Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo]naphthalenesulphonate

Endpoint summary

Description of key information

Skin irritation

The dermal irritation potential of test chemical was assessed in an experimental study conducted on rabbits. Also the predicted data for target chemical using the Danish QSAR database has also been compared with the experimental data. Based on the available data for the key and supporting studies, it can be concluded that the test chemical is unable to cause skin irritation and thus considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

 

Eye irritation

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

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Sustainability Support Services (Europe) AB, Sweden
- Lot/batch No.of test material: 0015
- Expiration date of the lot/batch: 13 Feb.; 2021
- Purity test date: No data

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: No data
- Specific activity: No data
- Locations of the label: No data
- Expiration date of radiochemical substance: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient Temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item was moistened with distilled water before application.
- Preliminary purification step (if any):No data
- Final dilution of a dissolved solid, stock liquid or gel: No data
- Final preparation of a solid: No data

FORM AS APPLIED IN THE TEST (if different from that of starting material) : No data

OTHER SPECIFICS:
Safety Precautions: Safety precautions included use of protective clothing, gloves, masks and eye protection (glasses).

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Age at study initiation: Young adult male and female rats aged between 6 – 9 weeks were used.
- Weight at study initiation: The weight ranges of approximately 210.2 to 245.4 grams at initiation of dosing were used.
Body weights at the start :
Male
Mean : 240.40 g (= 100 %)
Minimum : 234.6 g (- 2.41 %)
Maximum : 245.4 g (+ 2.08 %)
Total No. of animals : 5
Female
Mean : 215.98 g (= 100 %)
Minimum : 210.2 g (- 2.68 %)
Maximum : 220.6 g (+ 2.14 %)
Total No. of animals : 5
- Housing: The rats were individually housed in polycarbonate cages with paddy husk as bedding.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature was maintained at 20.1 to 21.9 degree centigrade.
- Humidity (%): Room humidity was maintained at 55.5% to 59.2%.
- Air changes (per hr): The animal room was independently provided with at least ten to fifteen air changes per hour of 100% fresh air that had been passed through the HEPA filters.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

IN-LIFE DATES: From: To: No data

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- Concentration (if solution): no

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): No data
- Concentration (if solution): No data

POSITIVE CONTROL
- Amount(s) applied (volume or weight): No data
- Concentration (if solution): No data

Duration of treatment / exposure:

24 hrs.

Observation period:

14 days

Number of animals:

10 (5/sex).

Details on study design:

TEST SITE
- Area of exposure: Dorsal surface and sides from scapular to pelvic area.
- % coverage: Approximately 10% of the total body surface area.
- Type of wrap if used: Porous gauze dressing and non-irritating tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Distilled water was used to remove residual test item.

OBSERVATION TIME POINTS
(Indicate if minutes, hours or days): Observations were conducted at 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day.

SCORING SYSTEM: Draize Method.

Irritation parameter:

overall irritation score

Basis:

animal: 1 - 10

Time point:

other: 0 - 14 d

Score:

0

Reversibility:

other: no data

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Overall result:
Sex : Male
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.

Sex : Female
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.

Other effects:

Clinical Signs of Toxicity and Mortality
Sex : Male
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.

Sex : Female
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.

Body Weight
Sex : Male
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 8.88% and 18.87% respectively.

Sex : Female
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.29% and 9.60% respectively.

Gross Pathological Findings
Gross pathological examination did not reveal any abnormalities in animals from 2000 mg/kg dose group.

Table No. I

 

Summary of Evaluation of Dermal Reaction

Test System : Sprague Dawley Rat

Sex : Male 

Group  No.	Dose mg/kg	Dermal Reaction	Total Number of Animals	Animal Nos.	Period of signs in days  From - to	Mortality
I	2000	No dermal reaction observed	5	1 - 5	Day 0 - Day 14	0/5

 

 

Sex : Female

 

Group  No.	Dose mg/kg	Dermal Reaction	Total Number of Animals	Animal Nos.	Period of signs in days  From - to	Mortality
I	2000	No dermal reaction observed	5	6 - 10	Day 0 - Day 14	0/5

 

 Table No. II

 

Individual Animal - Evaluation of Dermal Reaction

Test System : Sprague Dawley Rat

Sex : Male  

Group : I

Dose  : 2000 mg/kg body weight

Animal	Dermal	D A Y S
No.	Reaction	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14
1	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
3	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
4	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
5	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

 

 

Sex : Female  

Group : I

Dose  : 2000 mg/kg body weight

Animal	Dermal	D A Y S
No.	Reaction	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14
6	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
7	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
8	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
9	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
10	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Oedema	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Interpretation of results:

other: Not irritating

Remarks:

on the basis of EU Criteria

Conclusions:

From the study, it can be concluded that the test substance ‘Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalenesulphonate’ is nonirritant to the skin of Sprague Dawley rats when applied to the shorn skin of 5 male and 5 female animals at the tested dose level of 2000 mg/kg body weight. Also the erythema and edema score of rats was calculated as 0. Thus it can be concluded that the substance Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalenesulphonate can be classified under nonirritant category.

