L-aspartic acid, sodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

sodium 2-amino-3-carboxypropanoate

Cas Number:

323194-76-9

Molecular formula:

C4H7NO4.xNa

IUPAC Name:

sodium 2-amino-3-carboxypropanoate

Test material form:

solid: particulate/powder

Details on test material:

Name: L-aspartic acid, sodium salt monohydrate 1
Batch/Lot number: Z201125/ VG29848563
CAS number: 323194-76-9
Anhydrous substance name: L-aspartic acid, sodium salt, CAS: 17090-93-6
Appearance: Solid, white, crystalline powder
Purity: 98%
Expiry date: 24 November 2022
Storage conditions: Room temperature (15-25 ºC), protected from humidity (tight closed
container)

Specific details on test material used for the study:

Name: L-aspartic acid, sodium salt monohydrate
Lot No.: Z201125 / VG29848563
CAS No.: 323194-76-9
Appearance: white crystalline powder
Manufacturing date: 25 November 2020
Expiry date: 24 November 2022
Storage: At room temperature (15-25°C), protected from humidity
Safety precautions: According to the SDS

Test animals / tissue source

Species:

chicken

Strain:

other: Strain of chicken: ROSS 308

Details on test animals or tissues and environmental conditions:

Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni utca 129. Hungary
Only the eyes of healthy animals considered suitable for entry into the human food chain were used. Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal
(23.7 ºC to 23.9 ºC in first experiment and 19.3 ºC to 20.2 ºC in the additional experiment) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4 5 heads/box).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent negative control

Amount / concentration applied:

The test item was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test item, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eye.

Duration of treatment / exposure:

The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.

Observation period (in vivo):

na

Duration of post- treatment incubation (in vitro):

The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.

Number of animals or in vitro replicates:

3

Details on study design:

Treatment
After the zero reference measurements in each experiment, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the center of the cornea. The test item was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test item, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eye.
The three positive control eyes were treated in a similar way with 0.03 g of Imidazole.
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 µL from micropipette, in such a way that the entire surface of the cornea was covered with negative control, taking care not to damage or touch the cornea with the application equipment.
Based on the results of the experiment the test item showed negative outcome (GHS NC), consequently additional experiment was necessary to confirm or discard the negative outcome. All the steps in the additional experiment were the same as in the first experiment.


7.2.2 Test item removal
The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.

The minor amount of the test item, which did not prevent the observation process of the corneas, was stuck on the corneas’ surface in all eyes (three eyes) at 30 minutes after the post-treatment rinse in the first experiment. Gentle rinsing with 20 mL saline was performed in all test item treated eyes after the 30, 75, 120 and 180 minutes of observation. The test item treated cornea surfaces were not totally clear at 240 minutes after the post-treatment rinse in the first experiment. This effect was not observed during the additional experiment.

The positive control item Imidazole was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse in the first and additional experiments. Gentle rinsing with 20 mL saline was performed in all positive control treated eyes after the 30, 75, 120 and 180 minutes of observation. The positive control treated cornea surfaces were not totally clear at 240 minutes after the post-treatment rinse in the first and additional experiments.

7.2.3 Observation and assessment of corneal effects
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t = 0) and 30 minutes after the post-treatment rinse.

7.2.4 Retention of corneas
At the end of the procedures, the corneas were carefully removed from the eyes and placed individually into labeled containers of preservative fluid (4 % formaldehyde) for potential histopathology and stored, protected from light at room temperature.

7.2.5 Histopathology
Histopathology of the corneas was not performed. Corneas are discarded 2 months after the end of the experimental phase.


7.2.6 Demonstration of Proficiency
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.549.3229) using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

75 min scoring

Value:

>= 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

240 min scoring

Value:

>= 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

>= 1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

>= 0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Other effects / acceptance of results:

other effects: none

Any other information on results incl. tables

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity, fluorescein retention and other observation (morphological effect etc.) are given below. The experimental results are summarized in Tables 1.1-1.6 (Appendix I). The conclusion on eye irritancy was based on the criteria given in OECD guideline on quantitative assessments, shown in Appendices III and IV.

 

First experiment:

 

Test Item: L-aspartic acid, sodium salt monohydrate

 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	2%	I
Mean maximum corneal swelling at up to 240 min	2%	I
Mean maximum corneal opacity	1.0	II
Mean fluorescein retention	0.2	I
Other Observations	None
Overall ICE Class^1	2xI, 1xII

 

Positive Control: Imidazole

 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	32%	IV
Mean maximum corneal swelling at up to 240 min	36%	IV
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Corneal opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class^1	3xIV

 

The positive control Imidazole was classed as corrosive/severely irritating, UN GHS/CLP Classification: Category 1.

