L-Arginine, N2-(2,3-dihydroxypropyl)-, monohydrochloride

Endpoint summary

Description of key information

Skin irritation in vitro, OECD 439 (read-across): not irritating (Leuschner, 2012)

Eye irritation in vivo, OECD 405 (read-across): not irritating (Leuschner, 2012)

Eye irritation in vivo, HET-CAM: not irritating (Nitka, 2005)

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:

key study

Study period:

10 February 2012 - 03 March 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted according to international guideline and line with ECVAM recommendation under GLP albeit with slight deviation i.e. the optical density measurement was 540 nm instead of 570 nm.

Justification for type of information:

Refer to the read-across justification attached in Section 13.2.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24 August 2009

Deviations:

yes

Remarks:

The optical density measurement was 540 nm instead of 570 nm

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

The optical density measurement was 540 nm instead of 570 nm

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EST1000

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human derived epidermis keratinocytes

Source strain:

not specified

Details on animal used as source of test system:

N/A

Justification for test system used:

The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability was measured by dehydrogenase conversion of the vital dye MTT (3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue1 ; EINECS number 206-069-5, CAS number: 298-93-1), into a blue formazan salt that was quantitatively measured after extraction from tissues. Irritant substances were identified by their ability to decrease cell viability below defined threshold levels (i.e.≤ 50% for UN GHS category 2 irritants). Substances that produce cell viabilities above the defined threshold level will not be classified (i.e.> 50%, no category).
The reconstructed human epidermis model systems may be used to test solids, liquids, semi-solids and waxes. Solids may be soluble or insoluble in water. Whenever possible, solids should be tested as a fine powder. The test item is a solid white powder and therefore the test system was deemed suitable.

Vehicle:

unchanged (no vehicle)

Remarks:

However, the epidermis surface was moistened with Dulbecco's phosphate buffered saline (D-PBS) before application to ensure good contact with the skin.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Skin model EST1000 (CellSytems® Biotechnology GmbH, 53562 Katharinen, Germany), insert size 0.6 cm^2
- Tissue batch number(s): Batch no. EST-120213-001 (Catalogue no. CS-1001)
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified
- Date of initiation of testing: 10 February 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The models were cultivated at 21°C for 20 minutes according to the instructions of the EST1000 supplier CellSystems®.
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS).
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: N/A

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Yes
- Wavelength: 540 nm
- Filter: Not specified
- Filter bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT conversion assay. The optical density (OD) of the extracted (solubilised) dye from the tissue treated with the negative control (NC) should be at least 20-fold greater than the OD of the extraction solvent alone. The tissue treated with NC should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period. Viability was performed after a post-treatment incubation period of the rinsed tissues in fresh medium of 42 h. This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects. Each skin sample was placed in a MTT solution of 1 mg/mL (37 °C incubation temperature, 5 % CO2, 95 % humidity) for 3 h. The precipitated blue formazan product was extracted using the solvent propanol-2 , and the concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer. The measurements were made for each of the three tissues in duplicate.

- Barrier function: Stratum corneum was multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of the cytotoxic marker substance sodium dodecyl sulphate (SDS). The barrier function is assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration. Each batch of the epidermal model used meets defined production release criteria, set by the supplier, among which those for viability and for barrier function are the most relevant (MTT, 2 hours Triton X-100: target > 50%). The barrier properties of the tissues were verified by the supplier.

- Morphology: The test item was applied topically to a three-dimensional reconstructed human epidermis model, comprised of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The containment properties of the model prevented the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure.

- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.

- Reproducibility: Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range. The NC item was Dulbecco's phosphate buffered saline (D-PBS) and the PC item was 5% aqueous sodium dodecyl sulphate (SDS)

NUMBER OF REPLICATE TISSUES: 3 tissues in duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable): N/A
- No. of replicates: 3 tissues in duplicate
- Method of calculation used:
The MTT conversion assay was used to measure cell viability. The OD values obtained for each test sample were used to calculate a mean percentage viability relative to the negative control, which is arbitrarily set at 100%.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA
The test item is to be considered to be an irritant to skin in accordance with if the tissue viability after exposure and post-treatment incubation is ≤ 50%.
The test item is to be considered to have no category if the tissue viability after exposure and post-treatment incubation was > 50%.

