Nα-acetyl-DL-tryptophan

Endpoint summary

Description of key information

skin irritation (OECD 439, OECD 404): not irritating

Eye irritation (OECD 492, OECD 437, OECD 405): not 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:

key study

Study period:

2016-05-25 - 2016-05-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic™ RHE-model

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used; Test item was applied neat to the tissues

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model (Episkin/Skin Ethic Laboratories, Lyon, France)
- Tissue batch number: 16-RHE-052
- Expiration Date: May 30, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 42 ± 1 min at room temperature; thereafter at 37 °C for 42 ± 1 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS in order to remove any residual test material. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissues inserts with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the RHE model was assessed by undertaking a MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 1% Triton X-100. The ET-50 value was determined to be 6.9 h.
- Other: Absence of significant abnormalities after histological observations (HES stained vertical paraffin)

NUMBER OF REPLICATE TISSUES: The test item as well as the positive and negative control were tested in batch-triplicates. Therefore, a total number of nine tissues was used in this study.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test substance did not directly reduce MTT.

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

ACCEPTABILITY CRITERA
The negative control OD values for the RHE-model have to be in the range of ≥ 0.8 and ≤ 3.0.
The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean (1.417). The standard deviation value is considered valid if ≤ 18% of the group mean value.
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is three standard deviations above the current historical mean (3.26%).
The standard deviation between the three tissue replicates in each group shall be ≤ 18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg per tissue

NEGATIVE CONTROL
- Amount applied: 16 µL per tissue (Dulbecco`s Phosphate-Buffered Saline)

POSITIVE CONTROL
- Amount applied: 16 µL per tissue
- Concentration: 5% aqueous solution of sodium dodecyl sulfate in deionised water

Duration of treatment / exposure:

42 min (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

96

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The negative control OD values were 1.857, 1.830 and 1.822 and, thus, in the range of ≥ 0.8 and ≤ 3.0.

Acceptability of the Positive and Negative Control Data:
After treatment with the negative control (DPBS-buffer) the mean OD was 1.836 (standard deviation: 0.99%) and, thus, higher than the historically established threshold of 1.417.
After treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) the mean viability value was 1.25% (standard deviation: 2.51%) and, thus, lower than the historically established threshold of 3.26%.
The negative and positive control are acceptable as the difference compared with the historical data is minimal.

Variability of the Data:
The standard deviation between the three tissues replicates treated with the test item was 0.33% and, thus, ≤18%. The standard deviations between the three tissue replicates of the negative control and the positive control were 0.99% and 2.51%, respectively, and, thus, ≤18%.

The study met all acceptance criteria.

Table 1: Summary of Results

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.836	100.00
Positive Control	42	0.023	1.25
Test Material	42	1.763	96.00

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance did not show irritating properties towards reconstructed human epidermis tissue.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

07.02.-.22.02.1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

no

Species:

rabbit

Strain:

other: Russian Albino

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Asta Werke, Bielefeld
- Age at study initiation: about 4 month
- Weight at study initiation: 1,8 - 2,2 kg
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1 °C
- Humidity (%): 50 - 60 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12 h

Type of coverage:

occlusive

Preparation of test site:

other: left side shaved, right side clipped

Vehicle:

water

Controls:

not required

Amount / concentration applied:

0.5 g

Duration of treatment / exposure:

4 h

Observation period:

1, 24, 48 and 72 h

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: ~6 cm²
- Type of wrap if used: linen covered with synthetic tape

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize Scoring System

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of all 3 animals

Time point:

other: 1, 24, 48, 72 h

Score:

0

Max. score:

0

Table 1: Evaluation of all animals (mean score)

Day (after treatment)	1 (1 hour)	2 (24 hours)	3 (48 hours)	4 (72 hours)
Erythema	0	0	0	0
Edema	0	0	0	0

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13.02. - 28.08.1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

no

Species:

rabbit

Strain:

other: Russian Albino

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Asta Werke, Bielefeld
- Age at study initiation: about 5 - 6 month
- Weight at study initiation: 2,1 - 2,3 kg
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1 °C
- Humidity (%): 50 - 60 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12 h

