DL-alanine

Endpoint summary

Description of key information

DL-alanine is not be considered to have irritating or corrosive properties to human skin or eye nor to the respiratory system.

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:

August-September 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

98.5% purity

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult human derived epidermal keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 18 EKIN 035.
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Source: SkinEthic Laboratories, Lyon, France.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

14.7-18.1 mg

Duration of treatment / exposure:

15 +/- 0.5 min

Duration of post-treatment incubation (if applicable):

42 h at 37 deg C

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

102

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The positive control had a mean cell viability of 26% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 8%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

DL-alanine is non-irritant in the in vitro skin irritation test

Executive summary:

DL-alanine was tested for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

DL-alanine is a white crystalline powder with a purity of 98.5%. Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 102%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 26% after 15 ± 0.5 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 8%, indicating that the test system functioned properly.

In conclusion, DL-alanine is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-05 - 2018-03-22

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

The EpiDermTM model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

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 blue), 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 1 or Category 2). The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water.

Vehicle:

unchanged (no vehicle)

Remarks:

The model skin surface was moistened with Dulbecco’s phosphate buffered saline (D-PBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200, Lot no. 25876) MatTek
- Tissue batch number(s): 00267
- Quality analysis date: 2018-01-31
- Date of initiation of testing: 2018-01-08

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 35 min., follwed by room temperture for 25 min.
- Temperature of post-treatment incubation (if applicable): 42 hour, temperature not specified

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment with 2 measurements

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant or corrosive to skin if the viability is less or equal than 50%
- The test substance is considered to be non-corrosive to skin if the viability is greater than 50%

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg, the skin surface was moistened with 25 μL Dulbecco’s phosphate buffered saline

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 μL Dulbecco’s phosphate buffered saline
- Lot/batch no. (if required): 1894803

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): D-PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% aqueous sodium dodecyl sulphate (SDS)

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 , Mean viability (n=3), DL-alpha-Alanine

Value:

>= 86.6 - <= 97.8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

mean viability: 92.2 %

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control (vehicle): yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see table 'any other information on materials and methods incl. tables'

Table 1 Summarized Results of in vitro Skin Irritation

	Optical density [OD540] mean tissue (n = 3)	Mean Viability (n=3) [%]	CV [%]
D-PBS (negative control)	1.320	100.0	5.2
DL-alpha-alanine	1.217	92.2	5.6
5 % SDS (positive control)	0.099	7.5	9.3

CV : coefficient of variation

SDS : sodium dodecyl sulphate

D-PBS : Dulbecco's phosphate buffered saline

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions, DL-alanine tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to determine cytotoxic properties of DL-alanine to skin cells, which might lead to irritation of human skin, by using an artificial three-dimensional model of human skin according to OECD Guideline 439 and in compliance with GLP criteria.The EpiDermTM model was employed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2 - yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. DL-alanine was applied as solid test item to the model skin surface, which was moistened with Dulbecco’s phosphate buffered saline (D-PBS). D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

The mean viability of cells exposed to DL-alanine was 92.2% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. DL-alanine was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.320 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5. The viability of cells treated with the positive reference item, 5% SDS, was 7.5% of the negative control and fulfilled the acceptance criterion of ≤ 20%. The standard deviation determined for all triplicates was below the limit of acceptance of 18%. Hence, all acceptance criteria required were fulfilled.

Under the present test conditions, DL-alanine tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

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:

key study

Study period:

July 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

98.5% purity

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27438 Kit B).
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
Source
MatTek Corporation, Ashland MA, U.S.A.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

52.2-52.6 mg

Duration of treatment / exposure:

6 h +/- 15 min

Duration of post- treatment incubation (in vitro):

18 h +/- 15 min

Number of animals or in vitro replicates:

2

Details on study design:

No correction was made for the purity/composition of the test item.
The solid test item (52.2 to 52.6 mg) was applied directly on top of the skin tissue.
Amino acid FC-C 13387 was spread to match the size of the tissue. Any residual volumes were discarded.
The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with
50 µL Methyl Acetate (positive control) respectively.
At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C.
After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Irritation parameter:

other: cell viability (%)

Value:

82

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

DL-alanine is non-irritant in the EpiOcular(TM) test.

