Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., ammonium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2017 to 05 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, 4-sec-decyl, ammonium salts

Molecular formula:

C16H29NO3S

IUPAC Name:

Benzenesulfonic acid, 4-sec-decyl, ammonium salts

Constituent 2

Reference substance name:

Benzenesulfonic acid, 4-sec-undecyl, ammonium salts

Molecular formula:

C17H31NO3S

IUPAC Name:

Benzenesulfonic acid, 4-sec-undecyl, ammonium salts

Constituent 3

Reference substance name:

Benzenesulfonic acid, 4-sec-dodecyl, ammonium salts

Molecular formula:

C18H33NO3S

IUPAC Name:

Benzenesulfonic acid, 4-sec-dodecyl, ammonium salts

Constituent 4

Reference substance name:

Benzenesulfonic acid, 4-sec-tridecyl, ammonium salts

Molecular formula:

C19H35NO3S

IUPAC Name:

Benzenesulfonic acid, 4-sec-tridecyl, ammonium salts

Test material form:

other: Highly viscous light yellow liquid

Details on test material:

Storage Conditions: Highly viscous light yellow liquid

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: applied undiluted
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, applied as supplied
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: n/a

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Corneas from bovine eyes were obtained from a local abattoir.
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): Not reported
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility.
- Time interval prior to initiating testing: Same day
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: Penicillin at 100 IU/mL and streptomycin at 100 µg/mL added to Hanks Balanced Salt Solution during transportation.

Test system

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): 750 µL
- Concentration (if solution): undiluted

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

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

n/a

Duration of post- treatment incubation (in vitro):

Following rinsing, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied.

For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS

On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

QUALITY CHECK OF THE ISOLATED CORNEAS

The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

NUMBER OF REPLICATES

3 per treatment group

NEGATIVE CONTROL USED

The negative control substance was 0.9% sodium chloride solution (batch 17A09BB1C, expiry 13 May 2017) supplied by Baxter Healthcare Ltd., Thetford, UK.

SOLVENT CONTROL USED (if applicable)

N/A

POSITIVE CONTROL USED

The positive control substance was dimethyl formamide (DMF) (batch STBG0948, expiry March 2018) supplied by Sigma-Aldrich Company Ltd., Poole, UK.

APPLICATION DOSE AND EXPOSURE TIME

750 µL applied to each cornea and incubated at 32 ºC for 10 minutes.

TREATMENT METHOD

Closed chamber

POST-INCUBATION PERIOD

Following rinsing, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red
- POST-EXPOSURE INCUBATION: Following rinsing, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometer (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify) N/A

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As described in OECD 437.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

22.04

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control yields opacity and permeability values that are less than the established upper limits for these endpoints for bovine corneas as treated at the testing facility.
- Acceptance criteria met for positive control: The positive control yields an IVIS within 2 standard deviations of the historical control mean.
- Range of historical values if different from the ones specified in the test guideline: n/a

Any other information on results incl. tables

Table 1       Corneal Opacity

 

Test Substance	Cornea Number	Initial Opacity	Post Incubation Opacity	Change in Opacity	Mean Change in Opacity	Corrected Opacity	Mean Corrected Opacity
Test article	9	-5	4	9	N/A	10.7	7.0
	10	-4	2	6		7.7
	11	-4	-3	1		2.7
Negative	1	-4	-5	-1	-1.67	0.7	0.0
	2	-5	-7	-2		-0.3
	3	-4	-6	-2		-0.3
Positive	13	-3	66	69	N/A	70.7	73.0
	14	-5	69	74		75.7
	15	-5	66	71		72.7

 

Table 2       Corneal Permeability

 

Test Substance	Cornea Number	Mean Blank OD490	OD490	Corrected OD490	Mean Corrected OD490	Final Corrected OD490	Mean Group Corrected OD490
Test article	9		0.999	0.999	N/A	0.991	1.002
	10		0.871	0.0871		0.863
	11		1.162	1162		1.154
Negative	1		0.011	0.011	0.008	0.003	0.000
	2		0.006	0.006		-0.003
	3		0.008	0.008		0.000
Positive	13		0.121	0.121	N/A	0.113	0.416
	14		0.410	0.410		0.402
	15		0.741	0.741		0.733

 

Table 3       Calculated IVIS

 

Test Chemical	Mean Opacity	Mean Permeability	IVIS (Mean Opacity + (15 x Mean Permeability))
Test article	7.0	1.002	22.04
Negative control	0	0	0
Positive control	73.0	0.416	79.24

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

It was concluded that under the condition of this study, the test item, produced an IVIS score of 22.04 and therefore no prediction could be made in respect of its potential to cause eye irritation.

Executive summary:

OECD 437 (2017) - The Bovine Corneal Opacity and Permeability (BCOP) test was conducted using Benzenesulfonic acid, 4 -C10 -13 -sec-alkyl derivs., ammonium salts in accordance with 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" (2013).

Undiluted test item was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared.

Measurements for corneal opacity were made after 2 h incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 1 h and 25 min incubation in the vertical position with sodium fluorescein.

Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD490). The mean corrected opacity reading and permeability readings for the test item were 7.0 and 1.002, resulting in an In Vitro Irritation Score (IVIS) of 22.04.

As a result, no prediction could be made in respect to the test items potential to cause eye irritation.