(R)-2-(2,5-difluorophenyl)pyrrolidine (R)-2-hydroxysuccinate

Endpoint summary

Description of key information

Skin irritation (EpiSkin, OECD TG 439), Wingenroth 2021: not irritant

Eye irritation (EpiOcular, OECD TG 492); Leidenfrost 2021: irritant

Eye corrosion (BCOP, OECD TG 437); Leidenfrost 2021: not corrosive

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:

Sept-Nov 2021

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:

2021

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)

Specific details on test material used for the study:

-Purity (HPLC area %) 100
-Test item was used undiluted.
-For the solid test item 30 µl of 0.9% NaCl were used to moisten and ensure good contact with the epide1mis surface.

Test system:

human skin model

Source species:

human

Justification for test system used:

commercially available test method

Vehicle:

other: 0.9% NaCl

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (Henkel AG & Co, KGaA, Düsseldorf))

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2
gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION
- The optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA
- The mean optical density (OD) values obtained with the test item were used to calculate the
percentage of viability relative to the negative control, which is set at 100 %.
- According to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 0.9% NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% SDS in physiological saline

Duration of treatment / exposure:

20 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

cell viability, after 20 min [%]

Value:

100.58

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 EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No - according to the results of the pre-check
- Colour interference with MTT: No - according to the results of the pre-check

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Tabular summary of the results    

 

Sample No.	Test item	OD mean *	Std Dev	% Viability
1 - 3	Negative control NaCl 0.9 %	1.83	0.02	100.00
4 - 6	Positive control SDS 5 %	0.02	0.00	1.31
7 -9	test item (DFPP MALAT)	1.84	0.01	100.58

 

* 6 values

Interpretation of results:

GHS criteria not met

Conclusions:

not irritant, no classification warranted

Executive summary:

A study was performed for the assessment of the skin irritancy of the test item DFPP MALAT with reconstructed human epidermis (RhE). The experiment was carried out in vitro using the commercially available test method epiCS®.
The study was conducted in accordance with OECD TG 439 and EU Test Method B.46.
The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.
Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100%.
The results of the concurrent negative control (NC, 0.9 % NaCl) and positive control (PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.
The following value of cell viability was recorded for the test item: 101 % (rounded).
In conclusion the results of the assay used show no skin initant properties of the test item DFPP MALAT and thus, the test item requires no classification according to CLP.

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:

Oct - Nov 2021

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:

-Purity (HPLC area %) 100
- used as solid

Species:

other: reconstructed human cornea-like epithelium (RhCE)

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

JUSTIFICATION OF THE TEST METHOD AND CONSIDERATIONS REGARDING APPLICABILITY :
The EpiOcular™ eye irritation test (EIT) follows international OECD test guidelines. The EIT measures the ocular irritation potential of a test item by determination of cytotoxic effects on a reconstructed human cornea epithelium (RhCE) tissue model to discriminate chemicals not requiring classification for eye irritancy (UN GHS No Category) from those requiring classification. The EpiOcular™ EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation).

DESCRIPTION OF THE CELL SYSTEM USED, INCL. CERTIFICATE OF AUTHENTICITY AND THE MYCOPLASMA STATUS OF THE CELL LINE :
The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The model is standardized and commercially available. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier (MatTek Corporation, Slovakia).

ENVIRONMENTAL CONDITIONS :
The environmental conditions in the incubator were standardized as follows:
Incubator temperature: 37 +/- 2° C
CO2 gas concentration: 5 %
Humidity: 95 %
All incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode, Germany).

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 per tissue insert in duplicate
- Concentration (if solution): neat test item

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 μLper tissue insert in duplicate

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

tissue inserts were used in duplicate for test item, negative and positive control

Details on study design:

RhCE TISSUE CONSTRUCT USED, INCLUDING BATCH NUMBER :
EpiOcular RhCE tissue supplied by MatTek Corporation,

PRE-CHECK FOR POTENTIAL OPTICAL INTERFERENCES OF THE TEST ITEM :
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability.
The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment of potential direct MTT-reduction of the test item
In case of a direct MTT-reduction of the test item a killed tissue control (inserts, which were killed by freezing) was used in the main assay.
2. Assessment of potential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol
In case of an influence of test item color on OD measurement, a color control was used in the main assay.
The evaluation criteria for the pre-check were:
- For MTT reduction (visual assessment): If the MTT solution color turns blue/purple, the test item is presumed to have reduced the MTT. A killed control must be conducted.
- For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is >0.08 a Color Control must be conducted.

PROCEDURE FOR KILLED CONTROL OR COLOR CONTROL :
Killed control and color control not required.

DESCRIPTION OF THE METHOD USED TO QUANTIFY MTT FORMAZAN :
For viability testing the inserts were placed in new plates containing MTT solution (1 mg/ml in Maintenance medium at 37°C). The tissues were incubated for 180 ± 10 min. under standard culture conditions. The extraction of blue formazan was performed in isopropanol on a vertical shaker for 2-3 hours at room temperature. The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer (EL808, Bio-Tek; 96 well format, 200 μl). Data acquisition and evaluation were performed with the software "Gen5" (Bio-Tek).

ASSAY ACCEPTANCE CRITERIA :
The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.8
- mean relative viability of the positive control (PC) is < 50 % (relative to negative
control)
- the difference of viability between the two replicates is < 20 %.