Executive summary:

The study now reported was designed and conducted to determine the dermal reaction profile of Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalenesulphonate in Sprague Dawley rats.

The test item 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. 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.

From the study, it can be concluded that the test substance ‘Disodium 4-hydroxy-3-[(4-sulphona tonaphthyl)azo] naphthalenesulphonate’ is nonirritant to the skin of Sprague Dawley rats when applied to the shorn skin of 5 male and 5 female animals at the tested dose level of 2000 mg/kg body weight. Also the erythema and edema score of rats was calculated as 0. Thus it can be concluded that the substanceDisodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalenesulphonate can be classified under nonirritant category.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Principles of method if other than guideline:

The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected inthe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (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 Laboratories, Bratislava, Slovakien.
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 In Vitro Life Science Laboratories 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:

85.1

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

mean of OD :1.550 ;Non-irritant

Other effects / acceptance of results:

The MTT data show the assay quality controls were met.

Interpretation of results:

other: Not irritating

Conclusions:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance was determined to be 85.1%. Thus, substance was considered to be not irritating to the human eyes.

Executive summary:

The ocular irritation potential of test article Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo]naphthalenesulphonate (CAS no 3567 -69 -9) was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour 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 85.1 %. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human eyes and thus cannot be classified as eye irritant 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:

An experimental study has been investigated for the test chemical to observe the potential for dermal irritation to a greater or lesser extent. This study is based on in vivo experiments in rabbits. Also the predicted data for target chemical using the Danish QSAR database has also been compared with the experimental data that have been summarized as below;

 

 

The study reported was designed and conducted to determine the dermal reaction profile of Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalenesulphonate in Sprague Dawley rats. The test item 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. 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. From the study, it can be concluded that the test substance ‘Disodium 4-hydroxy-3-[(4-sulphona tonaphthyl)azo] naphthalenesulphonate’ is nonirritant to the skin of Sprague Dawley rats when applied to the shorn skin of 5 male and 5 female animals at the tested dose level of 2000 mg/kg body weight. Also the erythema and edema score of rats was calculated as 0. Thus it can be concluded that the substanceDisodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalenesulphonate can be classified under nonirritant category.

 

The above result was supported by Danish QSAR database. Based on the QSAR prediction done using the Danish (Q)SAR Database, the skin irritation was estimated to be negative on rabbits for Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo]naphthalenesulphonate. Thus it can be concluded that the substance Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo]naphthalenesulphonate has negative skin irritation effects and based on the CLP criteria for classification it can be classified as not irritating to skin irritation effects.

 

Based on the available data for key and supporting study, it can be concluded that test chemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

 

Eye irritation

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

 

The ocular irritation potential of test article Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo]naphthalenesulphonate (CAS no 3567 -69 -9) was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour 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 85.1 %. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human eyes

 

The above result was supported by an in-vivo experimental study conducted for the structurally similar read across substance in three female New Zealand White rabbits as per OECD Guidelines 405 and complying to the GLP procedures. About 0.1g of the undiluted test chemical was instilled in the conjunctival sac of rabbits after gently pulling the lower lid away from the eyeball. The other eye which remained untreated served as a control. The ocular lesions were evaluated at 1, 24, 48 and 72 hours after the treatment. The grades of ocular reactions (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animals were observed normally for 21 days. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weights before and during the study was observed. The overall irritation index of test chemical was 0.0 after 72 hours. Also did not produce any clinical signs of toxicity throughout the examination period of 21 days. Hence, under the test conditions, the chemical can be concluded to be not irritating to New Zealand White rabbit eyes.

 

The above results were further supported by another in-vivo eye irritation study conducted by authoritative database for similar read across substance to investigate the local eye irritative potential of test chemical. Each rabbit received 2 installation of 0.2 ml of a 10% test chemical aqueous suspension into the conjunctival sac of rabbits 5 times per week for 4 weeks. The chemical produced only minimal irritating effects after 2 hours which could not confirmed the irritation potential. Hence, on the basis of findings the Test chemical can be considered as not irritating to the rabbits’ eye.

 

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

Justification for classification or non-classification

The skin and eye irritation potential of test chemical and its structurally and functionally similar read across substanceswere observed in various studies. The results obtained from these studies indicate that the chemical is not likely to cause skin irritation and eye damage. Hence the test chemical can be classified under the category “Not Classified” for skin and eye as per CLP.