Sodium salts of substituted amino acid (2)

Endpoint summary

Description of key information

In vitro skin and eye irritation studies available.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Nov 2018 - 17 Dec 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)

Version / remarks:

July 2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™
- Tissue batch number: 18-EKIN-050
- Surface: 0.38 cm^2
- Expiration date: 17 December 2018

TEST FOR THE INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
- The test substance was checked for possible color interference and reduction of MTT before the study. No interference was found.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure to the test item: room temperature
- Temperature of incubations(°C): 37.0 ± 1.0°C (actual range 35.9 – 37.0°C)
- Humidity(%): 80 - 100% (actual range 70 - 90%)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: tissues were washed with phosphate buffered saline (1 washing step)
- Observable damage in the tissue due to washing: no

APPLICATION/TREATMENT OF TEST ITEM
- Number of replicate tissues: 3 for the test substance. Additionally, 3 tissues for the negative and the positive control each.
- Volume test item: 25 μL
- The positive control was re-spread after 7 minutes contact time.
- Exposure period: 15 minutes.
- Incubation period after exposure: 42 hours at 37°C.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 single run

MEASUREMENT OF CELL VIABILITY
- After incubation, tissues were dried and incubated with 2 mL MTT-solution (0.3 mg/mL in PBS).
- Incubation time: 3 hours at 37°C
- Formazan was extracted with 500 μL isopropanol (MatTek corporation)
- The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

INTERPRETATION (see Table 1)
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

ACCEPTABILITY CRITERIA
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
b) The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The liquid test item was applied undiluted (25 μL) directly on top of the tissue.

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37°C +3 hours with MTT

Number of replicates:

The test was performed on a total of 3 tissues per test item together with negative and positive controls.

Irritation / corrosion parameter:

% tissue viability

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- No visible damage on test system, no direct-MTT reduction and no colour interference with MTT was observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 11%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was < 7%, indicating that the test system functioned properly.

Table 2: Individual OD Measurements at 570 nm

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	1.1825 1.1440	1.1022 1.0589	1.2550 1.2064
Test item OD570measurement 1 OD570measurement 2	1.2464 1.2055	1.2406 1.1709	1.2199 1.1644
Positive control OD570measurement 1 OD570measurement 2	0.1414 0.1401	0.2036 0.2002	0.1645 0.1616

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of a skin irritation study, performed according to OECD guideline 439, Sodium salts of substituted amino acid (2) solution is non-irritant under the experimental conditions described in this report. The substance does not require classification according to GHS and according to Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jan 2019

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)

Version / remarks:

09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse Vitelco, 's-Hertogenbosch, The Netherlands.
- Storage, temperature, and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes in cMEM for opacity measurements and subsequently 90 ± 5 minutes in sodium-fluorescein for permeability determinations

Number of animals or in vitro replicates:

3 replicates for the negative, positive, and treatment group each.

Details on study design:

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µl of either the negative control, positive control or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea.
Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded.
- POST-EXPOSURE INCUBATION: 120 ± 10 minutes in cMEM for opacity measurements and subsequently 90 ± 5 minutes in sodium-fluorescein for permeability determinations

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (TECAN Infinite® M200 Pro Plate Reader). OD490 values of less than 1.500 were used in the permeability calculation.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

DECISION CRITERIA: (see table 1):
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤ 55, no prediction on irritant potency can be made.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 replicates

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean IVIS: 1.4

Positive controls validity:

valid

Remarks:

Mean IVIS: 49

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- The corneas treated with the test item showed opacity values of: -0.7, 0.6, 1.1 (corrected for negative control)
- Permeability values were: 0.010, 0.070, 0.014 (corrected for negative control)
- Individual IVIS scores were: -0.6, 1.7, 1.3

OTHER EFFECTS:
- The corneas were clear after the 10 minutes of treatment with the test item.
- A pH effect of the test item was observed on the rinsing medium

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within historical range (IVIS ranging from 0.7 to 1.8).
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 37 to 63).

Interpretation of results:

GHS criteria not met

Conclusions:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed according to OECD guideline 437 and GLP principles. Sodium salts of substituted amino acid (2) solution induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on available data the substance does not need to be classified for skin and eye irritation.