Bromoacetic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From may to June 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD, EPA, etc)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Monobromo acetic acid

IUPAC Name:

Monobromo acetic acid

Constituent 2

Reference substance name:

Bromoacetic acid

EC Number:

201-175-8

EC Name:

Bromoacetic acid

Cas Number:

79-08-3

Molecular formula:

C2H3BrO2

IUPAC Name:

2-bromoacetic acid

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Monobromoacetic acid
- Physical state: solid
- Analytical purity: white to yellow crystalline solid
- Purity test date: 98%
- Lot/Batch number: Not reported
- Storage condition of test material: ambient (20°C)

Test animals / tissue source

Species:

other: Chickens eye ex vivo

Strain:

other: ROSS, spring chickens

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Poultry slaughterhouse v.d. Bor, Amersfoortseweg 118, Nijkerkerveen, the Netherlands.
- Age at study initiation: 7 weeks old (male and female)
- Weight at study initiation: body weight range: 2.5-3.0 kg

HEAD SAMPLING PROCEDURE:
Twelve heads of these animals were cut off immediately after sedation of the animals by electric shock and incission of the neck for bleeding. The heads
were placed in small plastic boxes (3 heads per box) on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the
testing facility. During transportation, the heads were kept at ambient temperature.

EYE SAMPLING PROCEDURE:
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure:
First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium BP 2% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline of ambient temperature.
Next, the head with the fluorescein-treated cornea was examined with a slit-lamp
microscope , to ensure that the cornea was not damaged. If undamaged, the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 g applied to the cornea

Duration of treatment / exposure:

10 secondes

Observation period (in vivo):

The control eye and test eyes were examined at 30, 75, 120, 180, and 240 minutes after treatment, using the criteria and scoring system.

Number of animals or in vitro replicates:

The test substance was tested on five out of the six eyes; the sixth eye was treated in a similar way with isotonic saline only and served as a control. These six eyes comes from twelve chicken heads.

Details on study design:

STUDY DESIGN
The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The six chambers of the superfusion apparatus as well as the saline were temperature controlled at 32 ± 1.5 °C.
An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the six eyes, or eyes that were unacceptably stained with fluorescein (score higher than 0.5), indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and were replaced. After an equilibration period of 45-60 minutes, the corneal thickness of the six eyes was measured again to determine the zero reference value for corneal swelling calculations. At time t = 0, i.e. immediately after the zero reference measurement, the test substance was applied to the eye. For this purpose, the clamp holding the eye was placed on a paper tissue outside the chamber with the cornea facing upwards.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the corneal surface was rinsed thoroughly with 20 ml of isotonic saline of ambient temperature.
- Time after start of exposure: 10 secondes

EXAMINATION:
- The mean % swelling was calculated over all test eyes for the observation time points of 30, 75, 120, 180, and 240 minutes.
- The mean corneal opacity value was calculated over all test eyes for the observation time points of 30, 75, 120, 180, and 240 minutes.
- Fluorescein retention

SCORING SYSTEM:
* Corneal swelling:
Corneal swelling, expressed as a percentage, was calculated according to the following formula:

(corneal thickness at time t - corneal thickness at time t=0/corneal thickness at t=0) X 100%

The mean % swelling was calculated over all test eyes for the observation time points of 30, 75, 120, 180, and 240 minutes.

Interpretation:
mean corneal swelling (%) Category
-5 - 5 I
6 - 12 II
13 - 18 (>75 min. after treatment) II
(<75 min. after treatment) III
19 - 26 III
27 - 32 (>75 min. after treatment) III
(<75 min. after treatment) IV
>32 IV

* Corneal opacity: Opacity degree of density (area most dense taken for scoring)
0 = no opacity
1 = scattered or diffuse areas, details of iris clearly visible
2 = easily discernible translucent area, details of iris slightly obscured
3 = severe corneal opacity, no details of iris visible, size of pupil barely
discernible
4 = complete corneal opacity, iris invisible

Interpretation:
maximum mean opacity Category
score
0.0 - 0.5 I
0.6 - 1.5 II
1.6 - 2.5 III
2.6 4.0 IV

* Fluorescein retention:
0 = no fluorescein retention
1 = small number of cells retaining fluorescein
2 = individual cells and areas of the cornea retaining fluorescein
3 = large areas of the cornea retaining fluorescein

The mean fluorescein retention value was calculated over all test eyes for the observation time points of 30 minutes.

Interpretation:
mean fluorescein Category
retention score
0.0 - 0.5 I
0.6 - 1.5 II
1.6 - 2.5 III
2.6 4.0 IV

Assessment ex vivo eye irritancy grade
Classification combinations of the three categories
- A. Not irritating
3 x I
2 x I, I x II
- B. Slightly irritating
3 x II
2 x II, I x I
2 x II, I x III
2 x I, I x IV
I x I, I x II, I X 111 1
- C. Moderately irritating
3 x III
2 x III, I x II
2 x III, I x IV
2 x III, I x I
2 x II, I x IV
I x II, I x III, I x IV
- D. severely irritating
3 x IV
2 x IV, I X III
2 x IV, I x II
2 x IV, I x I

- Other investigations
* Morphological effects: These include "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test substance to the cornea. The severity of these findings can vary and combinations of effects can occur. The classification of these findings was subjective to the interpretation of the investigator.