Assay acceptability criteria: For each assay using valid batches, tissues treated with the NC exhibit OD reflecting the quality of the tissue that followed all shipment and receipt steps and all the irritation protocol processes. The OD values of controls should not be below historical established lower boundaries. Similarly, tissues treated with the PC, i.e. 5% aqueous SDS, should reflect the sensitivity retained by tissues and their ability to respond to an irritant substance in the conditions of each individual assay.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Three samples of 30 mg test item were applied to the skin model with a surface area of 0.6 cm^2 to uniformly cover the skin surface.
- Concentration (if solution): N/A

VEHICLE: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Dulbecco’s phosphate buffered saline (D-PBS) in triplicate (concentration not specified)
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): sodium dodecyl sulphate
- Concentration (if solution): 5 % aqueous

Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range.

Duration of treatment / exposure:

20 m

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3 tissues in duplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3 (mean)

Value:

134

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2 (mean)

Value:

76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1 (mean)

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: None reported
- Colour interference with MTT: None reported

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: N/A, model variability criteria not provided in OECD Guideline
- Range of historical values if different from the ones specified in the test guideline: N/A

The mean viability of the cells exposed to the test item was 98.0% of the mean negative control value. The OD540 values were well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50 % for a 20 minute exposure.
The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.
The viability of cells treated with the positive reference item, 5 % SDS, was 6.9 % of the negative controls and was below the cut-off value. Hence, 5 % SDS is predicted to cause pronounced skin irritation.

Table 1. Summary results of in vitro skin irritation optical density (OD)

Formazan extraction solution	Extinction# (n= 3 tissues)	SD	% extinction compared to control
Negative control D-PBS	0.548	0.027	-
Test item	0.537	0.152	98.0
Positive control 5 % SDS	0.038	0.002	6.9

# : at λ = 540 nm

D-PBS: Dulbecco's phosphate buffered saline

SD: standard deviation

SDS: 5 % aqueous deviation sodium dodecyl sulphate

 

Table 2. Individual values optical density (OD)

                                   Extinction at λ = 540nm

Replicates (duplicate measurements per tissue)	Negative control (D-PBS)	Test item	Postive control 5 % SDS
Tissue 1	0.592	0.412	0.035
	0.566	0.464	0.036
Tissue 2	0.537	0.447	0.042
	0.534	0.472	0.034
Tissue 3	0.533	0.727	0.040
	0.526	0.697	0.038
Mean	0.548	0.537	0.038
SD	0.027	0.152	0.002

D-PBS: Dulbecco's phosphate buffered saline

SD: standard deviation

SDS: 5 % aqueous deviation sodium dodecyl sulphate

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the conditions of the study, the test item was concluded not to be irritating to the skin.

Executive summary:

An in vitro study was performed in accordance with OECD 439 (2010), in order to assess the skin irritation potential of the test item.

The EST1000 three-dimensional reconstructed human epidermis skin model was utilised. Cell viability was determined by measuring the optical density (OD) at a wavelength of 540 nm.

The test item was applied to the model's skin surface for an exposure time of 20 minutes. D-PBS was used as a negative control and 5 % aqueous SDS was used as a positive reference control.

The mean viability of cells exposed to the test item was 98 % of the negative controls. The OD₅₄₀ value was above the percentage cut-off value for cell viability of 50 %, following 20 minutes exposure. The negative control and positive controls produced valid results (positive control: 6.9 % OD₅₄₀; i.e. below the 50 % cut off for cell viability).

It was concluded based on the conditions of the test, that the test item was not cytotoxic and not irritating to the skin and would not be classified under CLP Regulation (EC) 1272/2008.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer to the read-across justification attached in Section 13.2.

Reason / purpose for cross-reference:

read-across source

Details on animal used as source of test system:

N/A

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3 (mean)

Value:

134

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2 (mean)

Value:

76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1 (mean)

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: None reported
- Colour interference with MTT: None reported

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: N/A, model variability criteria not provided in OECD Guideline
- Range of historical values if different from the ones specified in the test guideline: N/A

The mean viability of the cells exposed to the test item was 98.0% of the mean negative control value. The OD540 values were well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50 % for a 20 minute exposure.
The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.
The viability of cells treated with the positive reference item, 5 % SDS, was 6.9 % of the negative controls and was below the cut-off value. Hence, 5 % SDS is predicted to cause pronounced skin irritation.

