues following exposure to the test article via MTT. Tissues 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 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 29.3%. 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 ‘’Irritating to eyes in Category 2” as per CLP Regulation

In another study performed to evaluate the ability of the test chemical to produce ocular irritation/corrosion in rabbits following a single application. The study was performed according to OECD 405 Guidelines and evaluated according to EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).3 young adult female New Zealand White rabbits were used for the study.

Before dosing the test substance was ground to a fine powder using a mortar and pestle. The bulk density of the test substance was determined by NOTOX and amounted to 0.698 g/ml. On the day of dose administration, three portions of 69±1.5 mg of the powder were dispensed in glass containers with screw caps. One portion of the dispensed amount (equivalent to 0.1 ml) was instilled into the conjunctival sac of the right eye of the rabbit using a spatula. The lids were then gently held together for two seconds and then released immediately after instillation of the test substance, the animals were observed and abnormalities were recorded. The eyes were examined approximately 1, 24, 48, 72 hours and 7 days after instillation of the test chemical. Immediately after treatment, the animals were transferred to metal cages. The left eye remained untreated and served as control. Immediately after instillation of the test substance, the animals were observed and abnormalities were recorded. The eyes were examined approximately 1, 24, 48, 72 hours and 7 days after instillation of the test chemical. Approximately 24 hours after instillation of the test substance (immediately after scoring the corneal opacity and the alterations of the iris and conjunctivae), a solution of 2% sodium fluorescien in water (pH adjusted to 7.0) was applied to both eyes of the test animals to examine quantitatively the potential for corneal injury. The brightly green staining area indicating epithelial damage was estimated as a percentage of total corneal area. Any observed local effects other than those indicated above were recorded. The ocular lesions were scored according to OECD 405 Guidelines and the results were evaluated according to the EEC criteria for classification and labeling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).

Immediately after dosing the (animals were observed to squeeze their eyelids together, and lacrimation was excessive. One hour later slight conjunctival redness was observed in all animals, as well as moderate to severe swelling of the eyelids. At this time lacrimation was still increased, but this effect disappeared during the next twenty-four hours. Forty-eight hours after dosing the blood vessels in the nictating membrane were injected, and in one animal (no. 251) this membrane was still slightly swollen. These effects were still present 72 hours after dosing, but disappeared completely during the next four days. Adverse effects on the cornea and iris were not observed in any of the rabbits during the entire observation period. Treatment of the eyes with fluorescein 24 hours after instillation of the test substance did not reveal any epithelial damage. Signs of systemic intoxication were not observed.

Based on these observations and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as an Eye Irritant.

Even though results of in-vitro study claim that the test chemical causes severe irritation to eyes, but in vivo experimental studies indicate a strong possibility that the test chemical can be not irritating to eyes.

Taking all these factors into consideration, the test chemical can be considered to be not irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Justification for classification or non-classification

Available results for the test chemical indicate a strong possibility that the test chemical was indeed not irritating to skin. Hence the test chemical can be classified under the category “Not Classified” as per CLP Classification.

Even though results of in-vitro study claim that the test chemical causes severe irritation to eyes, but in vivo experimental studies indicate a strong possibility that the test chemical can be not irritating to eyes.

Taking all these factors into consideration, the test chemical can be considered to be not irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.