3-Quinolinecarboxylic acid, 7-chloro-1-cyclopropyl-1,4-dihydro-8-methyl-4-oxo-, ethyl ester

Endpoint summary

Description of key information

Skin irritation (in vitro): Key study. Test method according to OECD 439, GLP study. The test substance can be considered as not irritant to skin as the mean percent viability of the treated tissues was found 78.2% in the in vitro RhE test.

Eye irritation (in vitro): Key study. Test method according to OECD 438, GLP study. The test item does not require classification for serious eye damage/eye irritation since the combination of the 3 endpoints assessed in the ICE test ( fluorescein retention, corneal opacity and corneal swelling) was 2x II and 1xI.

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:

02 July 2020 - 23 July 2020

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)

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)

Test system:

human skin model

Remarks:

(SkinEthic RHE® model)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Justification for test system used:

The SkinEthic RHE® model has been validated for irritation testing and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 20-RHE-080
- Production date: N/A
- Shipping date: 21.07.2020
- Delivery date: 21.07.2020
- Date of initiation of testing: 21.07.2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature.
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.1 (Acceptance criterion: >0.7). Historical negative control mean OD range = 0.530-1.211 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.5)
- Barrier function: 4.4 h (Acceptance criterion: 4.0h ≤ ET50 ≤10.0h)
- Morphology: 5.5 Cell layers (specification ≥ 4). Multi-layered, highly differenciated epidermis consisting of organized basal, spinous and granular layers and a multilayered stratum corneum.
- Contamination: No

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: there is no direct interaction between the test item and MTT.

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

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered as non-irritant to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32 mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 min at room temperature.

Duration of post-treatment incubation (if applicable):

41 hours at 37ºC, 5% CO2.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

78.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(distilled water)

Positive controls validity:

valid

Remarks:

2.2% viability (5% SDS)

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin model, plus a reduced validation with the SkinEthic RHE model, having into account that both models are very similar. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean OD = 0.918 (OD measured after 1:2 dilution in isopropanol; criterion for acceptability should be in the range ≥ 0.4 and ≤1.5).
- Acceptance criteria met for positive control: yes, mean viability = 2.2% (criterion for acceptability should be < 40%).
- Acceptance criteria met for variability between replicate measurements: yes. SD of negative, positive and test item replicates were 1.1, 0.5 and 13.5% respectively (criterion for acceptability, SD ≤ 18%).

Table 1. Table of results

	Well ID	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	SPL 1	0.885	0.930	0.918	101.3	100.0	1.1
		0.992
		0.913
	SPL 2	0.878	0.915		99.6
		0.927
		0.940
	SPL 3	0.920	0.910		99.1
		0.905
		0.906
Positive control	SPL 4	0.025	0.025	0.020	2.7	2.2	0.5	Irritant
		0.026
		0.024
	SPL 5	0.015	0.016		1.7
		0.016
		0.017
	SPL 6	0.019	0.019		2.1
		0.019
		0.02
Test item PH-20/0484	SPL 12	0.836	0.855	0.718	93.1	78.2	13.5	Non irritant
		0.873
		0.857
	SPL 13	0.624	0.614		66.9
		0.613
		0.605
	SPL 14	0.666	0.686		74.7
		0.699
		0.694

# mean of 3 values (triplicate of the same extract).

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The test substance can be considered as not irritant to skin as the mean percent viability of the treated tissues was found 78.2% in the in vitro RhE test.

Executive summary:

An in vitro skin irritation test was conducted for the test item in a reconstructed human epidermis model ( SkinEthic™) according to OECD TG 439 (GLP study). Three epidermis units, previously moistened with 10 μL of distilled water, were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated for the post-treatment incubation period for 19 hours and 41 minutes in maintenance medium (Episkin SA, batch No. 20 SMM 020) and for 21 hours and 19 minutes in fresh growth medium (Episkin SA, batch No. 20 SGM 051) at 37ºC, 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Under the test conditions, the mean percent viability of the treated tissues was 78.2%, versus 2.2% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is considered as not irritant to the skin.

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:

20 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

yes

Remarks:

Time from collection of the heads to enucleation was 2 h and 05 min instead of 2 h. The eyes were incubated for 55-75 min instead of 45-60 min. As the results obtained with controls were as expected, these deviations are considered as without impact.

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption.
- Number of animals: Not specified.
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old. 1.5 - 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: 2 hours and 05 min.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no

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):30 mg of test item

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

No post-treatment incubation is performed.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.9ºC and 32.0ºC.
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure using sodium fluorescein. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope.
Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that were not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes were rejected. (see table appendix No.4 on "Any other information on results incl. tables")
Once all eyes had been examined and approved, the eyes were incubated between 55 and 75 minutes to equilibrate them to the test system prior to dosing.

EQUILIBRATION AND BASELINE RECORDINGS:
Eyes were incubated between 55 and 75 minutes to equilibrate them to the test system prior to dosing (OECD TG 438 indicates approximately 45 to 60 min. As the results obtained with the eyes treated with the negative control, which has the longest time of acclimation, were conformed to what was expected, this deviation is considered as without impact on the conclusion of the study).
Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED
30 μL physiological saline - Dutscher Batch No. 3014011 (one eye)

SOLVENT CONTROL USED: not applicable.

POSITIVE CONTROL USED
Sodium hydroxide – Fisher Scientific, Batch No. 1550248 - 30 mg (three eyes)

APPLICATION DOSE AND EXPOSURE TIME
30 mg of the test item was applied for 10 seconds.

