4-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]-N-(3-ethoxypropyl)benzenesulphonamide

Endpoint summary

Description of key information

Disperse Red 092 is neither irritant nor corrosive to the skin as well as eyes.

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

Study period:

17 May 2016 to 19 May 2016

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:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Sponsor Batch Number PCR92X140707 (China)
- Expiration date of the lot/batch:14 October 2019
- Purity: 95.4 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4 °C in the dark

Test system:

human skin model

Source species:

human

Cell type:

other: target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium

Cell source:

other: No specified as study used an EpiDerm™ Reconstructed Human Epidermis Model Kit from MatTek

Source strain:

other: Not applicable

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recognised in vitro test for corrosivity

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 23337
- Delivery date: 17 May 2016
- Date of initiation of testing: 17 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable):

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates: 2
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)

- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %.]

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg, used as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Sterile distilled water
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): 8.0N Potassium hydroxide

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

Duplicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

107.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 Minute exposure

Value:

98.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Other effects / acceptance of results:

Quality Criteria
The mean OD562 for the negative control treated tissues was 1.701 for the 3 Minute exposure period and 1.906 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied. The relative mean tissue viability for the positive control treated tissues was 3.4% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied. In the range 20 to 100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30 %. The acceptance criterion was therefore satisfied.

The relative mean ciabilities for each treatment group were:

Exposure period	Percentage viability
	Negative control	Positive control	Test item
3 minutes	100*	3.5	107.3
60 minutes	100*	3.4	98.7

 

*The mean viability of the negative control tissues is set to 100 %.

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 40444/B was considered to be non-corrosive to the skin.

Executive summary:

The purpose of this test was to evaluate the corrosivity potential of FAT 40444/B 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. The study was carried out according to OECD guideline 431 and EU method B.40. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. The test item produced a sufficient amount of color that may interfere with the end point optical density readings. Therefore, additional tissues were incorporated into the testing for color correction purposes. These tissues were treated identically with the exception of not being placed onto MTT. 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 triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viabilities for each treatment group were as follows:

 

Exposure period	Percentage viability
	Negative control	Positive control	Test item
3 minutes	100*	3.5	107.3
60 minutes	100*	3.4	98.7

*The mean viability of the negative control tissues is set to 100 %.

The quality criteria required for acceptance of results in the test were satisfied. So based on the study results, FAT 40444/B was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2016 to 27 June 2016

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:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
PCR92X140707 (China)
- Expiration date of the lot/batch:
14 October 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
4 °C in the dark

Test system:

human skin model

Source species:

human

Cell type:

other: adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen

Cell source:

other: EpiSkinTM Tissues (0.38cm²) lot number 16-EKIN-025

Source strain:

other: not applicable

Details on animal used as source of test system:

Not applicable

Justification for test system used:

Recognised in vitro test for skin irritation

Vehicle:

unchanged (no vehicle)

Details on test system:

Study Design
Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT

MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 10 mg of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made. The test item was considered to have the potential to cause color interference with the MTT endpoint. Therefore, an additional procedure was performed using viable tissues to assess for the possibility of color interference. 10 mg of the test item was applied to three viable tissues in parallel to the main test with the exception that the MTT incubation period was replaced by incubation with maintenance medium (therefore with out MTT). Three viable tissues were also used for negative control purposes which remained untreated. The optical density measurements from these tissues were then assessed for possible quantitative correction of the results.

Pre-incubation (Day 0: Tissue Arrival)

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 Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 well s 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
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12 well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and t he epidermis. Approximately 10 mg (26.3 mg/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 Ca2+ and Mg2+. 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.

MTT Loading/Formazan Extraction (Day 3)
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. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination. 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.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored 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 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10µL, used as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10µL DPBS
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µL SDS
- Concentration (if solution): 5 % w/v

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 Minute exposure

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item  

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.648			96.7
	0.611	0.670	0.072	91.2	100*	10.7
	0.750			111.9
Positive Control Item	0.099			14.8
	0.059	0.077	0.020	8.8	11.5	3.0
	0.074			11.0
Test Item	0.518			77.3
	0.565	0.544	0.024	84.3	81.2	3.6
	0.550			82.1

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 40444/B was classified as non-irritant to skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of FAT 40444/B using the EPISKIN^TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 h. The study was carried out according to OECD guideline 439 and EU method B.46. 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 colorimetric 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 test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was shown to produce a colored solution therefore additional color correction tissues were included in parallel to the main test. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labeled microtubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of 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 labeled 96 well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 81.2 % after the 15 minute exposure period and 42 h post exposure incubation period. Quality criteria: The quality criteria required for acceptance of results in the test were satisfied. Based on the study results, FAT 40444/B was classified as non-irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 June 2016 to 19 September 2016

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:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

EC 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PCR92X140707 (China)
- Expiration date of the lot/batch: 14 October 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4 °C in the dark

