Ammonium 3-nitrobenzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-26 - 2018-01-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 438 (Isolated Chicken Eye 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:

OECD Guideline for the Testing of Chemicals No. 438 (2017): “Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classifications for Eye Irritation or Serious Eye Damage”

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

EU Method B.48.: “Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage”. Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium 3-nitrobenzoate

EC Number:

242-965-2

EC Name:

Ammonium 3-nitrobenzoate

Cas Number:

19328-56-4

Molecular formula:

C7H5NO4.H3N

IUPAC Name:

ammonium 3-nitrobenzoate

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: licensed slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice
- Characteristics of donor animals (e.g. age, sex, weight): healthy approximately 7-week-old chickens whose body weight ranged from 1.5 to 2.5 kg killed for consumption
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): After sedation of the chickens by electric shock and incision of the neck for bleeding, their heads were transported to the laboratory in a plastic container at ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container.
- Time interval prior to initiating testing: The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 30 minutes. All eyeballs used in the test came from the same group of eyes collected on a specific day.
- indication of any existing defects or lesions in ocular tissue samples: After a careful excision of the eyelids so as not to damage the cornea, the eyeballs were checked for the presence of damage. The surface of the eyeballs was treated with a 2% fluorescein solution (w/v) for a few seconds in order to evaluate the corneal integrity. After the removal of the dye by rinsing the corneal surface with a physiological salt solution, fluorescein retention and corneal opacity scores were determined using the slit-lamp microscope BP 900 LED (HAAG STREIT) to ensure that the cornea was undamaged. Only eyeballs without damage were dissected.
- Indication of any antibiotics used: none stated

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): 0.03 g
- Concentration (if solution): undiluted, ground to a powder

Duration of treatment / exposure:

240 min

Duration of post- treatment incubation (in vitro):

30 min

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Only eyeballs without damage were dissected.
The enucleated eyeball was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to the superfusion apparatus so that the entire cornea was supplied with the physiological saline drip.
After the insertion of the eyeballs to the clamp, another evaluation of fluorescein retention, corneal opacity and corneal swelling was performed. Results of corneal opacity and fluorescein retention for all examined eyeballs were equal to or less than 0.5. Deviation of corneal thickness for all examined eyeballs was less than 10%. The corneal thickness was determined using of the depth measuring device no. 1 on the slitlamp microscope and using of SP-100 pachymeter (TOMEY). The slit-width on slitlamp microscope was set at 0.095 mm. The pachymeter measures the period of time from emitting ultrasounds to receiving the same ultrasound, reflected by corneal posterior part. Applying a calculation formula shown below, corneal thickness was calculated.
L=V x t/2, where L- corneal thickness; V-ultrasound velocity trough cornea; t-measured time.
Prior to the application of the test item and the control items, all examined and approved eyeballs were incubated at temperature of 32°C ± 1.5°C for 45-60 minutes in order to equilibrate them to the test system. During the incubation, the eyeballs were continuously supplied with physiological saline at constant temperature of 33 ± 0.5°C and in the average volume of 0.10-0.15 mL/min.
After that, a zero reference measurement for corneal thickness and opacity was recorded. It served as a baseline (i.e. time = 0). The fluorescein score determined at dissection of eyeball served as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
The test item and the control items were tested on three eyeballs each.

NEGATIVE CONTROL USED
0.03 ml physiological saline

POSITIVE CONTROL USED
0.03 g imidazole

APPLICATION DOSE AND EXPOSURE TIME
The test item and the control items were uniformly applied to the corneal surface for 10 seconds. Then, they were rinsed from the eye with 20 mL of physiological saline at ambient temperature. Next, the eyeballs in their holders were placed in the superfusion apparatus in the original upright position. The additional rinsing was not necessary.

OBSERVATION PERIOD
The corneas treated with the test item and the control items were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The test item and the control items were uniformly applied to the corneal surface for 10 seconds. Then, they were rinsed from the eye with 20 mL of physiological saline at ambient temperature. Next, the eyeballs in their holders were placed in the superfusion apparatus in the original upright position. The additional rinsing was not necessary.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: see below, scoring system
- Damage to epithelium based on fluorescein retention: see below, scoring system
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: see below, scoring system
- Macroscopic morphological damage to the surface: Gross evaluation of the treated corneas: The aim of gross evaluation was to determine whether any morphological effects, e.g. pitting of corneal epithelial cells, roughening of the corneal surface, and sticking of the test item to the cornea were visible.
- Others (e.g, histopathology): Histopathological evaluation of the treated corneas: Following the final evaluation of the treated eyeballs (240 minutes after the application of the test item and the control items), the eyeballs were fixed in a 4% solution of formaldehyde for at 24 hours. Next, specimens were collected (one specimen in the plane including the cornea, lens with the surrounding sclera, and optic nerve). The tissue material was dehydrated (2 hours in a mixture of 4% phenol/70% ethanol; 2 hours in 95% ethanol; 3 hours in 100% ethanol; 2 hours in equal volumes of xylene and 100% ethanol; 4 hours in xylene) and prepared using a paraffin technique. Paraffin blocks were cut into smaller parts, whose thickness was 5 μm, with a microtome and stained using Hematoxylin and Eosin. The histological preparations were evaluated under a light microscope. The following layers of the cornea were evaluated: the anterior corneal epithelium, the anterior elastic lamina (Bowman’s membrane), the corneal stroma, the posterior elastic lamina (Descemet’s membrane), the posterior corneal epithelium. All treated eyeballs were subject to this evaluation

SCORING SYSTEM:
- Mean corneal swelling (%) :
The degree of corneal swelling was determined by measuring corneal thickness using a slit-lamp microscope and a pachymeter (30 minutes after treatment only pachymeter was used). It was expressed as a percentage and calculated according to the following formula:
corneal swelling = [(corneal thickness at time t – corneal thickness at time t 0) / corneal thickness at time t 0] * 100
The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time
points. Based on the highest mean score for corneal swelling, as observed at any time point, the final
category score for the test item and the control items was given.