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.1 g

Observation period (in vivo):

1, 24, 48 and 72 hours after administration

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM: DRAIZE

TOOL USED TO ASSESS SCORE: hand-slit lamp

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 4 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of all 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

of animal #1 and #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

iris score

Basis:

mean

Remarks:

of animal #1 and #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 48 h

Irritant / corrosive response data:

Slight conjunctivae (score 1) was observed in 1/3 rabbits at the 1-h reading time point, which persisted until 24 h after installation. The conjunctivae had cleared completely within 48 h. Swelling (score 2) was observed in 1/rabbits at the 1-h reading time point, but cleared completely within 24 h. The iris in 1/3 rabbits showed congestion, swelling and moderate circumcorneal hyperaemia of score 1 at the 24-h reading time point. This effect was totally reversible within 48 h. In 2/3 rabbits the corneas showed scattered or diffuse areas of opacity of score 1 at the 1-h reading time point which was fully reversible within 24 h and 48 h, respevtively. In 1/3 rabbits a score of 2 was obtained for the cornea at the 1-h reading time point, which persisted until 24 h after installation. In this animal, a score of 1 was observed at the 48-h and 72-h reading time point.

Table 1: Results of the in vivo eye irritation study

Rabbit #	Time [h]	conjunctivae		iris	cornea
		redness	swelling
1	1	0	0	0	1
	24	0	0	0	0
	48	0	0	0	0
	72	0	0	0	0
	average	0,0	0,0	0,0	0,0
2	1	0	0	0	1
	24	0	0	0	1
	48	0	0	0	0
	72	0	0	0	0
	average	0,0	0,0	0,0	0,3
3	1	1	2	0	2
	24	1	0	1	2
	48	0	0	0	1
	72	0	0	0	1
	average	0,3	0,0	0,3	1,3

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

CLP: not classified

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:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species:

human

Strain:

other: EpiOcular™

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):

18 h

Number of animals or in vitro replicates:

in duplicates for each treatment and contol group

Details on study design:

- RhCE tissue construct used, including batch number: EpiOcular™ tissue (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia), batch number: 23757
- Viability: The quality of the final tissue was assessed by undertaking an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 0.3% Triton X-100. The ET-50 value was determined to be 20.43 min.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a preexperiment. Since the MTT solution colour did not turn blue/purple, the test substance is not presumed to have reduced the MTT. An additional test with freeze-killed tissues did not have to be performed.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength: 570 nm
- Evaluation criteria: The test substance is considered to be not irritating to eye if the test substance-treated tissue viability is >60%.
- Acceptance criteria: The results are acceptable if the negative control OD is >0.8 and <2.5, if the mean relative viability of the positive control is below 50% of the negative control viability and if the difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items).
- Reference to historical data of the RhCE tissue construct: Historical control data was used to assess the validity of the test.

Irritation parameter:

other: % tissue viability mean value of 2 tissues

Run / experiment:

6 h exposure

Value:

107.7

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 OBSERVATIONS:
- Visible damage on test system: The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test substance in water or isopropanol did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.384 and 1.417) was in the range of > 0.8 and < 2.5 (please refer to table 2 under "any other information on results incl. tables").
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (18.2%) (please refer to table 2 under "any other information on results incl. tables").

Table 1 Results after treatment for 6 h with the test substance and the controls

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 minus 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]**
Blank	0.038	0.038	0.038	0.000
Negative Control	1.384	1.361	1.373	1.335	1.355	98.5	3.0	100.0
	1.417	1.410	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 substance	1.387	1.462	1.425	1.387	1.460	102.3	10.8	107.7
	1.562	1.579	1.571	1.533		113.1

* Mean of two replicate wells after blank correction

** Relative absorbance [rounded values]: 100 × (absorbance test substance/positive control) / (absorbance negative control)

