Hydroxylammonium chloride

Endpoint summary

Description of key information

Skin irritation (OECD 439 and rabbit): irritating

In vivo study: RA from source substance bis(hydroxyammonium) sulfate (CAS 10039-54-0)

Eye irritation ( OECD 437 and 492): irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Dec 2015 - 19 Feb 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

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

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: SkinEthicTM RHE-model RHE/S/17

Details on animal used as source of test system:

TEST SKIN MODEL
- Source:Episkin/SkinEthic Laboratories, Lyon, France

TEST METHOD
The RHE-model is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. lt is used for skin irritancy testing which involves topical application of test item to the surface of the skin and the subsequent assessment of its effects on cell viability. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 6-well plates containing 1 mL prewarmed (room temperature) assay medium per well and preincubated in an incubator (37 °C, 5% CO2) before use on next day.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37
- CO2 gas concentration (%): 5

Justification for test system used:

To reduce animal testing, this alternative in vitro method was used. The human skin RHE-model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE-model RHE/S/17
- Tissue batch number(s): 15-RHE-151, 16-RHE-007, 16-RHE-018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 h (± 5 min)
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: all 3 RHE models showed viabilities (OD = 1.0 - 1.2) in range of the acceptance criteria of OECD 439 (OD = 0.8 - 3)
- Barrier function: all 3 RHE models showed a barrier function (ET50 = 4.7 - 5.4) in range of the acceptance criteria of OECD 439 (ET50 = 4 - 10)
- Morphology:all 3 RHE models showed 6 - 6.5 cell layers, absence of significant histological abnormalities and a well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum, which is according to OECD 439
- Contamination: no information
- Reproducibility: The results of the positive and negative control showed reproducibility over time.

NUMBER OF REPLICATE TISSUES: triplicates were used per dose

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 runs were performed

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant or corrosive to skin if the viability after 42 minutes exposure (and 42 h of post incubation time) is less or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure (and 42 h of post incubation time) is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (10 µl of deionised water was applied before treatment)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 100%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% in deionized water

Duration of treatment / exposure:

42 ± 1 min

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1, mean value of negative controls (DPBS)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

(% of negative control)

Run / experiment:

1, mean value of positive control (5% SDS salt)

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

(% of negative control)

Run / experiment:

1, mean value of the test item (100%)

Value:

32.68

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2, mean value of negative controls (DPBS)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

(% of negative control)

Run / experiment:

2, mean value of positive control (5% SDS salt)

Value:

1.59

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

(% of negative control)

Run / experiment:

2, mean value of the test item (100%)

Value:

50.52

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3, mean value of negative controls (DPBS)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

(% of negative control)

Run / experiment:

3, mean value of positive control (5% SDS salt)

Value:

1.18

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3, mean value of the test item (100%)

Value:

6.76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: no information available from the study report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: The acceptance criteria for the variability of ≤ 18% was met for the positive and negative control measurements, but not for the test item measurements (in run 1 and 3 it was 62.77 and 142.17%, respectively)

Table 1: MTT assay after 42 min exposure

	Negative Control			Positive Control			Test item
Tissue sample	1	2	3	1	2	3	1	2	3
OD570	2.111	1.911	1.781	0.023	0.034	0.022	0.911	1.016	0.023
	2.004	1.895	1.924	0.022	0.031	0.021	0.935	0.965	0.327
	2.105	2.02	1.784	0.023	0.028	0.022	0.187	0.963	0.022
OD570 (mean)	2.074	1.942	1.83	0.023	0.031	0.022	0.678	0.981	0.124
Relative viability (mean) [%]	100	100	100	1.1	1.59	1.18	32.68	50.52	6.76
SD [%]	2.9	3.51	4.48	2.22	9.77	3.21	62.77	3.04	142.17
Viability (mean values of all replicates) [%]	100			1.29			29.98666667

Interpretation of results:

other: irritating potential (Skin Irrit. 2 or Skin Corr. 1 according to Regulation (EC) No 1272/2008)

Conclusions:

CLP: Skin Irrit, Cat.2 (Annex VI harmonized classification)

Under the conditions of the test, the test substance was shown to have an irritating or corrosive potential towards reconstructed human epidermis tissue in the SkinEthic™ model. The result does not allow for the classification of the test substance as irritant or corrosive and therefore further evaluation and/or data generation is required.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 24-48 h

Score:

2.5

Max. score:

4

Reversibility:

fully reversible within: 8 days

Remarks on result:

other: exposure time: 20 h; scale was seen on day 6

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 24-48 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 8 days

Remarks on result:

other: exposure time: 20 h; scale was seen on day 6

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 24-48 h

Score:

0

Max. score:

4

Reversibility:

other: no effects

Remarks on result:

other: exposure time: 20 h

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 24-48 h

Score:

0

Max. score:

4

Reversibility:

other: no effects

Remarks on result:

other: exposure time: 20 h

Irritation parameter:

erythema score

Time point:

24/48/72 h

Remarks on result:

not determinable because of methodological limitations

Remarks:

