Potassium 2,5-dihydroxybenzenesulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 January to (date to be inserted)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

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)

Deviations:

no

GLP compliance:

no

Remarks:

Lab was not set up to do GLP for these specific assays. Studies were performed in accordance with the principles of GLP and relevant OECD guideline. They were monitored by QA in accordance with GLP.

Test material

Test material information

Constituent 1

Reference substance name:

Potassium 2,5-dihydroxybenzenesulphonate

EC Number:

244-584-7

EC Name:

Potassium 2,5-dihydroxybenzenesulphonate

Cas Number:

21799-87-1

Molecular formula:

(HO)2C6H3SO3K

IUPAC Name:

potassium 2,5-dihydroxybenzenesulphonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: The Dow Chemical Company
- Lot No.of test material: YY00G7P285
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from exposure to light
- Stability under test conditions: Assumed stable for the durstion of the study
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was administered to the test system as a 20% (w/v) dilution in sterile, deionized water. Stability not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item (white crystalline powder) diluted to 20% (w/v) dilution in sterile, deionized water.
- Final dilution of a dissolved solid: 20% (w/v) dilution in sterile, deionized water.

FORM AS APPLIED IN THE TEST (if different from that of starting material) . As instructed by the Sponsor, the test substance was administered as a 20% (w/v) dilution in sterile, deionized water. The test article dilution was prepared by weighing the test article into a prelabeled conical tube. Sterile, deionized water added until a 20% (w/v) dilution ws acheived, and the vial vortexed mixed prior to application.

Test animals / tissue source

Species:

other: Bovine corneas, obtained as a by-product from freshly slaughtered animals.

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals (J.W. TREUTH & SONS, Inc., Baltimore, MD) (United States)
- Number of corneas: Three corneas were used in the presence of the positive or negative control. Five corneas were used in the presence of the test substance.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
- Time interval prior to initiating testing: The eyes were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice packs. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated.

- indication of any existing defects or lesions in ocular tissue samples: The eyes were grossly examined for damage and those exhibiting defects were discarded.
- Indication of any antibiotics used: The eyes were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice packs.

Test system

Vehicle:

water

Remarks:

sterile deinionized water

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume with unit): 750 µL of the test substance
- Concentration (if solution): The solid test substance, K-Salt, was tested as a 20% (w/v) dilution in sterile, deionized water.

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL of negative control, sterile, deionized water (Quality Biological).

Duration of treatment / exposure:

4 hour exposure at 32 ± 1ºC

Duration of post- treatment incubation (in vitro):

90 minutes at 32 ± 1ºC

Number of animals or in vitro replicates:

Three corneas were incubated in the presence of the positive or negative control at 32 ± 1ºC for approximately 4 hours.
Five corneas were incubated in the presence of the test substance at 32 ± 1ºC for approximately 4 hours.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS : The eyes were examined for damage, any exhibiting defects were discarded. The tissue surrounding the eyeball was carefully removed and the cornea was excised such that a 2 to 3 mm rim of sclera was present around the cornea. The isolated corneas were then stored in a Petri dish containing HBSS until they were mounted in a corneal holder. The corneas were mounted in holders with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and the screws tightened. Starting with the posterior chamber, the two chambers were then filled with Minimum Essential Medium (EMEM) without phenol red, containing 1% fetal bovine serum and 2 mM L glutamine (Complete MEM (without phenol red)). Each corneal holder was uniquely identified with a number in permanent marker, on both the anterior and posterior chambers. The corneal holders were incubated at 32 ± 1ºC for a minimum of 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS: The eyes were grossly examined for damage and those exhibiting defects were discarded.

NUMBER OF REPLICATES : Three corneas were incubated in the presence of the positive or negative control at 32 ± 1ºC for approximately 4 hours. Five corneas were incubated in the presence of the test substance at 32 ± 1ºC for approximately 4 hours.

