Poly(substitutedmethyl)amino acid

Endpoint summary

Description of key information

In vitro skin and eye irritation studies are available.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Oct 2018 - 08 Oct 2018

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)

Version / remarks:

20 July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes

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).

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™
- Tissue batch number: 18-EKIN-040
- Surface: 0.38 cm^2
- Expiration date: 8 October 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 36.8 – 37.3°C)
- Humidity(%): 80 - 100% (actual range 71 - 86%)

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.
- Volumen 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 in air containing 5% CO2.
- 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.

Duration of treatment / exposure:

15 minutes

Irritation / corrosion parameter:

% tissue viability

Value:

82

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean cell viability: 100%

Positive controls validity:

valid

Remarks:

Mean cell viability: 17%

Table 2:  Individual OD Measurements at 570 nm

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570 measurement 1 OD570 measurement 2	1.2691 1.4125	1.1130 1.2929	1.3486 1.6808
Test item OD570 measurement 1 OD570 measurement 2	0.9845 1.1103	1.1447 1.1547	1.1466 1.1748
Positive control OD570 measurement 1 OD570 measurement 2	0.4137 0.4675	0.2014 0.2271	0.1325 0.1521

OD = Optical density

Triplicate exposures are indicated by A, B and C.

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: yes, the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.

- Acceptance criteria met for positive control: yes, the mean relative tissue viability was 17 %

- Acceptance criteria met for variability between replicate measurements: yes, the variability between tissues treated with the test item was ≤ 12%

- Since the mean relative tissue viability for Substituted amino acid (2) solution FC-C 13587 was above 50% is considered to be non-irritant.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the test substance is not irritating in the in vitro skin irritation test.

Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Small model (EPISKIN-SM ™)), the influence of Substituted amino acid (2) solution FC-C 13587 on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin. After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 7% whereas the test substance showed cell viability of 82%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test. Based on these results, Substituted amino acid (2) solution FC-C 13587 needs not to be classified for skin irritating properties.

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:

06 November 2018

Reliability:

1 (reliable without restriction)

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

Specific details on test material used for the study:

No correction was made for the purity/composition of the test item.

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 and those exhibiting defects were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
750 μL directly on top of the corneas.

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes
90 ± 5 minutes with Na-fluorescein cMEM solution

Number of animals or in vitro replicates:

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

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
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. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed.

- POST-EXPOSURE INCUBATION: 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 passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values
of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
- Other: possible pH effects of the test substance on the corneas were recorded.

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.

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).
Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

DECISION CRITERIA: According to the guideline (see table 1)

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 replicates

Value:

110

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean IVIS: 0.1

Positive controls validity:

valid

Remarks:

Mean IVIS: 44

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- The corneas treated with the test item showed opacity values of 88, 90 and 80.
- Permeability values of the test item were 1.725, 1.225 and 1.867.
- Individual IVIS scores of the corneas treated with the test item were 114, 109 and 108 (n=3).

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

ACCEPTANCE OF RESULTS (see table 2) :
- Acceptance criteria met for negative control: yes, results were within historical range (IVIS ranging from -0.7 to 1.3).
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 30 to 52).

 Table 2: In Vitro Irritancy Score

Treatment	Final Opacity(A)	Final OD4902	In vitro Irritancy Score(B)
Negative control	-0.3	-0.002	-0.4
	1.2	0.009	1.3
	-0.8	0.006	-0.7
Positive control	22	1.869	50
	14	1.034	30
	18	2.259	52
The test item	88	1.725	114
	90	1.225	109
	80	1.867	108

(A)In vitro irritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

(B)Positive control and test item are corrected for the negative control.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed according to OECD guideline 437 and GLP principles. Substituted amino acid (2) solution FC-C 13587 induced serious eye damage through both endpoints resulting in an average IVIS > 55. Therefore Substituted amino acid (2) solution FC-C 13587 is classified as Category 1 according to GHS criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

This substance does not need classification for skin irritation. It should be classified for eye irritation, cat. 1. This may be related to the low pH of 1.6 of the solution.