Reaction products of naphthalene, benzyl alcohol, sulphuric acid, formaldehyde and ammonia

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 30, 2018 to February 14, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium 1,1'-methylenebis[(phenylmethyl)naphthalene-2-sulphonate]

EC Number:

284-353-8

EC Name:

Diammonium 1,1'-methylenebis[(phenylmethyl)naphthalene-2-sulphonate]

Cas Number:

84852-41-5

Molecular formula:

C35H38N2O6S2.2H3N

IUPAC Name:

Diammonium 1,1'-methylenebis((phenylmethyl)naphthalene-2-sulphonate)

Constituent 2

Reference substance name:

Benzyl methylene-bis-napthalene-2-sulphonic acid ammonium salt

IUPAC Name:

Benzyl methylene-bis-napthalene-2-sulphonic acid ammonium salt

Constituent 3

Reference substance name:

Isomers of Benzyl methylene-bis-naphthalene trisulphonic acid ammonium salt

Molecular formula:

Not applicable

IUPAC Name:

Isomers of Benzyl methylene-bis-naphthalene trisulphonic acid ammonium salt

Constituent 4

Reference substance name:

Ammonium sulphate

EC Number:

231-984-1

EC Name:

Ammonium sulphate

Cas Number:

7783-20-2

Molecular formula:

(NH4)2SO4

IUPAC Name:

diammonium sulfate

Constituent 5

Reference substance name:

Unidentified isomers formed during the reaction

Molecular formula:

Not applicable

IUPAC Name:

Unidentified isomers formed during the reaction

Constituent 6

Reference substance name:

Formaldehyde

EC Number:

200-001-8

EC Name:

Formaldehyde

Cas Number:

50-00-0

Molecular formula:

C H2 O

IUPAC Name:

formaldehyde

Test material form:

solid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Isolated corneas from the eyes of freshly slaughtered cattle.
- Age at study initiation: 3.5 to 4.8 years.

Test system

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL / VEHICLE
- Amount applied: Volume of 750 µL applied to each cornea consisting of 20% w/v of test item in distilled water (Mysore Research Chemical Laboratories; Batch number: 7787).

Duration of treatment / exposure:

3 hours and 58 minutes.

Observation period (in vivo):

After the incubation period the corneas were washed with EMEM without phenol red and incubated in a horizontal position for 95 minutes.

Number of animals or in vitro replicates:

Nine eyes were selected, with three corneas apportioned to each of the three treatment groups (positive control, negative control, and test item).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Upon arrival to the test facility, eyes were examined for defects including opacity, scratches and neovascularization. Only corneas free of such defects were used in the experiment. Before the start of experiment, empty cornea holder’s opacity was measured and the mean opacity value obtained was considered as I0. Corneas free of defects were dissected with 2 to 3 mm rim of sclera.

QUALITY CHECK OF THE ISOLATED CORNEAS
Isolated corneas were mounted in designated corneal holders by placing the endothelial side of the cornea against the O-ring of the posterior chamber. The anterior chamber was placed over the cornea and both the chambers were joined together by tightening the chamber screws. The corneal holders were equilibrated for at least 1 hour by allowing the corneas to equilibrate and to achieve normal metabolic activity with Minimum Essential Medium (MEM) with 1% Fetal Bovine Serum and supplemented with 1% antibiotics - Penicillin and Streptomycin (MEM) in an incubator at 32±1ºC. Fresh pre-warmed MEM was added to both the chambers of cornea holders after completion of equilibrium period and baseline opacity were recorded for each cornea.
Opacity of each cornea was calculated by using a formula I0/I and opacity value was calculated for initial readouts (Before Treatment) by using the formula [(I0/I-b)/a] where a=0.0251, b=0.9894.
Corneas lesser than 7 opacity units, after an initial 1-hour equilibration period were used for the experiment.

NUMBER OF REPLICATES: Triplicate.

NEGATIVE CONTROL USED: Distilled water only from Mysore Research Chemical Laboratories.

POSITIVE CONTROL USED: Imidazole from VETEC.

APPLICATION DOSE AND EXPOSURE TIME
750 µL of distilled water, imidazole (20 % w/v), and test item (20 % w/v).

TREATMENT METHOD: Sealed chamber during exposure.