Irritation parameter:

mean percent tissue viability 

Value:

2.92

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No - according to the results of the pre-check
- Colour interference with MTT: No - according to the results of the pre-check

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS :
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

 

	Final Cell viability	OD mean (n=4)
Compound	[%]
DFPP MALAT	2.92	0.054
Positive control	22.47	0.418
Negative control	100	1.861

 

Conclusions:

irritating properties; further tests warranted

Executive summary:

An in vitro study for assessing ocular irritation properties of the test item DFPP MALAT was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular™. The model used is standardized and commercially available.
The EpiOcular™ Eye Irritation Test (EIT) was conducted in accordance with OECD 492.
The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period.
Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %.
The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model.
The final mean percent tissue viability recorded for the test item is 3% (rounded).
According to the results of this study the test item DFPP MALAT was identified as  irritant to the eyes and further test are required for classification according to CLP.

Reference 2

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

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:

2020

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 100%
used as solid

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- Number of animals: n/a
- Characteristics of donor animals (e.g. age, sex, weight): not reported
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transport: 1 L containers with 500 mL HSS and 1 % penicillin / streptomycin solution, transport of the containers in coolers on ice
- Indication of any existing defects or lesions in ocular tissue samples: Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter. Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin / streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.
- Selection and preparation of corneas: On the next day the containers with the eyes were transferred in an incubator at 32 °C (± 1 °C) for about 2 hours. For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed in a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour into the incubator at 32 °C (± 1 °C).
- Quality check of the isolated corneas: After approximately 1 hour, the MEM medium was aspirated, and the chambers were filled with fresh MEM medium. For each cornea the reference opacity value was measured then.

Vehicle:

physiological saline

Remarks:

The test item was suspended with a glass rod, a whirl mixer and shaking shortly prior to application in isotonic saline solution to achieve a concentration of 20 % (w/v). The preparation was visually described as suspension.

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% (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

4 h

Number of animals or in vitro replicates:

three corneae each treatment and /or control

Details on study design:

NUMBER OF REPLICATES: three

NEGATIVE CONTROL USED: yes physiological saline

SOLVENT CONTROL USED (if applicable) yes physiological saline

POSITIVE CONTROL USED yes imidazole 20% solution

APPLICATION DOSE AND EXPOSURE TIME 750 µL of a 20% suspension (w/v)

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Three

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort 3.4 SP6 software from Testo AG, Lenzkirch).
- Corneal permeability: The medium in anterior chamber of each holder was replaced by 1ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes.
After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 µL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluorescein solution was prepared and also filled into the 96-well plate, in triplicates.
The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader (Bio-Tek EL 808, Software Gen5).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
Same decision citreria used as specified in the guideline.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

-7.6

Vehicle controls validity:

valid

Remarks:

-1.7

Positive controls validity:

valid

Remarks:

139.7

Irritation parameter:

other: permeability value

Run / experiment:

mean

Value:

-0.002

Vehicle controls validity:

valid

Remarks:

0.049

Positive controls validity:

valid

Remarks:

1.193

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

7.6

Vehicle controls validity:

valid

Remarks:

-2.4

Positive controls validity:

valid

Remarks:

121.8

Opacity score

	Cornea No.	Initial opacity	final opacity	Opacity change	Mean	SD
Negative control isotonic saline solution	1	7.9	6.4	-1.5
	2	7.4	4.5	-2.9	-2.4	0.8
	3	7.2	4.4	-2.9
	Cornea No.	Initial opacity	final opacity	Opacity change	Corrected opacity change	Mean	SD
Positive control Imidazole	4	7.0	199.9	192.9	195.3
	5	6.6	108.6	102.0	104.4	121.8	66.5
	6	6.5	69.8	63.3	65.7
Test item	7	6.4	8.1	1.7	4.2
	8	6.3	14.2	7.9	10.3	7.6	3.1
	9	6.1	12.0	5.9	8.3

 

Permeability score

	Cornea No.	Mean OD490	Mean	SD
Negative control isotonic saline solution	1	0.050
	2	0.046	0.049	0.002
	3	0.051
	Cornea No.	Mean OD490	Mean corrected OD490	Mean	SD
Positive control Imidazole	4	1.111	1.062
	5	1.538	1.490	1.193	0.257
	6	1.076	1.028
Test item	7	0.046	-0.002
	8	0.050	0.002	-0.002	0.003
	9	0.044	-0.005

 

Tabular In vitro irritancy score

	Cornea No.	Opacity per cornea	Permeability per cornea	per cornea	IVIS per group
					mean	SD
Negative control isotonic saline solution	1	-1.5	0.050	-0.7
	2	-2.9	0.046	-2.3	-1.7	0.8
	3	-2.9	0.051	-2.1
Positive control Imidazole	4	195.3	1.062	211.2
	5	104.4	1.490	126.8	139.7	66.0
	6	65.7	1.028	81.1
Test item	7	4.2	-0.002	4.1
	8	10.3	0.002	10.3	7.6	3.1
	9	8.3	-0.005	8.3

 

 

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on OECD TG 437 and the experimental conditions reported the test item is not classified as seriously damaging the eye (no Category).

Executive summary:

This in vitro study was performed to assess them corneal irritation and damage potential of DFPP MALAT by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 2020.

 

The corneae were incubated with the test substance and controls for 4 h. After exposure the corneae were rinsed with phenol red containing MEM. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) was calculated as mean opacity value + (15 x mean corrected OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

A 20% dilution of the test substance in isotonic saline solution caused no increase of the corneal opacity and permeability. 

The positive control (Imidazole) increased the opacity and permeability of the corneae in this experiment.

With the negative control (isotonic saline solution) neither an increase of opacity nor permeability of the corneae could be observed in the experiment.

Since the mean in vitro irritancy score of the test substance was 7.6, DFPP MALAT is not considered to be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the study results no classification is warranted for skin irritation and classification Cat. 2 (H319) is warranted for eye irritation according to Regulation (EC) No. 1272/2008 (CLP), ANNEX I.