- Mean maximum opacity score :
Corneal opacity was determined by assigning appropriate values to opaque areas. The mean corneal opacity value for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, the final category score for the test item and the control items was given.
The corneal opacity scores are presented in the table below.
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent areas; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment :
The mean fluorescein retention value for all test eyeballs was calculated on the grounds of the observations made 30 minutes after the end of the exposure. This value was used to determine the general category score for the test item and the control items. The fluorescein retention scores are presented in the table below.
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: The decision criteria as indicated in the TG were used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

fluorescein retention score

Run / experiment:

test item

Value:

2.7

Vehicle controls validity:

not applicable

Negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

other: ICE class IV

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

test item

Value:

2.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

test item

Value:

8.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Results 4

Irritation parameter:

other: gross evaluations

Run / experiment:

test item

Remarks on result:

other: The deposits of the test item were observed on the cornea surface.

Results 5

Irritation parameter:

histopathological observations

Run / experiment:

test item, eyeball No. 1

Remarks on result:

other: vacuolation, necrosis and erosions of the anterior corneal epithelium

Results 6

Irritation parameter:

histopathological observations

Run / experiment:

test item, eyeball No. 2

Remarks on result:

other: necrosis and erosions of the anterior corneal epithelium, proliferation of the anterior corneal epithelium cells

Results 7

Irritation parameter:

histopathological observations

Run / experiment:

test item, eyeball No. 3

Remarks on result:

other: vacuolation, necrosis and erosions of the anterior corneal epithelium, vacuolation of the posterior corneal epithelium

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

According to UN GHS classification criteria “no prediction can be made” (1 x IV, 1 x III, 1 x II)

Conclusions:

The study was conducted under GLP according to OECD guideline 438 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating / corrosive potential of the test substance to the eye in vitro.
On the grounds of the study results described in this Report and the overall in vitro Irritancy Classification, it may be stated that the test item, i.e. Ammonium-3-nitrobenzoate, probably did not induce serious eye damage, but it can have negative effect on cornea. According to UN GHS classification criteria “no prediction can be made”
On the basis of the results obtained in the course of the histopathological evaluation it can be concluded that the test item can have a negative effect on the chicken cornea in the ICE test.
Hence, no conclusion can be drawn whether the substance needs to be classified as GHS category 1 or 2, for a clear distinction an in vivo study would be required based on the current state of the art. However, based on the tonnage band of the substance, in vivo testing is not required, and with regard to animal welfare no further testing will be performed and the result of the present study will be considered inconclusive. Taking into account the fact that the substance is not corrosive but irritating to the skin, a precautionary classification as Eye Irr. Cat. 2 will be made.

Executive summary:

The Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classifications for Eye Irritation or Serious Eye Damage was performed in order to obtain information on health hazards resulting from a superficial influence of the test item, i.e. Ammonium-3-nitrobenzoate, on the eye, according to OECD 438 under GLP.

The eyeballs were collected from chickens obtained from a licensed slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice where they were killed for human consumption.

In the isolated chicken eye test (in vitro), toxic effects to the cornea were measured by a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention), a qualitative assessment of opacity, a quantitative measurement of increased thickness (swelling), and qualitative macroscopic and histopathological evaluations of morphological damage to the surface.

The study was conducted on nine eyeballs. The second run was not necessary. In order to control the quality of the procedure, the eyeballs used for the purpose of the experiment were assessed for potential damage (corneal opacity, corneal thickness and fluorescein retention). Result of corneal opacity and fluorescein retention for all examined eyeballs was equal to or less than 0.5. Deviation of corneal thickness for all examined eyeballs was less than 10%. Before the application of the test item and the control items, all examined and approved eyeballs were incubated at temperature of 32 ± 1.5°C for 45-60 minutes in the superfusion apparatus in order to equilibrate them to the test system. After that, a zero reference measurement for corneal thickness and opacity was recorded.

The test item (ground to a powder) and the item used in the positive control (imidazole) were applied in the amount of 0.03 g, whereas the item used in the negative control (physiological saline) was applied in a volume of 0.03 mL. Three eyeballs were used for the test item and three for each control item. Every time, the test item and the control items were applied to the corneal surface for 10 seconds and kept at temperature between 20 – 23º C. Then, they were rinsed from the eye with 20 mL of physiological salt solution at ambient temperature. Next, the eyeballs in their holders were placed in the superfusion apparatus in the original upright position.

The corneas treated with the test item and the control items were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse. At all observation time points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure. Following the final evaluation of the treated eyeballs, i.e. 240 minutes after the application of the test item and the control items, the eyeballs were fixed in a 4% solution of formaldehyde in order to allow histopathological examinations to be conducted.

 

Results of the studies:

For eyeballs treated with the test item:

- the mean fluorescein retention value was equal to 2.7 (ICE class IV),

- the maximal mean corneal opacity value was equal to 2.3 (ICE class III),

- the maximal mean corneal swelling value was equal to 8.8 % (ICE class II),

- the test item was observed on the cornea surface

- histopathological examinations revealed: vacuolation (eyeballs no. 1, no. 3), necrosis and erosions of the anterior corneal epithelium (eyeballs no. 1, no. 2, no. 3), proliferation of the anterior corneal epithelium cells (eyeball no. 2), vacuolation of the posterior corneal epithelium (eyeball no. 3). These results can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method.

 

Interpretation of the study results:

For test item (Ammonium-3-nitrobenzoate) the prediction cannot be made (1 x IV, 1 x III, 1 x II) according to UN GHS classification criteria.