4-anilinobenzenediazonium hydrogen sulphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 16th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

October 9th, 2017

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)

Version / remarks:

February 14th, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-anilinobenzenediazonium hydrogen sulphate

EC Number:

224-756-8

EC Name:

4-anilinobenzenediazonium hydrogen sulphate

Cas Number:

4477-28-5

Molecular formula:

C12H11N3O4S

IUPAC Name:

4-anilinobenzene-1-diazonium hydrogen sulfate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

The test item suspension for the test was prepared just before the use and protected from light to avoid the degradation of the sample.

Test animals / tissue source

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany.
- Number of animals: not specified.
- Characteristics of donor animals (e.g. age, sex, weight): the cattle were between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): the eyes were removed as soon as possible after death of the cattle and immersed in Hanks’ Balanced Salt Solution with 1 % Penicillin-Streptomycin solution (Penicillin 100 U/ml, Streptomycin 100 μg/ml) in a suitable cooled container. Then, they were transported to the laboratory within 1 hour where they were immediately used for the test.
- Indication of any existing defects or lesions in ocular tissue samples: after the arrival of the corneas at the laboratory, they were examined and only corneas which were free from damages were used. Before performing the test, the baseline opacity was measured by placing the holder with the cornea in an opacitometer and recording the illuminance (unit: LUX). None of the corneas showed tissue damage.
- Indication of any antibiotics used: 1 % Penicillin-Streptomycin solution (Penicillin 100 U/ml, Streptomycin 100 μg/ml).

Test system

Vehicle:

Hank's balanced salt solution

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): test item is a solid non-surface-active substance. It was tested as a pipettable suspension with a concentration of 20 % in Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10).

VEHICLE: Hank’s Balanced Salt Solution (HBSS).

Duration of treatment / exposure:

4 hours at 32 ± 1 °C.

Number of animals or in vitro replicates:

3 replicates for test item, for positive control and for negative control (total 9 treatments).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C. On the day of the assay, the MEM without phenol red was supplemented with sodium bi-carbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS:
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. The baseline opacity was measured by placing the holder with the cornea in an opacitometer and recording the illuminance (unit: LUX). None of the corneas showed tissue damage; therefore, all corneas were used.

NUMBER OF REPLICATES: 3 replicates of test item, of positive control and of negative control (total 9 treatments).

NEGATIVE CONTROL USED: Hank’s Balanced Salt Solution (HBSS).

POSITIVE CONTROL USED: Imidazole, 20 % solution in Hank’s Balanced Salt Solution (HBSS).

APPLICATION DOSE AND EXPOSURE TIME: 750 µl negative control solution, 750 µl test item suspension and 750 µl positive control solution were applied to each replicate.

TREATMENT METHOD: open chamber method.
In order to apply the test item, the nut was unscrewed to remove the glass disc. 750 µl of the test item were tested as suspension at 20 % concentration in HBSS. The test item was given directly on the epithelium that the cornea was completely covered with test item. Exposure time on the corneas was 4 hours at 32 ± 1 °C.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE and OPACITY/PERMEABILITY MEASUREMENTS:
After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded. After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 ml sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas. For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/ml was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.
Opacity = [I0 /I )-b] / a
a = 0.0251 and b = 0.9894 being Opacitometer-specific empirically determined variables
I0 = the empirically determined illuminance through a cornea holder with windows and Medium, here: Io= 1066.73
I = the measured illuminance (unit: LUX)
- Corneal permeability: the corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference + (15 x corrected OD492 value)
The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]
Note: All calculations are performed with unrounded values. Therefore, recalculation with rounded values may lead to slightly different results.

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

ACCEPTANCE CRITERIA:
The experiment is considered valid if:
- the mean value of the IVIS of the positive control should be within 2 standard deviations of the current historical mean;
- the negative control shows an IVIS ≤ 3.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

test item

Value:

ca. 279.76

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean of 3 replicates

Other effects / acceptance of results:

RESULTS:
In vitro irritancy score for test item was 279.76 (mean of three replicates). The test item 4-anilinobenzenediazonium hydrogen sulphate induced serious eye damage on the cornea of the bovine eye that should be classified as UN GHS category I. The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.
In the negative control, no signs of eye irritation were observed.
The positive control induced serious eye damage, which would be classified as GHS category I.

ACCEPTANCE OF RESULTS:
- Acceptance criteria was met for negative control: IVIS negative control as mean of 3 replicates: 0.16 (required ≤ 3).
- Acceptance criteria was met for positive control: IVIS positive control mean value of 3 replicates: 94.01 (required within 2 standard deviations of the current historical mean, i.e within 70.56 - 157.04).

Any other information on results incl. tables

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS %
Negative Control HBSS	0.24	0.16	233.96*
	0.49
	-0.25
Test Item 4-anilinobenzenediazonium hydrogen sulphate	301.50	279.76	7.00
	263.52
	274.26
Positive Control 20% imidazole solution	87.71	94.01	5.87
	96.33
	98.00

*the high relative standard deviation of theIVISof negative control is due to mathematical rea-sons, as the respective means are very small.

Applicant's summary and conclusion

Interpretation of results:

other: causes serious eye damage according to the CLP Regulation (EC n.1272/2008).

Conclusions:

4-anilinobenzenediazonium hydrogen sulphate causes serious eye damage according to the CLP Regulation (EC n.1272/2008).

Executive summary:

The potential to induce eye damage of 4-anilinobenzenediazonium hydrogen sulphate was examined according to OECD TG 437 (Bovine Corneal Opacity and Permeability - BCOP).

The test item 4-anilinobenzenediazonium hydrogen sulphate was incubated on the cornea of a bovine eye for 4 hours at 32 ± 1 °C.

After removal of the test item, opacity and permeability values were measured. Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) is 0.16.

20 % imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 94.01.

Under the conditions of this study, the test item 4-anilinobenzenediazonium hydrogen sulphate induced serious eye damage on the cornea of the bovine eye. The calculated IVIS is 279.76.

According to OECD TG 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage and it is classified as UN GHS Category I or H318 (eye damage cat.1) according to the CLP Regulation (EC n.1272/2008).