72-h reading time point was not determined

Irritation parameter:

edema score

Time point:

24/48/72 h

Remarks on result:

not determinable because of methodological limitations

Remarks:

72-h reading time point was not determined

Interpretation of results:

other: Category 2 based on CLP/EU GHS criteria, according to Regulation (EC) No 1272/2008

Conclusions:

CLP: Skin Irrit, Cat.2, corresponding to the harmonized classification according to Annex VI of CLP Regulation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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

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

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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

 

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Nov - 16 Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcularTM Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcularTM human cell construct (OCL-200-EIT, MatTek Corporation) is used in this assay. It is composed of human-derived epidermal keratinocytes, consisting of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. All biological components of the EpiOcularTM tissue and the kit culture medium have been tested and are free of the presence of viruses, bacteria and mycoplasma.
Analysis for tissue functionality and quality was performed and passed.

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): 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

25 min (Post-exposure immersion incubation)
18 h (Post-treatment incubation)

Number of animals or in vitro replicates:

2 replicates

Details on study design:

- Details of the test procedure used
The in vitro eye test using the human cornea model EpiOcularTM and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential. Cell viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT in control and test item treated cultures. The percent reduction of cell viability in comparison to untreated negative controls is used to predict eye irritation potential.

- RhCE tissue construct used, including batch number
The EpiOcularTM human cell construct (OCL-200-EIT, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot number: 23757)

- Doses of test chemical and control substances used
50 mg test item; 50 μL positive control (methyl acetate) and 50 μL negative control (deionised water)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
exposure: 6 h min at 37 ± 1.5 °C
post-exposure immersion: 25 min at room temperature
post-exposure incubation: 18 h at 37 ± 1.5 °C

- Description of any modifications to the test procedure
The equilibration period was 18 min instead of 15 min. The used Assay Medium was not warmed to 37 °C prior to 1 hour pre-incubation, but used at the same temperature as the tissues.

- Number of tissue replicates used per test chemical and controls
2 replicates each

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
570 nm

- Description of the method used to quantify MTT formazan
Inserts were removed from the 24-well plate after the incubation time inn MTT solution (3 h), the bottom of the insert was blotted on absorbent material and transferred to a 6-well plate containing 2 mL isopropanol per well in a manner avoiding the isopropanol to flow into the insert. The plate was sealed with parafilm. MTT was extracted overnight (about 19.5 hours) at 2-8 °C in the dark and shaken for 2.25 hours at room temperature afterwards. 200 μL samples were placed into the wells of a 96-well plate and the absorbance /optical density was measured in a 96-well plate spectrophotometer to determine cell viability.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
The results obtained for the respective negative and positive controls are in the range of the historical control data.

- Complete supporting information for the specific RhCE tissue construct used
A copy of the test kit quality control sheet and of the certificate of analysis provided by the manufacturer are included in the study report.

- Reference to historical data of the RhCE tissue construct
yes, no further information available

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
no information provided

- Positive and negative control means and acceptance ranges based on historical data
negative control mean OD: 1.49, range: 1.24 - 2.05
positive control mean OD: 0.48, range: 0.22 - 0.64

- Acceptable variability between tissue replicates for positive and negative controls
Acceptable variability between tissue replicates for positive and negative controls < 20%.

- Acceptable variability between tissue replicates for the test chemical
Acceptable variability between tissue replicates for the test chemical < 20%.

Irritation parameter:

other: mean viability (%)

Run / experiment:

negative control

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: mean viability (%)

Run / experiment:

positive control

Value:

18.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: mean viability (%)

Run / experiment:

test item

Value:

2.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no information provided

DEMONSTRATION OF TECHNICAL PROFICIENCY: no information provided

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, as the mean OD value (1.355) of the two negative control tissues lies between 0.8 and 2.5.
- Acceptance criteria met for positive control: Yes, as the mean percentage viability for the positive control (18.2%) lies below 50% of the control viability.

Table 1: MTT results of eye irritation study 

Dose Group	Absorbance Well 1 (Tissue 1/2)	Absorbance Well 2 (Tissue 1/2)	Mean Absorbance* (Tissue 1/2)	Mean Absorbance* Tissue 1 and 2 (- Mean Blank)	Mean Absorbance of 2 Tissues*	Rel. Absorbance [%] Tissue 1 and 2**	Difference of the Rel. Absorbances [%] Tissue 1 and 2	Viability [% of Negative Control]**
Negative Control	1.384	1.361	1.373	1.335	1.355	98.5	3	100
	1.417	1.41	1.414	1.376		101.5
Positive Control	0.249	0.258	0.253	0.215	0.247	15.9	4.6	18.2
	0.318	0.314	0.316	0.278		20.5
Test Item	0.065	0.068	0.066	0.028	0.031	2.1	0.4	2.3
	0.072	0.072	0.072	0.034		2.5