Executive summary:

The eye hazard potential of DL-alanine was tested. For this purpose DL-alanine was topically applied on the Reconstructed Human EpiOcular™ Model. The possible eye hazard potential was tested through topical application for 6 hours. The study procedures described in this report were based on the most recent OECD guideline.

DL-alanine is a white crystalline powder. The test item (52.2 to 52.6 mg) was applied directly on top of the tissue for 6 hours±15 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 21% after 6 hours±15 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 11%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours±15 minutes treatment with the test item compared to the negative control tissues was 82%. Since the mean relative tissue viability for DL-alanine was above 60% after 6 hours±15 minutes treatment the test item is considered to be non-irritant.

In conclusion, DL-alanine is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-05 - 2018-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse
- Characteristics of donor animals (e.g. age, sex, weight): 6 to 12 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no

Vehicle:

physiological saline

Remarks:

0.9% sodium chloride solution (w/v)

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% suspension

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9% sodium chloride solution

Duration of treatment / exposure:

240 min.

Duration of post- treatment incubation (in vitro):

Not reported.

Number of animals or in vitro replicates:

3 per treatment group (test item, negative control and positive control)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.

After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups.

NUMBER OF REPLICATES
3 per treatment group

SOLVENT CONTROL USED (if applicable)
yes

POSITIVE CONTROL USED
yes

APPLICATION DOSE AND EXPOSURE TIME

TREATMENT METHOD: [closed chamber / open chamber]
The open-chamber method was used. The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) , while preventing bubble formation. The corneal holder was equilibrated at 32 °C ± 1 °C for at least one hour.

POST-INCUBATION PERIOD: yes/no. If YES please specify duration
no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of 1 mL sodium fluorescein dye (5 mg/mL in 0.9% sodium chloride solution) measured with the aid of microtiter plate reader (OD490) .The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

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

Irritation parameter:

cornea opacity score

Run / experiment:

20 % DL-alpha-Alanine, mean of group

Value:

>= 2.869 - <= 8.925

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

20% DL-alpha-Alanine, mean of group

Value:

>= 2.816 - <= 8.818

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Following treatment with DL-alanine a mean permeability value of 0.005 (> 0.001) compared to the negative control were determined.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: no

Table 1 Opacity Values

	Cornea No.	Opacity [Opacity Units]	Corrected Opacity
				Mean of group	Standard deviation
0.9 % NaCl	1	0.478	0.292	0.292	0.256
	2	0.399
	3	0.000
20 % Imidazol	4	63.586	63.294	68.420	4.491
	5	71.952	71.660
	6	70.598	70.306
20 % DL-alpha-Alanine	7	9.562	9.270	5.897	3.028
	8	5.299	5.007
	9	3.706	3.414

Table 2 Permeability OD Values (490 nm)

	Cornea no.	Permeability [OD]	Mean of Triplicates	Corrected Permeability [OD]
					Per Cornea		Per Group
					Mean	SD	Mean	SD
0.9 % NaCl	1	0.010	0.009	-			0.010	0.006
		0.010		-
		0.008		-	0.009	0.001
	2	0.004	0.005	-	0.005	0.001
		0.006		-
		0.006		-
	3	0.011	0.016	-	0.016	0.005
		0.016		-
		0.021		-
20 % Imidazol	4	1.839	1.814	1.829	1.804	0.029	1.377	0.414
		1.821		1.811
		1.782		1.772
	5	1.389	1.359	1.379	1.349	0.029
		1.332		1.322
		1.356		1.346
	6	0.987	0.988	0.977	0.978	0.002
		0.987		0.977
		0.990		0.980
20 % DL-alpha-Alanine	7	0.005	0.003	-0.005	-0.007	0.002	-0.005	0.002
		0.003		-0.007
		0.002		-0.008
	8	0.003	0.004	-0.007	-0.006	0.001
		0.005		-0.005
		0.003		-0.007
	9	0.006	0.007	-0.004	-0.003	0.001
		0.006		-0.004
		0.008		-0.002