Table 1. Summary results of in vitro skin irritation optical density (OD)

Formazan extraction solution	Extinction# (n= 3 tissues)	SD	% extinction compared to control
Negative control D-PBS	0.548	0.027	-
Test item	0.537	0.152	98.0
Positive control 5 % SDS	0.038	0.002	6.9

# : at λ = 540 nm

D-PBS: Dulbecco's phosphate buffered saline

SD: standard deviation

SDS: 5 % aqueous deviation sodium dodecyl sulphate

 

Table 2. Individual values optical density (OD)

                                   Extinction at λ = 540nm

Replicates (duplicate measurements per tissue)	Negative control (D-PBS)	Test item	Postive control 5 % SDS
Tissue 1	0.592	0.412	0.035
	0.566	0.464	0.036
Tissue 2	0.537	0.447	0.042
	0.534	0.472	0.034
Tissue 3	0.533	0.727	0.040
	0.526	0.697	0.038
Mean	0.548	0.537	0.038
SD	0.027	0.152	0.002

D-PBS: Dulbecco's phosphate buffered saline

SD: standard deviation

SDS: 5 % aqueous deviation sodium dodecyl sulphate

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the conditions of the study, the test item was concluded not to be irritating to the skin.

Executive summary:

An in vitro study was performed in accordance with OECD 439 (2010), in order to assess the skin irritation potential of the test item.

The EST1000 three-dimensional reconstructed human epidermis skin model was utilised. Cell viability was determined by measuring the optical density (OD) at a wavelength of 540 nm.

The test item was applied to the model's skin surface for an exposure time of 20 minutes. D-PBS was used as a negative control and 5 % aqueous SDS was used as a positive reference control.

The mean viability of cells exposed to the test item was 98 % of the negative controls. The OD₅₄₀ value was above the percentage cut-off value for cell viability of 50 %, following 20 minutes exposure. The negative control and positive controls produced valid results (positive control: 6.9 % OD₅₄₀; i.e. below the 50 % cut off for cell viability).

It was concluded based on the conditions of the test, that the test item was not cytotoxic and not irritating to the skin and would not be classified under CLP Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

01 April 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Detail on test item is not clear

Qualifier:

according to guideline

Guideline:

other: The Hen's Egg Test- Utilizing the Chorioallantonic Membrane (HET-CAM)

Version / remarks:

Kemper & Luepke

Deviations:

not specified

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL: Not specified
- Source and lot/batch No. of test material:
- Expiration date of the lot/batch:
- Purity test date:

RADIOLABELLING INFORMATION (if applicable): N/A
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL: Not specified
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: Applied diluted
- Treatment of test material prior to testing: diluted to 15% in distilled water
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material):N/A

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS: N/A
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is

Species:

chicken

Strain:

other: White Leghorn

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EGGS
- Source: Moyer’s Chicks, Inc. Quakertown Pennsylvania
- Number of eggs: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): Not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Fresh eggs stored in test facility up 7 days at 500 F, the eggs were incubated in a Kuhl incubator which rotated automatically once every hour. Incubation temperature was controlled at 990 F (+/- 10 F) with relative humidity of 60 – 70% for the 10 days of incubation. On day 8, the eggs were turned so that the acutely angled end faced down. On day 10 the eggs were removed from the incubator and placed in preheated and humidified Plexigals work encloser.

Vehicle:

water

Remarks:

Distilled water

Controls:

other: Nivea moisturing lotion 50% and vaseline dermatology formula lotion 50%

Amount / concentration applied:

0.3 ml

Duration of treatment / exposure:

20 seconds

Observation period (in vivo):

30 seconds, 2 and 5 minutes

Duration of post- treatment incubation (in vitro):

Not applicable

Number of animals or in vitro replicates:

4 CAMs

Details on study design:

Fresh eggs stored in test facility up 7 days at 500 F, the eggs were incubated in a Kuhl incubator which rotated automatically once every hour. Incubation temperature was controlled at 990 F (+/- 10 F) with relative humidity of 60 – 70 % for the 10 days of incubation. On day 8, the eggs were turned so that the acutely angled end faced down. On day 10 the eggs were removed from the incubator and placed in preheated and humidified Plexigals work encloser. Forceps were then used to remove the shell down to the shell-membrane junction. The inner egg membrane was then hydrated with a warm, physiological saline solution. The saline was removed after 2 – 5 minutes exposure. Utilizing point forceps, the inner egg membrane was then carefully removed to reveal the CAM.
The test or reference article, at a dosage of three-tenth of one millilitre (0.3ml) of liquid was administered to each of four CAM’s. Twenty second later, the test or control article was rinsed from each CAM with 5 ml of physiological saline. All CAMs were observed immediately prior to test article administration, at 30 seconds, 2 and 5 minutes after exposure to the test article. The reaction of the CAM, the blood vessels, including the capillaries, and the albumin were examined and score for initiation effects as detailed below.
Effects score
Time (min) 0.5 2 5
Hyperemia 5 3 1
Minimal Haemorrhage (‘Feathering) 7 5 3
Haemorrhage (Obvious Leakage) 9 7 5
Coagulation and/or Thrombosis 11 9 7
The numerical, time dependant scores were tolerated for each CAM. Each reaction type can be recorded only once for each CAM, therefore the maximum score per CAM is 32. The mean score was determined for all CAM’s similarly tested.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

1.75

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

not specified

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The reference substances have historically been categorised as being practically non-irritating, eliciting score approaching 0, at 24 hours, when dosed at 100% and tested using Draize occular irritation methodologies (Draize Scale: 0 - 110). At 50%, the score of 1.00 and 2.00 all within the acceptability range.