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir as a by-product from freshly slaughtered animals
- Characteristics of donor animals (e.g. age, sex, weight): adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): excised eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics
- Time interval prior to initiating testing: They were transported to the test facility over ice packs on the same day of slaughter.
- indication of any existing defects or lesions in ocular tissue samples: The corneas were prepared immediately on arrival.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution): 20 % w/v solution in 0.9 % w/v sodium chloride

Duration of treatment / exposure:

240 mins

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES
3 per treatment

NEGATIVE CONTROL USED
0.9 % w/v solution

POSITIVE CONTROL USED
Imidazole, 20 % w/v solution in 0.9 % w/v sodium chloride

APPLICATION DOSE AND EXPOSURE TIME
20 % w/v solution, 240 mins

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea (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
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD492)
- Others (e.g., pertinent visual observations, histopathology): (please specify): Histopathology.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. Yes

Irritation parameter:

in vitro irritation score

Value:

4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	4.0
Negative Control	1.8
Positive Control	98.4

Interpretation of results:

GHS criteria not met

Conclusions:

No prediction of eye irritation can be made.

Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (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 was carried out according to OECD guideline 437 and EU method B.47.

The test item was applied at a concentration of 20 % w/v in 0.9 % w/v sodium chloride solution for 240 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 test item is classified according to the prediction model as follows:

IVIS	Classification
≤ 3	No category. Not requiring classification to UN GHS or EUCLP
> 3; ≤55	No prediction of eye irritation can be made
> 55	Category 1. UN GHS or EU CLP Causes serious eye damage

Results

The in vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	4.0
Negative Control	1.8
Positive Control	98.4

Conclusion

No prediction of eye irritation can be made.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Skin corrosion in vitro with Disperse Red 092

The skin corrosion potential of the test substance was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Duplicate tissues were treated. The test item produced a sufficient amount of color that may interfere with the end point optical density readings. Therefore, additional tissues were incorporated into the testing for color correction purposes. These tissues were treated identically with the exception of not being placed onto MTT. 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 triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. The optical density (OD) was measured at 562 nm (OD562). The relative mean viabilities for the groups exposed for 3 minutes and 60 minutes were determined to be 107.3 and 98.7 %, respectively. Hence, the test item was considered to be non-corrosive to the skin.

Skin irritation in vitro with Disperse Red 092

The skin irritation potential of the test item was evaluated using the EPISKIN^TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was shown to produce a colored solution therefore additional color correction tissues were included in parallel to the main test. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of 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 labeled 96 well plate. The optical density was measured at 562 nm. The relative mean percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues) of the test item treated tissues was 81.2 % after the 15 minute exposure period and 42 hours post exposure incubation period. The test item was classified as non-irritant.

As discussed above, Disperse Red 092 (FAT 40444) was found to be neither corrosive nor irritating in in vitro tests, hence considered to be not an irritant to skin.

Eye irritation

Disperse Red 092 (FAT 40444) was tested in an bovine corneal opacity test, however based on the results of this study, no prediction on the irritation potential of the substance could be made. Hence, studies with read across substances Disperse Red 086 and Disperse Red 086:1 were used to complete the assessment.

Bovine corneal opacity test with Disperse Red 092

In a bovine corneal opacity test, the test item was applied at a concentration of 20 % w/v in 0.9 % w/v sodium chloride solution for 240 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 Score for test item treated tissues was 4.0, which comes to second assessment category of >3 and ≤55, which indicates that no prediction of eye irritation can be made for the substance.

In vivo eye irritation with Disperse Red 086 (Read Across)

An in vivo study was conducted to evaluate the acute eye irritation potential of Disperse Red 086 (of ca. 93 % purity) in rabbits according to OECD Guideline 405 and EU method B5 in compliance with GLP. 0.1 mL (47 mg) of the test substance was placed into the conjunctival sac of the left eye of three male rabbits without rinsing. The right eye remained untreated and was used for control purposes. Observation were made at 1, 24, 48 and 72 h following treatment. No corneal or iridial effects were noted. However, slight conjunctival irritation was observed in all the treated animals which was reversible within the observation period of 72 h. Under the study conditions, the test substance produced irritation of the conjunctiva which was reversible within 72 h, hence, the substance is considered as not an eye irritant.

In conclusion, Disperse Red 092 was tested in a bovine corneal opacity test, in which IVIS (in vitro irritation score) was calculated to be 4.0, which comes to second assessment category of >3 and ≤55, which indicates that no prediction of eye irritation can be made for the substance. This was regarded as a borderline case as the IVIS was close to the lower limit of 3. However, the read across substance Disperse Red 086 when tested in an in vivo eye irritation study, was considered to be not an eye irritant. Hence, based on the above discussion, Disperse Red 092 was also considered to be not an eye irritant.

Justification for classification or non-classification

Based on the above discussion, the substance does not warrant the classification for irritation according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.