NEGATIVE CONTROL USED : Sterile, deionized water (Quality Biological)

POSITIVE CONTROL USED : 20% dilution of imidazole in complete MEM

APPLICATION DOSE AND EXPOSURE TIME : An aliquot of 750 µL of test substance, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea, and incubated at 32 ± 1ºC for approximately 4 hours.
After the 4-hour exposure period, the controls or test substance treatments were removed. The epithelial side of each cornea was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the controls or test substance. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior and the posterior chambers were refilled with fresh Complete MEM (without phenol red) and a final opacity measurement was performed immediately (with no further post-exposure incubation).

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: yes, corneas were then incubated for approximately 90 minutes at 32 ± 1ºC.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After 4-hour exposure, the controls or test substance treatments were removed. The epithelial side of each cornea was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the controls or test substance. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior and the posterior chambers were refilled with fresh Complete MEM (without phenol red) and a final opacity measurement was performed immediately (with no further post-exposure incubation).

- POST-EXPOSURE INCUBATION: No post-exposure incubation after test substance removal.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Opacity measurement: The change in opacity for each cornea (including the negative control corneas) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of each cornea for that treatment condition.

- Corneal permeability:
Permeability Measurement: The mean OD490 value for the blank wells was calculated. The mean blank OD490 value was then subtracted from the raw OD490 value of each well (corrected OD490). Any dilutions that were made to bring the OD490 readings into the linear range of the platereader (OD490 should be less than 1.500), had each diluted OD490 reading multiplied by the dilution factor. The final corrected OD490 values of the test substance and the positive control were then calculated by subtracting the average corrected OD490 value of the negative control corneas from the corrected OD490 value of each treated cornea.

- Others (e.g, pertinent visual observations, histopathology): None

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) : For regulatory purposes, the In Vitro Score (IVIS) cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category I) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are found in the table below (OECD 437, adopted 26 July 2013). This guidance on categorization applies only to test substances evaluated using the appropriate standard protocols as described in OECD 437 .

IVIS UN GHS

<3 No Category
>3; <55 No prediction can be made
>55 Category I

For non-regulatory purposes, the following classification system was established by Sina et al. (1995) based on studies with a wide range of test materials. While this classification system provides a good initial guide to interpretation of these in vitro data, these specific ranges may not be applicable to all classes of materials or other exposure times. Whenever possible, results should be compared to “benchmark” materials tested under similar exposure conditions.

In Vitro Score:

≤ 25 = mild irritant
from 25.1 to 55 = moderate irritant
from 55.1 and above = severe irritant

In addition, Vanparys et al. (1993) proposed a modification of the Sina classification system based on an evaluation of 50 pharmaceutical and commercially available industrial compounds. The modification allows for the identification of a non-irritant classification.

In Vitro Score:
from 0 to 3 = non irritant
from 3.1 to 25 = mild irritant
from 25.1 to 55 = moderate irritant
from 55.1 and above = corrosive/severe irritant


DECISION CRITERIA:
Criteria for Determination of a Valid Test: The BCOP assay was accepted when the positive control (imidazole) caused an in vitro score that fell within two standard deviations of the historical mean. The results of the positive control fell within two standard deviations of the historical mean (within a range of 66.8 to 127.5) and the assay was considered valid.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1 run

Value:

80.7

Vehicle controls validity:

not specified

Positive controls validity:

valid

Remarks:

The results of the positive control fell within two standard deviations of the historical mean (range: 66.8 to 127.5). The assay was therefore considered valid.

Any other information on results incl. tables

Assay Date	IIVS Test substance No.	Sponsor’s Designation	Conc.	Exposure Time	Opacity Value	OD490Value	In VitroScore	pH
9 Jan 18	17AJ00	K-Salt	20%	4 hours	-3.0	-0.007	-3.1	2.5
9 Jan 18	Positive control	Imidazole	20%	4 hours	62.3	1.224	80.7	-

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

According to the current OECD TG 437 the test article is predicted to not require classification or labelling for ocular irritation according to the Globally Harmonized System (GHS No Category).

Conclusions:

Based on the in vitro score of -3.1, the test substance, K-Salt, would be classified as a non-irritant according to the classification system established by Vanparys et al. (1993), where a substance that induces an in vitro score 0 to 3 would be predicted to be a non-irritant to the eye.
According to the current OECD TG 437 the test article is predicted to not require classification or labelling for ocular irritation according to the Globally Harmonized System (GHS No Category).