Hydroxylammonium chloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 Nov 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

/ no raw (uncorrected) and no mean values for opacity and permeability

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

26 July 2013

Deviations:

yes

Remarks:

/ no raw (uncorrected) and no mean values for opacity and permeability

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:

08 Dec 2010

Deviations:

yes

Remarks:

/ no raw (uncorrected) and no mean values for opacity and permeability

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxylammonium chloride

EC Number:

226-798-2

EC Name:

Hydroxylammonium chloride

Cas Number:

5470-11-1

Molecular formula:

ClH.H3NO

IUPAC Name:

hydroxyammonium chloride

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: tightly closed, in the dark, at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid: 20% (w/v) in a 0.9% sodium chloride solution

FORM AS APPLIED IN THE TEST (if different from that of starting material)
liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, Brensbach, Germany
- Characteristics of donor animals (e.g. age, sex, weight): 17 - 43 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The isolated eyes were transported in HBSS (with streptomycin/ penicillin (0.01%)), ice cooled.
- Time interval prior to initiating testing: The comeas were prepared immediately upon arrival.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: streptomycin/ penicillin

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL (= 150 mg)
- Concentration (if solution): 20% w/v in vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 0.9% w/v
- Lot/batch no. (if required): 144440 16
- Purity: 0.9%

Duration of treatment / exposure:

4 h at 32 ± 1 °C

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

Number of eyes for the test item/ negative control/ positive control: 3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: A careful macroscopic examination was performed on all eyes to detect the presence of any defects. Only corneas free of damage were used. The cornea was removed from the eye leaving a rim of about 2 to 3 mm of sclera. The isolated corneas were immersed in pre-warmed HBSS (32 ± 1°C) until they were mounted in BCOP holders. A pre-treatment opacity reading was taken for each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS: Yes, the eyes were free of defects.

NUMBER OF REPLICATES: 3

SOLVENT CONTROL USED: Yes, sodium chloride (0.9% w/v)

POSITIVE CONTROL USED: Yes, imidazole (20% in sodium chloride (0.9% w/v))

APPLICATION DOSE AND EXPOSURE TIME: 750 μl test item (20%), 4 h

TREATMENT METHOD: closed chamber: The test item solution, negative control and positive control were introduced into the anterior chamber through the dosing holes of the chamber. Afterwards, the corneas were incubated for 4 h in an incubator in a horizontal position.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The cornea was rinsed three times with EMEM containing phenol red and one time with EMEM without phenol red.
- POST-EXPOSURE INCUBATION: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A baseline opacity was determined as well as the post-exposure opacity.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany) at 490 nm
- Others (e.g, pertinent visual observations, histopathology): In addition each cornea was visually observed.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG (OECD 437) and in "Any other information on materials and methods incl. tables" was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

negative control - cornea 1

Value:

-0.062

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

negative control - cornea 2

Value:

1.816

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

negative control - cornea 3

Value:

0.706

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

positive control - cornea 1

Value:

70.793

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

positive control - cornea 2

Value:

71.315

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 6

Irritation parameter:

cornea opacity score

Run / experiment:

positive control - cornea 3

Value:

79.27

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

test item - cornea 1

Value:

57.755

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 8

Irritation parameter:

cornea opacity score

Run / experiment:

test item - cornea 2

Value:

47.875

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 9

Irritation parameter:

cornea opacity score

Run / experiment:

test item - cornea 3

Value:

25.708

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 10

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

negative control - mean out of all 3 eyes

Value:

-0.001

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 11

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

positive control - cornea 1

Value:

2.142

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 12

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

positive control - cornea 2

Value:

2.303

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Results 13

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

positive control - cornea 3

Value:

2.119

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 14

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

test item - mean out of all 3 eyes

Value:

-0.002

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 15

Irritation parameter:

in vitro irritation score

Run / experiment:

negative control - mean out of all 3 eyes

Value:

0.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 16

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control - mean out of all 3 eyes

Value:

106.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 17

Irritation parameter:

in vitro irritation score

Run / experiment:

test item - mean out of all 3 eyes

Value:

43.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, using Phenylbutazone (not classified) and Dibenzoyl-L- tartaric acid (Cat. 1)

ACCEPTANCE OF RESULTS:
The test is acceptable if the positive control produces an IVIS which falls within two standard deviations of the current historical mean (for this testing facility: 75.5 - 137.3).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (for this testing facility: -1.4 - 3.5).
A single test run with three corneas should be suffcient for a test item when the resulting classification is unequivocal.

- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1: Results of BCOP test

Parameter	Opacity	Permeability [OD490]	IVIS
			per cornea	per group (mean)	SD
Negative control (physiological saline)	-0.062	-0.001	-0.082	0.8	0.9
	1.816	-0.001	1.801
	0.706	-0.001	0.696
Positive control (lmidazole (20%))	70.793	2.142	102.928	106.6	4.1
	71.315	2.303	105.860
	79.270	2.119	111.060
Test item	57.755	-0.002	57.725	43.7	16.4
	47.875	-0.002	47.850
	25.708	-0.002	25.673

 

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive (Eye Irrit. 2 or not classified according to Regulation (EC) No 1272/2008)

Conclusions:

Under the conditions of the test, the test substance was shown to have no corrosive potential in the Bovine corneal opacity and permeability (BCOP) test prediction model. The result does not allow for the non-classification or classification as irritant of the test substance and therefore further evaluation and/or data generation is required.