Table 2: Historical control data

Positive Control		Negative Control [OD570]
Mean Viability	32.47%	Mean Absorption	1.49
Rel. Standard Deviation	10.29%	Rel. Standard Deviation	0.24
Range of Viabilities	15.90% - 42.30%	Range of Absorbance	1.24 - 2.05
Mean Absorption	0.48
Rel. Standard Deviation	0.14
Range of Absorbance	0.22- 0.64

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

Under the conditions of the conducted EpiOcular the test substance did not possess irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 26, 2016 - August 12, 2016

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 Ocular Corrosives and Severe Irritants)

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)

Specific details on test material used for the study:

Designation: Art. 112488
Synonym: N-Acetyl-DL-tryptophan
Batch: K46889088
Purity: 99.9%
(alkalimetric,
calculated on dried
weight)
Appearance: White to almost white, crystalline powder or colorless crystals
Minimum shelf life: July 31, 2020
Storage: Tightly closed, dark at room temperature (15 to 25°C)

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

750 µL (i.e. 150mg/750µL) of test item preparation, negative or positive control.

The test item Art. 112488 (N-Acetyl-DL-tryptophan) was prepared as a 20% (w/v) suspension in a 0.9% sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

in vitro: triplicate design

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice. The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded.
Each cornea was mounted in a cornea holder (CiToxLAB, Veszprém, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.
For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgeräte GmbH, Asslar, Germany) in a vertical position at 32 ± 1°C for about one hour.
At the end of the incubation period, the incubation medium was replaced by fresh pre-warmed (32 ± 1°C) incubation medium in both compartments. The baseline opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). The light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values).

QUALITY CHECK OF THE ISOLATED CORNEAS
Any corneas that showed macroscopic tissue damage (e.g.scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

NUMBER OF REPLICATES
Three corneas were used per group (negative control, positive control or test item group).

NEGATIVE CONTROL USED
0.9% sodium chloride solution, B. Braun Meldungen AG, Batch: 15385012, Released until: August 2018

POSITIVE CONTROL USED
Art. 814223 (Imidazole), Merck KGaA, Batch S6746923, Purity (GC): 99.8% (a/a), Released until: August 31, 2018. Imidazole was dissolved with 0.9% sodium chloride solution to a concentration of 20% (w/v).

APPLICATION DOSE AND EXPOSURE TIME

TREATMENT METHOD:
Fresh incubation medium was filled into the posterior compartment, while the surface of the cornea in the anterior compartment was treated with 750 µL of either the test item preparation, negative or positive control. The negative and positive control preparations were introduced with the closed-chamber method through the dosing holes of the chamber, whereas the test item was applied to the anterior chamber with the open-chamber method. For this, the window locking-ring and the glass window were removed from the anterior chamber prior to treatment.
After application, the corneas were incubated in an incubator in a horizontal position at 32 ± 1°C for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation period, the negative and positive control preparations were removed from the anterior chamber without opening the chamber. The corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. Fresh incubation medium was replaced in both compartments prior to reading the opacity value after treatment.
To remove the test item from the epithelium, the window locking-ring and the glass window from the anterior chamber were removed. The corneas were gently rinsed with wash medium using a syringe. Before measurement of the opacity value after treatment, fresh incubation medium was replaced in both compartments. Each cornea was observed visually and pertinent observations were recorded (e.g.tissue peeling, residual test chemical, non-uniform opacity patterns).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity value of each individual cornea was corrected for background opacity by subtracting the initial baseline opacity reading from the post treatment opacity reading. In addition, the opacity values of both the treatment and positive control groups were corrected for the mean negative control opacity values.
- Corneal permeability: For each cornea either treated with the positive control or the test item, an individual corrected OD490 value was calculated by subtracting the average negative control permeability value from each individual permeability reading. From the individual corrected permeability values, a mean corrected permeability value was calculated for each group.
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = corrected opacity value + (15 x corrected permeability OD490 value)

DECISION CRITERIA: IVIS ≤ 3 results in no classification, IVIS >55 results in GHS-classification as “inducing serious eye damage, category 1”, IVIS between >3 and ≤55 results in ‘no prediction can be made’ .