SD : standard deviation

OD : optical density

Table 3 In vitro irritancy score (IVIS)

	Cornea No.	Opacity	Permeability	IVIS
				Per Cornea	Per Group
					Mean	SD
0.9 % NaCl	1	0.478	0.009	0.613	0.442	0.189
	2	0.399	0.005	0.474
	3	0.000	0.016	0.240
20 % Imidazol	4	63.294	1.804	90.354	89.075	3.632
	5	71.660	1.349	91.895
	6	70.306	0.978	84.976
20 % DL-alpha-Alanine	7	9.270	-0.007	9.165	5.817	3.001
	8	5.007	-0.006	4.917
	9	3.414	-0.003	3.369

SD : standard deviation

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the present test conditions DL-alanine tested in the in vitro BCOP test method, had an IVIS value of 5.817, which is just above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55 (identifying test substances as inducing serious eye damage; UN GHS Category 1). Consequently no conclusion concerning irritant or severely irritant potential of the test item can be made from this study

Executive summary:

The purpose of this study was to determine a possible potency of DL-alanine of being 'ocular corrosive and severe irritant' employing an in vitro Bovine Corneal Opacity and Permeability Assay (BCOP)according to OECD Guideline 437 and in compliance with GLP criteria.

Three corneas were used for each treatment group (test item, solvent control and positive control). The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% DL-alanine as recommended in the test guideline 437 for non-surfactant solids.0.9% NaCl solutionwas used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item.

The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 240 minutes. The optical density (OD) was measured at a wavelength of 490 nm.The acceptance criteria of validity were fulfilled in this test.

The corneas treated with the negative control item 0.9 % sodium chloride solution revealed a mean opacity value of 0.292 ± 0.256 and a mean permeability value of 0.010 ± 0.006. The calculated IVIS value of 0.442 ± 0.189 was well below the cut-off value of 3 (UN GHS no category).

The corneas treated with the positive control item 20% Imidazole in 0.9% NaCl solution revealed a mean opacity value of 68.420 ± 4.491 and a mean permeability value of 1.377 ± 0.414 compared to the solvent control. The calculated IVIS value of 89.075 ± 3.632 was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Following treatment with DL-alanine a mean opacity of 5.897 ± 3.028 and a mean permeability value of <0.001compared to the negative control were determined. The calculated IVIS of 5.817 ± 3.001 is just above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55 (identifying test substances as inducing serious eye damage; UN GHS Category 1). Consequently no conclusion concerning irritant or severely irritant potential of the test item can be made from this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two in vitro skin irritation studies (OECD 439) carried out with DL-alanine did not show skin irritation.

In one in vitro eye irritation study (OECD 437) with DL-alanine, the calculated IVIS score of 5.817 ± 3.001 was slightly above the cut-off value of 3 (UN GHS no category) but far below the cut-off value of 55 (serious eye damage (UN GHS Category 1)), so based on this OECD 437 study no prediction on eye irritation potential could be made. However, the other in vitro eye irritation study (OECD 492) did not show eye irritation.

The absence of skin and eye irriation was corroborated by negative in vivo tests in rabbits using the following other amino acids: L-valine, L-tert-leucine, L-isoleucine and L-alanyl-L-glutamine.

·       L-alanine like L-valine, L-tert. leucine, L-isoleucine is a non-polar natural amino acid.

·       The pH-value of L-alanine like that of L-valine, L-tert.-leucine, L-isoleucine is in the neutral range.

These common features are indications for common properties as to skin irritation/corrosion (ECETOC, 1995) as well as eye irritation/corrosion (ECETOC, 1998).

See also section 13.   

Respiratory System

There are no data available which would indicate that DL-alanine is irritating to the respiratory system.

 

Justification for classification or non-classification

Available in vitro data and read-across to relevant in vivo data with other amino acids provide substantial weight of evidence that DL-alanine does not show irritating or corrosive properties both to skin and eye.