Table 3. Experimental scoring results for irritation

Test Group	CAM #	Score@
		0.5 min.	2 min.	5 min.	Total
N-Glyceryl Arginine HCL (15% in distilled water)	1	0	0	0	0
	2	0	0	1	1
	3	0	3	0	3
	4	0	3	0	3 Average: 1.75
Nivea Moisturizing Lotion (50%)	1	0	0	1	1
	2	0	0	1	1
	3	0	0	1	1
	4	0	0	1	1 Average: 1.00
Vaseline Dermatology Formula Lotion (50%)	1	0	0	1	1
	2	0	0	1	1
	3	5	0	0	5
	4	0	0	1	1 Average: 2.00

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study, the test article was not considered an occular irritant

Executive summary:

The test or reference article, at a dosage of three-tenth of one millilitre (0.3ml) of liquid was administered to each of four CAM’s. Twenty seconds later, the test or control article was rinsed from each CAM with 5 ml of physiological saline.  All CAMs were observed immediately prior to test article administration, at 30 seconds, 2 and 5 minutes after exposure to the test article. The reaction of the CAM, the blood vessels, including the capillaries, and the albumin were examined and score for initiation effects.

 

The numerical, time dependant scores were tolerated for each CAM. Each reaction type can be recorded only once for each CAM, therefore the maximum score per CAM is 32. The mean score was determined for all CAM’s similarly tested.

The reference substances have historically been categorised as being practically non-irritating, eliciting score approaching 0, at 24 hours, when dosed at 100% and tested using Draized ocular irritation methodologies (Draize Scale: 0 - 110).  at 50%, the score of 1.00 and 2.00 all within the acceptability range.

 

The average score for the test article was 1.75, falling within the classification for no irritation. Under the condition of the study, the results indicate that N-Glyceryl Arginine HCl, at 30%, would have practically no occular irritation potential in vivo.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 February 2012 - 17 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed to test guideline, according to GLP and the study is well-reported.

Justification for type of information:

Refer to the read-across justification attached in Section 13.2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

24 April 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

Himalayan

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: Approx. 6.5 - 8.5 months
- Weight at study initiation (at dosing):
Animal no. 1: 2.4 kg
Animal no. 2: 2.5 kg
Animal no. 3: 2.7 kg
- Housing: For 8 h following test item application, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn, wiping of the eyes with the paws and excluded irritation of the eye by excrements and urine. During the acclimatisation period and after the 8-hour period in restrainers, the animals were kept singly in cages with dimensions of 380 mm x 425 mm x 600 mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany)
- Diet (e.g. ad libitum): Commercial diet, ssniff7 K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany, served as food.
- Water (e.g. ad libitum): Tap water was offered daily ad libitum before and after the exposure period.
- Acclimation period: 20 adaptation days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature of 20°C ± 3°C (maximum range).
- Humidity (%): Relative humidity amounted to 30% - 70% (maximum range; aim was 50% - 60%).
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Rooms were lit (150 lux at approx 1.5 m room height) and darkened on a 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 10 Feb 2012 To: 17 Feb 2012

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Single instillation of 100 mg test item into the conjunctival sac of the right eye of each animal (left eye served as untreated control).
The test item was placed into the conjunctival sac of the right eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the material.

Duration of treatment / exposure:

1 h

Observation period (in vivo):

72 h (observations at 1, 24, 48 and 72 h after administration)

Duration of post- treatment incubation (in vitro):

N/A

Number of animals or in vitro replicates:

3 animals (male)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): One hour after administration the eyes were rinsed with portions of 20 mL 0.9% aqueous NaCl solution, each.
- Time after start of exposure: 1 h

SCORING SYSTEM:

CORNEA
Opacity: degree of density (area most dense taken for reading)
no ulceration or opacity = 0
scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible = 1
easily discernible translucent area, details of iris slightly obscured = 2
nacreous areas, no details of iris visible, size of pupil barely discernible = 3
opaque cornea, iris not discernible through the opacity = 4

IRIS
normal = 0
markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, iris reactive to light (a sluggish reaction is considered to be an effect) = 1
haemorrhage, gross destruction, or no reaction to light = 2