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Table 1: Summary of Results

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	SD
Negative control	0.9% NaCl Solution	0.131	0.005	0.211	0.1	0.4
		0.391	0.004	0.451
		-0.456	0.003	-0.416
Positive control	20% Imidazole solution	75.832	2.276	109.977	111.2	1.6
		88.094	1.160	112.999
		80.965	1.969	110.505
Test item	Art. 112488	3.149	0.001	3.164	4.0	1.0
		5.200	-0.002	5.170
		3.783	0.001	3.793

Interpretation of results:

other: a prediction regarding the eye hazard potential according to OECD 437 after exposure to the test substance is not possible.

Conclusions:

Under the conditions of the conducted test, a prediction regarding the eye hazard potential according to OECD 437 after exposure to the test substance is not possible.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are reliable data available on skin and eye irritating properties of N-acetyl-DL-tryptophan (CAS 87-32-1).

Skin irritation

In vitro

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a human skin model according to OECD Guideline 439 and in compliance with GLP (reference 7.3.1-1). In this study the test substance did not show skin damaging properties towards human-derived epidermal keratinocytes. Therefore, the test substance is not predicted to show any skin irritation potential.

In vivo

Skin irritancy potential of N-acetyl-DL-tryptophan was determined in an in vivo skin irritation study performed according to OECD 404 (reference 7.3.1-2). The test substance was topically applied to the shaved skin (left side) and the clipped skin (right side) of three Russian Albino rabbits for 4 h. Local skin irritation reactions were scored at 1, 24, 48 and 72 h after substance application. The scores for all reading time points in all animals for erythema and oedema were 0. The test substance does not cause skin irritation.

Based on the results of the conducted in vitro and in vivo studies, N-acetyl-DL-tryptophan is considered as not irritating to the skin.

Eye irritation

In vitro

The corrosive properties towards the eyes were determined using a bovine corneal opacity and permeability (BCOP) test according to OECD Guideline 437 and in compliance with GLP (reference 7.3.2-2). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 4.0, thus a prediction of the eye-damaging potential was not possible. To exclude the eye irritation potential of the test substance, an in vitro eye irritation test using a human cornea model according to OECD Guideline 492 and in compliance with GLP (reference 7.3.2-1) was conducted. In this study the test substance did not show irritating properties towards human-derived epidermal keratinocytes. The mean tissue viability was 107.7%, and the acceptance criteria were met for the positive and negative controls. Assessing the results of the 2 in vitro tests, the test substance does not meet the classification criteria for eye irritation/corrosion.

In vivo

The eye irritation potential of the test substance was determined by an in vivo eye irritation test in rabbits according to OECD guideline 405 and in compliance with GLP (reference 7.3.2-3).0.1 g N-acetyl-DL-tryptophan (unchanged)was instilled into one eye of each of three male rabbits. The eyes were scored for eye irriation paramters 1, 24, 48 and 72 hours after instillation. Slight conjunctivae (score 1) was observed in 1/3 animals at the 1-h reading time point; persisting until the 24-h reading time point. The conjunctivae had cleared within 48 h. The scores for chemosis were 0 at all the reading time points in all animals. Effects on the iris were observed in 1/3 animals at the 24-h reading time point with a score of 1, which had cleared within 48 h. Effects on the cornea were observed in 2/3 rabbits with a score of 1 at the 1-h reading time point, which was fully reversible within 24 and 48 h, respectively. In 1/3 rabbits a cornea score of 2 was observed at the 1-h and 24-h reading time point, being not fully reversible within 72 h (score 1).

Under the experimental conditions the mean chemosis score for each animal was 0.00. The mean conjuctivae scores and mean iris scores for the 3 animals were 0.00, 0.00 and 0.33, respectively. The mean cornea scores for the 3 animals were 0.00, 0.33 and 1.33, respectively.

Based on the results of the conducted studies, N-acetyl-DL-tryptophan is considered as not irritating to the eyes.

Justification for classification or non-classification

The available data on skin and eye irritation with N-acetyl-DL-tryptophan do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.