Results and discussion

In vivo

Irritant / corrosive response data:

During application of the test substance to the cornea, severe corneal opacity occurred. The severity of this opacity hampered accurate measurement of corneal thickness. Therefore, an estimation of the corneal swelling values are given in the table (see below).
After treatment with the undiluted test substance, the thickness of the cornea of all 5 test-eyes increased considerably. The maximum thickness detectable with the measurement device was already reached at 180 minutes after treatment.
Severe complete corneal opacity was observed in all 5 test eyes at all observation time points. In addition, loosening of epithelium was observed in all test eyes.
The determination of fluorescein retention at 30 minutes after treatment, revealed severe fluorescein retention of the test eyes. The control eye did not show any unusual effects. The categories for corneal swelling, corneal opacity, and fluorescein retention were IV, IV, and IV.

Any other information on results incl. tables

Individual values for corneal swelling (approximate values), corneal opacity and fluorescein retention values of the control and test eyes obtained with undiluted Bromoacetic acid.

Eye	Corneal		Fluorescein
No.	Swelling	Opacity	Retention
After 30 min
1	12.3	4.0	3.0
2	25.0	4.0	3.0
3	16.1	4.0	3.0
4	23.9	4.0	3.0
5	23.9	4.0	3.0
6 (control)	0.0	0.0	0.0
After 75 min
1	13.8	4.0
2	32.8	4.0
3	29.0	4.0
4	34.3	4.0
5	25.4	4.0
6 (control)	0.0	0.0
After 120 min
1	76.9	4.0
2	79.7	4.0
3	80.6	4.0
4	71.6	4.0
5	79.1	4.0
6 (control)	-3.1	0.0
After 180 min
1	>84.6	4.0
2	>87.5	4.0
3	>93.5	4.0
4	>79.1	4.0
5	>79.1	4.0
6 (control)	-3.1	0.0
After 240 min
1	>84.6	4.0
2	>87.5	4.0
3	>93.5	4.0
4	>79.1	4.0
5	>79.1	4.0
6 (control)	-3.1	0.0

Mean values for corneal swelling (approximate values), corneal opacity and fluorescein retention values of the test eyes treated with Bromoacetic acid, and the irritancy categories based on the maximum mean scores, and the final EC-classification.

Time intervals	Corneal		Fluorescein retention
	Swelling	Opacity
30	20.2 (2.5)	4.0 (0.0)	3.0 (0.0)
75	27.1 (3.7)	4.0 (0.0)
120	77.6 (1.6)	4.0 (0.0)
180	>84.8 (2.7)	4.0 (0.0)
240	>84.8 (2.7)	4.0 (0.0)
Parameter
Cornel swelling	>84.8	IV
Corneal capacity	4.0	IV
Fluorescein retenson	4.0	IV

 

Applicant's summary and conclusion

Interpretation of results:

highly irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

On the basis of the results obtained with this ex vivo bioassay and according to the scheme for (EC-) classification applied, it can be concluded that undiluted Bromoacetic acid is severely irritating and possibly corrosive to eyes.
On the basis of these results, Bromoacetic acid is proposed to classify the test substance according to the EEC-standards (mentioned in EECDirective 83/467/EEC and published in the Official Journal of the European Communities, L 257, Volume 26, 16 September 1983), as R41: risk of serious damage to eyes. The classification according to Regulation 1927/2008 is Eye irrtation category 1.

Executive summary:

Bromoacetic acid is a corrosive substance, for reason of animal welfare an alternative test; the Chicken Enucleated Eye Test (CEET) has been performed.

A sample of Bromoacetic acid was examined for eye irritating/corrosive potential in an ex vivo bioassay, namely the Enucleated Eye Test with chicken eyes. The eyes were obtained from slaughter animals, which had to be killed anyway. This bioassay was carried out because the test substance was expected to cause severe effects in the standard eye irritation/corrosion study with rabbits.

In this ex vivo bioassay, three parameters were measured to disclose possible adverse eye effects, namely corneal thickness (expressed as corneal swelling), corneal opacity, and fluorescein retention. The measurement of corneal swelling in this assay guaranteed a highly

objective parameter, which enables the investigator to discriminate the damaging effects of test materials very precisely, this in contrast to the conventional rabbit test, which uses subjective gross measurements only. In combination with the measurement of corneal opacity and fluorescein retention, though assessed by subjective observation, but being very accurately measured by the use of the slit-lamp microscope, a reliable evaluation of the eye irritation potential of test materials is achieved. The study was carried out because the test substance was expected to cause severe effects in the standard eye irritation/corrosion study with rabbits.

The following criteria and scoring systems were applied for the assessment of possible effects:

- Corneal swelling

- Corneal opacity

- Fluorescein retention

- Morphological effects

Scoring is based on the combination of the three categories, which allow the interpretation of the results in relation to the EU Directive

83/467/EEC Part II (B) of Appendix 6, by scientific judgement.

After treatment with the undiluted test substance, the thickness of the

cornea of all 5 test-eyes increased considerably. The maximum

thickness detectable with the measurement device was already reached

at 180 minutes after treatment.

Severe complete corneal opacity was observed in all 5 test eyes at all

observation time points. In addition, loosening of epithelium was

observed in all test eyes.

The determination of fluorescein retention at 30 minutes after treatment,

revealed severe fluorescein retention of the test eyes. The control eye

did not show any unusual effects.

The categories for corneal swelling, corneal opacity, and fluorescein

retention were IV, IV, and IV.