POST-INCUBATION PERIOD: Yes, 95 minutes at 32 ± 1 ºC.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Treated corneas were washed with EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was measured with an aid of opacitometer.
- Corneal permeability: The passage of sodium fluorescein dye was measured after 95 minutes of incubation with the aid of VIS spectrophotometry as optical density at 490 nm (OD490).
- Others (e.g, pertinent visual observations, histopathology): Histopathological examination.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
- Test item of IVIS score, ≤ 3 would be considered as UN GHS no category.
- Test item of IVIS score, >3 and ≤ 55 would be considered as 'no prediction can be made', subsequently testing with any other adequate method remains at the discretion of the sponsor.
- Test item of IVIS score, > 55 would be considered as severe irritant causing serious eye damage and classified as UN GHS category 1 without further testing.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

4-hour exposure

Value:

46.54

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: corneal permeability

Run / experiment:

4-hour exposure

Value:

1.488

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

4-hour exposure

Value:

68.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Opacity and permeability values were less than the established upper limits of background opacity and permeability values for bovine corneas.
- Acceptance criteria met for positive control: Yes. The IVIS score was within two standard deviations of the historical mean.

Any other information on results incl. tables

Summary of In Vitro Opacity Scores

Group & Treatment	Initial Opacity Reading (I)	Opacity Reading (After Treatment)	Change in Opacity Value	Mean Change in Opacity Value	Corrected Opacity Value	Average of Permeability Value	Corrected Permeability Value	Mean of Corrected Opacity Value	Corrected Permeability Value	IVIS Value
G1 & Vehicle Control	3.6	6.9	3.3	2.4	2.4	0.078	0.099	-	-	-
	3.0	4.2	1.2			0.124
	1.2	3.9	2.6			0.094
G2 & Positive Control	3.5	100.9	97.4	129.4	95.0	1.767	1.668	127.00	1.634	151.5
	3.4	156.1	152.8		150.4	1.710	1.611
	1.2	139.2	138.0		135.6	1.720	1.622
G3 & Test Item	3.4	56.2	52.8	48.9	50.5	1.679	1.580	46.54	1.488	68.9
	3.2	47.0	43.8		41.4	1.656	1.557
	2.8	52.9	50.1		47.7	1.427	1.328

IVIS: In Vitro Irritancy Score.

 

Note:

Change in Opacity Value = Opacity reading (after treatment) - Initial opacity reading (I)

Corrected Opacity Value = Change in opacity value of positive/negative control/test item – mean change in opacity value of vehicle control.

Corrected Permeability Value = Average of permeability value of positive/negative control/test item - mean average of permeability value of vehicle control.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Following a Bovine Corneal Opacity and Permeability (BCOP) test for aryl sulphonate condensate, a mean corneal opacity and permeability score of 46.54 and 1.488 was calculated, respectively, and an In Vitro Irritancy Score (IVIS) of 68.9. The IVIS score is greater than the threshold value of 55 and indicative of corrosivity and severe irritancy to bovine corneas. Subsequently, the registered substance can be regarded as a Category 1 eye irritant (irreversible effects on the eye) (CLP Regulation (EC) No 1272/2008).

Executive summary:

A study was undertaken for aryl sulphonate condensate to ascertain its eye irritation potential. This was achieved in line with Good Laboratory Practice (GLP) and 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). Nine cattle eyes collected from a slaughter house that were free of defects and with opacity units <7 were selected for use in the experiment. Aryl sulphonate condensate was applied to corneas within a distilled water vehicle at a volume of 750 µL (20% w/v) (n = 3). A negative control consisting of 750 µL distilled water and a positive control consisting of 750 µL imidazole (20 % w/v) was prepared (n = 3). All corneas were incubated within anterior chambers, in designated cornea holders, for a period of approximately 4 hours at 32 ± 1 ºC. Opacity was measured with an opacitometer and permeability was measured spectrophotometrically at 490 nm (OD490) following an addition post-exposure incubation of 90 minutes at 32 ± 1 ºC. Baseline opacity and permeability values obtained for blank (media) treated corneas were used for correction.

All acceptance criteria were fulfilled. The mean corrected opacity and permeability values of the test were determined to be 46.54 and 1.488, respectively. An In Vitro Irritancy Score (IVIS) of 68.9 was calculated in addition, which is suggestive of a severe irritating / corrosive effect to the corneas. Histopathological examination revealed that the registered substance had resulted in mild and minimal focal vacuolation that was especially pronounced in the wing layer of the epithelium. A classification of serious eye damage / irritation with irreversible effects on the eye (Category 1) has subsequently been assigned to aryl sulphonate condensate (CLP Regulation (EC) No 1272/2008).