5-bromo-1,3-dichloro-2-fluorobenzene

Endpoint summary

Description of key information

Not corrosive to skin (in vitro) (GLP, OECD TG 431)

Irritating to skin (in vitro) (GLP, OECD TG 439)

Mildly irritating to the eyes (in vivo) (GLP, OECD TG 437, OECD TG 405)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek In Vitro Life Science Laboratories, Bratislava - Slovakia

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-incubation of EpiDerm tissues
The assay medium was brought to room temperature before use. The assay medium (0.9 mL) was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 and 60-minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium and were pre-incubated (37 °C, 5% CO2) for approximately
1 hour before dosing.

Main Test
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3 and 60-minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 µL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. Duplicate tissues were used for each treatment group. For the 60-minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator
(37 °C, 5% CO2) for the 60 minute exposure period.
The same procedure was repeated for the 3 minute exposure period. Because the exposure time was only 3 minutes, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. Tissues were blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3-hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extract (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution.
3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre-labeled
96-well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL of test substance

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes of exposure

Value:

121.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes of exposure

Value:

113.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control
The absolute OD570 of the negative control treated tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive control
Potassium Hydroxide 8.0N solution was used as a positive control. An assay meets the acceptance criteria if mean relative tissue viability of the 60 minute exposure for the positive control is < 15%.
Coefficient of Variation
In the range 20 and 100% viability, the coefficient of variation between tissue replicates should be ≤ 30%.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean percent cell viability for the substance at 3 and 60 minutes was 121.1% and 113.3%, respectively, therefore under the conditions of this assay the test item was considered to be non-corrosive to the skin.

Executive summary:

The purpose of this test is to evaluate the skin corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period.  At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and three aliquots of 200 µL were transferred to the appropriate wells of a pre-labelled 96 well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage cell viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean percent viability for each treatment group was:

3 minutes exposure period: Negative control 100% cell viability; Positive control 2.9% cell viability; Test substance: 121.1% cell viability

60 minutes exposure period: Negative control 100% cell viability; Positive control 2.4% cell viability; Test substance: 113.3% cell viability

The quality criteria required for acceptance of results in the test were satisfied.

Reference 2

Endpoint:

skin 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-incubation of EpiSkin tissues
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Colour Satisfactory: Yes
Agar Medium Colour Satisfactory: Yes

These checks verified the tissues were acceptable for use in the study. The tissue model supplier also demonstrated the batch of tissues was acceptable by determining the barrier function and also performed histological examination demonstrating appropriate morphology of the tissues. 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
2 mL of maintenance medium, warmed to approximately 37 oC, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the substance for an exposure period of 15 minutes. The substance was applied topically to the corresponding tissues ensuring uniform covering. Approximately 10 µL (26.3 µL/cm2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The substance was applied topically to the corresponding tissues ensuring uniform covering. Approximately 10 µL (26.3 µL/cm2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

15.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control treated tissues and the standard deviation value of the viability was 1.2%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.702 and the standard deviation value of the viability was 7.7%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 7.6%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of this EPISKINTM assay conducted on the substance, the results indicate that the test item is an irritant to skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the substance using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours.  The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Triplicate tissues were treated with the substance for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals from the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate

200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate.  The optical density was measured at 570 nm.

Data are presented as percentage tissue viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the substance treated tissues was 15.8% compared to the negative control after the 15-minute exposure period and 42-hours post-exposure incubation period. This is below the 50% threshold therefore the substance was considered to be an irritant to the skin under the conditions of this assay.

The quality criteria required for acceptance of results in the test were met therefore the study is considered valid.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of test substance was applied to the corneas

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3

Details on study design:

Preparation of corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects.
Selection of corneas and opacity reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.
Treatment of corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.
Application of sodium fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
Permeability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of medium representing each cornea was applied to a designated well on a 96 well plate and the optical density at 492 nm (OD492) was measured using the LabTech LT-4500 microplate reader.
Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
No histopathology was performed in this study.
Data evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
Opacity measurement
The change in corneal opacity (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
In vitro irritancy score
The following formula was used to determine the IVIS:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.
Visual observation
The condition of the cornea was visually assessed post treatment and post incubation.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

For the assay to be considered acceptable, the following positive control criterion should be met:
Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2017 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 65.1 (actual result was 44.7).
For the assay to be considered acceptable, the following negative control criteria should be met:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤2.3 and for permeability ≤0.041 (actual result was 1.8).

Interpretation of results:

GHS criteria not met

Conclusions:

The IVIS score for the substance at 120 minutes was 3.0. Therefore, the substance was found to fall into "No category". Not requiring classification to UN GHS or EU CLP.

Executive summary:

The purpose of this test was to evaluate the eye irritation/ corrosivity potential of the test item using the bovine corneal opacity (BCOP) method.  

The BCOP test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro.  In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method is used to identify test items (both chemicals and mixtures) which are potential serious eye corrosives or irritants as well as those which are non-irritant, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1.  Items that are non-irritants are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Undiluted test substance was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently.  The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The in vitro irritancy scores after 120 minutes were:

Test substance: 3.0

Negative control: 1.8

Positive control: 44.7

Reference 2

Endpoint:

eye irritation: in vivo

Remarks:

The study was conducted to meet the national regulatory requirements in a non-EEA country.