CONJUNCTIVAE
Redness (refers to palpebral and bulbar conjunctivae, excluding cornea and iris)
normal = 0
some blood vessels hyperaemic (injected) = 1
diffuse, crimson colour; individual vessels not easily discernible = 2
diffuse beefy red = 3

CHEMOSIS
Swelling: refers to lids and/or nictitating membranes
normal = 0
some swelling above normal = 1
obvious swelling with partial eversion of lid = 2
swelling with lids about half-closed = 3
swelling with lids more than half-closed = 4

TOOL USED TO ASSESS SCORE:
The eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48 and 72 hours after the administration. The eye reactions were observed and registered.
24 hours after administration, fluorescein (fluorescein SE Thilo drops (ALCON PHARMA GmbH, 79108 Freiburg, Germany)) was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

other: 1 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 24 h

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

1

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

other: 1 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 24 h

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

1

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

other: 1 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 24 h

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

1

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Under the present test conditions, a single instillation of 100 mg test item per animal into the conjunctival sac of the right eye of three rabbits caused the following changes:
Conjunctival redness (grade 1) was observed in all animals 1 hour after instillation.
There were no systemic intolerance reactions. The effects observed were fully reversible within 24 hours of instillation.
For the 24 h fluorescein test, there were no pathological findings in all animals.

Other effects:

N/A

Table 1. Examination of the treated (right) eye: 100 mg test item per eye

(Animal no. 1/2/3)

Time after administration	Cornea	Iris	Conjunctivae
	Opacity		Redness#	Chemosis##
Before dosing	0/0/0	0/0/0	0/0/0	0/0/0
60 minutes	0/0/0	0/0/0	1/1/1	0/0/0
24 hours	0/0/0	0/0/0	0/0/0	0/0/0
48 hours	0/0/0	0/0/0	0/0/0	0/0/0
72 hours	0/0/0	0/0/0	0/0/0	0/0/0

# refers to palpebral and bulbar conjuntivae (excluding cornea and iris)

## swelling refers to lids and/or nictitating membrane

24 h fluorescein test - all animals - no pathological findings

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the conditions of the study, the test item is deemed to be non-irritating to the eye.

Executive summary:

A study was performed in accordance with OECD 405 (2002), in order to determine the eye irritation potential of the test item.

A single installation of 100 mg test item was applied to the right eye (into the conjunctival sac) of each of 3 male rabbits. The left eye was left untreated, serving as a control. After 1 hour of exposure, the eyes were rinsed with portions of 20 mL 0.9% aqueous NaCl solution. The animals were examined using a slit-lamp prior to administration and at 1, 24, 48 and 72 h after administration. 24 h after administration, fluorescein was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions. Scores were assigned based on the severity of effects (for cornea, iris, conjunctivae and chemosis).

Conjunctival redness (grade 1) was observed in all animals 1 h after instillation. There were no systemic intolerance reactions. The effects observed were fully reversible within 24 hours of instillation.

Under the conditions of this study, the test item is not irritating to the eye and would not be classified under CLP Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vitro based on read-across substance (OECD 439; Leuschner, 2012): The test item was applied to the model's skin surface for an exposure time of 20 minutes. This resulted in the cellular mean viability of 98 % of the negative controls and OD₅₄₀ value above the percentage cut-off value for cell viability of 50 %/. It was concluded based on the conditions of the test, that the test item was not cytotoxic and not irritating to the skin and would not be classified under CLP Regulation (EC) 1272/2008.

Eye irritation ex vivo, HET-CAM (Nitka, 2005): The test or reference article, at a dosage of three-tenth of one millilitre (0.3ml) of liquid was administered to each of four CAM’s. The average score for the test article was 1.75, falling within the classification for no irritation.  Under the condition of the study, the results indicate that N-Glyceryl Arginine HCl, at 30%, would have practically no ocular irritation potential in vivo.

Eye irritation in vivo based on read-across substance (OECD 405; Leuschner, 2012): A single installation of 100 mg test item applied to the right eye (into the conjunctival sac) of each of 3 male rabbits. This resulted into conjunctival redness (grade 1) in all animals 1 h after instillation. There were no systemic intolerance reactions. The effects observed were fully reversible within 24 hours of instillation. Under the conditions of this study, the test item is not irritating to the eye and would not be classified under CLP Regulation (EC) 1272/2008.

 

Justification for classification or non-classification

Based on the results of the skin and eye irritation studies performed on the substance and on the analogue of the substance (used for read-across), it is concluded that the substance is not irritating to the skin or eyes and would not be classified under CLP Regulation (EC) 1272/2008.