* Mean of two replicate wells after blank correction

** Relative absorbance [rounded values]: (100 x absorbance (Test Item/ Positive Control))/(absorbance Negative Control)

Interpretation of results:

other: irritating potential (Eye Irrit. 2 or Eye Dam. 1 according to Regulation (EC) No 1272/2008)

Conclusions:

Under the conditions of the test, the test substance was shown to have an irritating or corrosive potential towards reconstructed human epidermis tissue in the EpiOcular™ EIT prediction model. The result does not allow for the classification of the test substance as irritant or corrosive and therefore further evaluation and/or data generation is required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Irritation

Justification for read-across

There is data available regarding skin irritation for Hydroxylammonium chloride (CAS 5470-11-1). In addition, read-across from an appropriate substance is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.1. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

As the data on skin irritation which is available for Hydroxylammonium chloride (CAS 5470-11-1) is not sufficient, information available for the analogue substance Bis(hydroxyammonium) sulfate (CAS 10039-54-0) are taken into account to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.1.

Skin Irritation

CAS 5470-11-1

An in vitro skin irritation study was performed with the test substance according to OECD guideline 439 (reference 7.3.1-1). The SkinEthicTM RHE-model was treated with 16 mg of the test substance for 42 min, after which the test substance was removed by rinsing. The viability of the cells was determined by a MTT test. Three independent experiments resulted in viabilities of 32.68 %, 50.52 % and 6.76 % and a mean viability of 30%,. Thus, under the conditions of the test, the test substance was shown to have an irritating or corrosive potential towards reconstructed human epidermis tissue in the Skin Ethic model. Due to the high variability of the three results and the type of the study (positive result does not allow distinction between Cat 1 and 2), no conclusive decision can be made regarding classification of the test substance.

CAS 110039-54-0

A skin irritation study was performed with the test substance in a valid study using an internal protocol in four Vienna White rabbits (reference 7.3.1-2). The clipped skin was exposed to ~2 g of the test substance for 1, 5, 15 min (in 2 animals) and for 20 hours (2 animals) under occlusive conditions. The rabbits were observed for 8 days. Skin reactions were assessed 24 and 48 h after dosing. The mean Draize scores for erythema formation were 2.5 in the male and 2 in the female after the 20 h expopsure, both fully reversible within 8 days. No signs of edema were observed at any time point. In addition, readings of the two animals after exposure times of 1, 5 and 15 min resulted in no effects after 1 and 5 min and a slight erythema (scored 1) after 15 min in both animals. As 50% of the animals showed a positive response (after 20 h exposure), Bis(hydroxyammonium) sulfate (CAS 110039-54-0) and therefore its target substance Hydroxylammonium chloride (CAS 5470-11-1) are considered to be skin irritant category 2. The used experimental conditions may be regarded as worst case assessment (20 hrs exposure, occlusive conditions). Under the conditions of this study, the test substance has to be considered to be irritating to the skin.

Based on the results of both studies and according to CLP classification criteria, the test substance is considered to be a skin irritant category 2.

Eye irritation

CAS 5470-11-1

The potential for eye irritation and serious eye damage of Hydroxylammonium chloride (CAS 5470-11-1) was tested in two reliable In-Vitro assays in accordance with OECD guidelines and GLP compliance.

A reliable in vitro eye irritation/corrosion study was performed with the test substance according to OECD guideline 437 (reference 7.3.2-1). The isolated cattle corneas were treated with 750 µl of the test substance (20 % in physiological saline) for 4 h, after which the test substance was removed by rinsing. The corneal opacity and permeability were measured and the IVIS (In-Vitro Irritancy Score) calculated. Three replicates resulted in a mean IVIS of 43.7. 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 is required.

Another reliable eye irritation study is available performed with the test substance according to OECD guideline 492 (reference 7.3.2-2). The EpiOcularTM Model was treated with 50 mg of the test substance for 6 h, after which the test substance was removed by rinsing. The viability of the cells was determined by an MTT test. Two independent experiments resulted in a mean viability of 2.3% after treatment with the test substance, meaning a positive test result.

Under the conditions of the test, the test substance was shown to have an irritating or corrosive potential towards reconstructed human epidermis tissue in the EpiOcular™ EIT prediction model.

Taking into consideration the results of both studies, the test substance is considered to be an eye irritant Category 2.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Hydroxylammonium chloride, data will be generated from information on reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

 

Applying the RA-A approach and in consistency with the harmonized classification, which is available for the target and the source substance bis(hydroxyammonium) sulfate (CAS 10039-54-0) according to Regulation (EC) 1272/2008, Annex VI (both with the Index No. 612-123-00-2), the target substance Hydroxylammonium chloride meets the criteria for classification as Skin Irrit Cat. 2, H315.

 

The available data on eye irritation/corrosion with the test substance hydroxylammonium chloride meet the classification criteria as Eye Irrit Cat 2, H319, according to Regulation (EC) 1272/2008, and in consistency with the harmonised classification of Hydroxylammonium chloride (CAS 5470-11-1) as Eye Irrit Cat 2, H319, according to Annex VI of Regulation (EC) No 1272/2008 (Index No. 612-123-00-2).