 

¹ Details of data interpretation for Isolated Chicken Eye Class are given in Appendices III and IV.

 

Negative Control: NaCl (9 g/L saline)

 

Observation	Value	ICE Class^1
Maximum corneal swelling at up to 75 min	0%	I
Maximum corneal swelling at up to 240 min	0%	I
Maximum corneal opacity	0.5	I
Fluorescein retention	0.5	I
Other Observations	None
Overall ICE Class^1	3xI

 

The negative control NaCl (9g/L saline) had no significant effects on the chicken eye in this experiment.

 

Additional experiment:

 

L-aspartic acid, sodium salt monohydrate

 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	1%	I
Mean maximum corneal swelling at up to 240 min	2%	I
Mean maximum corneal opacity	0.5	I
Mean fluorescein retention	1.0	II
Other Observations	None
Overall ICE Class^1	2xI, 1xII

 

 

Positive Control: Imidazole

 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	36%	IV
Mean maximum corneal swelling at up to 240 min	37%	IV
Mean maximum corneal opacity	3.8	IV
Mean fluorescein retention	3.0	IV
Other Observations	Corneal opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class^1	3xIV

 

The positive control Imidazole was classed as corrosive/severely irritating, UN GHS/CLP Classification: Category 1.

 

¹ Details of data interpretation for Isolated Chicken Eye Class are given in Appendices III and IV.

 

Negative Control: NaCl (9 g/L saline)

 

Observation	Value	ICE Class^1
Maximum corneal swelling at up to 75 min	2%	I
Maximum corneal swelling at up to 240 min	2%	I
Maximum corneal opacity	0.0	I
Fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class^1	3xI

 

The negative control NaCl (9g/L saline) had no significant effects on the chicken eye in this experiment.

 

Positive and negative control values were within the corresponding historical control data ranges (Appendix II) in the first and the additional experiments.

 

¹ Details of data interpretation for Isolated Chicken Eye Class are given in Appendices III and IV.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this ICET, the overall ICE classes for the test item was 2xI and 1xII in the first and the additional experiments.
According to the guideline OECD 438, L-aspartic acid, sodium salt monohydrate is categorized as “No Category”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item L-aspartic acid, sodium salt monohydrate by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (0.03 g/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS/CLP)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test item exposure) were determined at each of the above time points.

 

Based on the results of the first experiment the test item showed a negative outcome (GHS NC). According to the Study Plan an additional experiment was necessary to confirm or discard the negative outcome.

 

The positive control Imidazole were grounded before the use in the study. The test item and positive control were applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item or positive control, respectively. Three test item treated eyes, three positive control treated eyes and one negative control treated eye (which was treated with 30 µL of NaCl 9g/L saline solution) were used in the first and additional experiments.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

 

The minor amount of the test item, which did not prevent the observation process of the corneas, was stuck on the corneas’ surface in all eyes (three eyes) at 30 minutes after the post-treatment rinse in the first experiment. The test item treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse in the first experiment. This effect was not observed during the additional experiment.

 

Imidazole was stuck on the corneas’ surface in all eyes (three eyes) at 30 minutes after the post-treatment rinse in both cases (first and additional experiments). The Imidazole treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse in the first and the additional experiments.

 

 

The overall ICE classes of the test item treated corneas were twice I (based on corneal swelling of 2 % within 240 minutes and based on the fluorescein retention of 0.2) and once II (based on the corneal opacity score of 1.0) in the first experiment and the overall ICE classes of the test item treated corneas were twice I (based on corneal swelling of 2 % within 240 minutes and based on the corneal opacity score of 0.5) and once II (based on the fluorescein retention of 1.0) in the additional experiment.

 

The positive control Imidazole was classed as corrosive/severely irritating, UN GHS/CLP Classification: Category 1 and the negative control had no significant effects on the chicken eye in in the first and in the additional experiments. So, the positive and negative controls showed the expected results in the first and in the additional experiment. The experiments were considered to be valid.

 

In this ICET, the overall ICE classes for the test item was 2xI and 1xII in the first and the additional experiments.

According to the guideline OECD 438, L-aspartic acid, sodium salt monohydrate is categorized as “No Category”.