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Sex: Male
Number of animals: 3
Age/weight at dosing: ~16-18 weeks (young adult); 4056 – 4440 g
Source: S&K-LAP Kft., 2173 Kartal, Császár út 135, Hungary
Housing: Individual caging
Acclimatisation period: 49 / 51 / 61 days
Diet: UNI diet for rabbits, Cargill Takarmány Zrt., H-5300 Karcag, Madarasi út, Hungary, ad libitum (The batch numbers of the lots used in the study were 0005454439, 0005517191, and 0005606701, expiry dates: 12 March 2019, 10 April 2019, and 25 May 2019, respectively.)
Water: Tap water, from an automatic system, ad libitum, fit for human consumption
Environmental conditions
Temperature: 18.6 – 22.5 °C
Humidity: 32 – 59%
Air changes: 15-20 air exchanges/hour
Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
In-life dates: Start: 06 March 2019; End: 25 March 2019

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

The test substance was not removed from the treated eye, i.e. no washing of the treated eye occurred.

Observation period (in vivo):

1 week

Number of animals or in vitro replicates:

3

Details on study design:

Animal assignment and treatment: Before the start of the test, both eyes of the provisionally selected test rabbits were examined with fluorescein staining for evidence of ocular irritation or defect using a hand-held slit-lamp.
The first animal (1487) showed scores of above zero at 1, 24, 48, and 72-hour observation points after treatment. This animal showed minimal symptoms after 24 hours, thus a second animal (1496) was treated after the 48-hour observation of the first animal.
The second animal showed scores of above zero at 1, 24, 48, and 72-hour observation points after treatment. This animal became symptom-free at the 1-week observation point, thus a third animal (1480) was treated after the 1-week observation of the second animal.
The third animal showed scores of above zero at 1, 24 and 48 and 72-hour observation points after treatment. This animal became symptom-free at the 1-week observation point.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Remarks:

discharge

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Remarks:

discharge

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Remarks:

discharge

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritant / corrosive response data:

Some signs of irritation were seen in the treated eye after one hour following treatment, including medium redness of the conjunctivae, mild chemosis or significant conjunctival discharge. The majority of effects were generally reversible within one day, except for redness of the conjunctivae being reversible within 7 days.

Interpretation of results:

Category 2B (mildly irritating to eyes) based on GHS criteria

Conclusions:

The test item was graded as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to the modified Kay and Calandra classification system.

Executive summary:

The primary eye irritation effect of the test item was evaluated according to OECD test guideline no.: 405 (2017), EPA OPPTS 870.2400 (1998), EU No 2017/735, B.5 (2017). The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (09 October 2017). CA5528 was administered as an instillation of a single dose of 0.1 mL into the conjunctival sac of the left eye of 3 young adult male New Zealand White rabbits. The untreated right eye served as the control. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, plus 1 week after test material instillation. Observations with fluorescein staining were made approximately 24 hours before treatment and then 24, 48 and 72 hours, plus 1 week after treatment. Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Results obtained from these three animals were used to classify the test item for irritation potential.

No Initial Pain Reaction/Pain reaction (IPR/PR) was observed in any of the animals.

Animal (no. 1487) clinical observation

One hour after the application, redness (score 2), chemosis (score 1), and discharge (score 1) were noted in the animal. No remaining test item was noted in the treated eye of the animal. At 24, 48, and 72 hours after the treatment, redness (score 1), was noted in the animal. Fluorescein staining was negative. At 1 week after the treatment, no conjunctival or corneal effects were observed in this animal. Fluorescein staining was negative.

Animal (no. 1496) clinical observation

One hour after the application, redness (score 2), chemosis (score 1), and discharge (score 3) were noted in the animal. No remaining test item was noted in the treated eye of the animal. At 24, 48, and 72 hours after the treatment, redness (score 1), was noted in the animal. Fluorescein staining was negative. At 1 week after the treatment, no conjunctival or corneal effects were observed in this animal. Fluorescein staining was negative.

Animal (no. 1480) clinical observation

One hour after the application, redness (score 2), chemosis (score 1), and discharge (score 2) were noted in the animal. No remaining test item was noted in the treated eye of the animal. At 24 hours after the treatment, redness (score 2), was noted in the animal. Fluorescein staining was negative. At 48 and 72 hours after the treatment, redness (score 1), was noted in the animal. Fluorescein staining was negative. At 1 week after the treatment, no conjunctival or corneal effects were observed in this animal. Fluorescein staining was negative.

The study was terminated one week after the treatment of the third rabbit. The control eye of each animal was symptom-free during the study. No mortality occurred during the study. The body weights of all rabbits were considered to be within the normal range of variability.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance exhibited effects in a valid and reliable in vitro study on the skin irritation potential. It meets the criteria for classification for skin irritation (Cat. 2) in accordance with CLP-Regulation (EC) No. 1272/2008. No adverse effects were seen in the available in vitro and in vivo eye irritation studies, and the substance does not require a classification for this endpoint.