OBSERVATION PERIOD:Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: NO

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points (see table 4)
- Damage to epithelium based on fluorescein retention: Fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item (see table 5)
- Swelling: optical pachymeter on a slit-lamp microscope ((HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I). The slit-width was set at 9 1/2 equalling 0.095 mm. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item (see table 3).
- Macroscopic morphological damage to the surface: The aim of this evaluation was to determine whether any “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea were visible.These findings can vary in severity and may occur simultaneously.

SCORING SYSTEM:
- Mean corneal swelling: It was expressed as a percentage and was calculated from corneal thickness measurements according to the following formula:
(corneal thickness measurement at time t - corneal thickness at time=0 / corneal thickness at ime=0 )*100

- Mean maximum opacity score:
0 - No opacity,
0.5 -Very faint opacity
1- Scattered or diffuse areas; details of the iris clearly visible
2- Easily discernible translucent area; details of the ris are slightly obscured,
3-Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4-Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment :
0-No fluorescein retention,
0.5-Very minor single cell staining,
1-Single cell staining scattered throughout the treated area of the cornea,
2-Focal or confluent dense single cell staining,
3-Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: Decision criteria was used as indicated in the TG.

Irritation parameter:

cornea opacity score

Run / experiment:

Highest mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE Class I

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean at 30 minutes post-treatment

Value:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class II

Irritation parameter:

percent corneal swelling

Run / experiment:

Highest mean

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class II

Irritation parameter:

morphological effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No effects

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: NO, no morphological effects were noted, whatever the examination time.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES, the combination of the three endpoints for the negative control, physiological saline, was 3xI classified as “No Category”
- Acceptance criteria met for positive control: YES, the combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV,classified as “Corrosive/Severe Irritant ”

Appendix No. 4: Selected eyes for the performance of the ICE test

Chamber	Fluorescein retention	Corneal opacity	Morphological effects	Corneal thickness (e)
n°1	0.5	0	N.t.R.	0.50
n°2	0.5	0	N.t.R.	0.48
n°3	0.5	0	N.t.R.	0.49
n°4	0.5	0	N.t.R.	0.51
n°5	0.5	0	N.t.R.	0.51
n°6	0.5	0	N.t.R.	0.53
n°7	0.5	0	N.t.R.	0.53
n°8	0.5	0	N.t.R.	0.53
n°9	0.5	0	N.t.R.	0.49
n°10	0.5	0	N.t.R.	0.48
n°11	0.5	0	N.t.R.	0.51
n°12	0.5	0	N.t.R.	0.52
n°13	0.5	0	N.t.R.	0.45
n°14	0.5	0	N.t.R.	0.50
n°15	0.5	0	N.t.R.	0.47
n°16	0.5	0	N.t.R.	0.48
Mean corneal thickness value =				0.50
Range of accepted thickness :				0.45 ≤ e ≤ 0.55

N.t.R: Nothing to report

Table 9: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Test item

Endpoint measured	Eye No.	Time (min
		0	30	75	120	180	240
Corneal opacity	10	0	0	0	0	0	0
	11	0	0	0	0	0	0
	12	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class			I
Fluorescein retention	10	0.5	1	-	-	-	-
	11	0.5	0.5	-	-	-	-
	12	0.5	0.5	-	-	-	-
Mean		0.5	0.7	-	-	-	-
ICE class			II
Corneal thickness	10	0.48	0.48	0.50	0.53	0.57	0.57
	11	0.51	0.51	0.51	0.54	0.54	0.54
	12	0.52	0.52	0.52	0.55	0.55	0.55
Corneal swelling (%)	10	-	0	4	10	19	19
	11	-	0	0	6	6	6
	12	-	0	0	6	6	6
Mean		-	0	1	7	10	10
ICE class			II
Combination of the 3 Endpoints		2 x II, 1 x I
CLASSIFICATION		No Category

Note: No morphological effects were noted, whatever the examination time.

Table 8: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Positive control

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	4	4	4	4	4	4	4
	5	4	4	4	4	4	4
	6	4	4	4	4	4	4
Mean		4.0	4.0	4.0	4.0	4.0	4.0
ICE class			IV
Fluorescein retention	4	0.5	3	-	-	-	-
	5	0.5	3	-	-	-	-
	6	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class			IV
Corneal thickness	4	0.51		-	-	-	-
	5	0.51	-	-	-	-	-
	6	0.53	-	-	-	-	-
Corneal swelling (%)	4	(-)	(-)	(-)	(-)	(-)	(-)
	5	(-)	(-)	(-)	(-)	(-)	(-)
	6	(-)	(-)	(-)	(-)	(-)	(-)
Mean		-	-	-	-	-	-
ICE class			IV
Combination of the 3 Endpoints		3 x IV
CLASSIFICATION		Category 1 : Corrosive / Severe irritant

Note:

( - ): evaluation of corneal swelling not possible (Corneal opacity = 4 at each examination time)

Table 7: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Negative control

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	16	0.0	0.0	0.0	0.0	0.0	0.0
ICE class			I
Fluorescein retention	16	0.5	0.5	-	-	-	-
ICE class			I
Corneal thickness	16	0.48	0.48	0.48	0.48	0.48	0.48
Corneal swelling (%)	16	-	0	0	0	0	0
ICE class			I
Combination of the 3 Endpoints		3 x I
CLASSIFICATION		No Category

Note: No morphological effects were noted, whatever the examination time.

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The test item was determined to not cause severe damage or irrritation for eyes in the ICE test.

Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hidroxide (positive control) or 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. According to CLP Regulation EC no. 1272/2008 the test item does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No category), since the combination of the 3 endpoints for the test item was 2xII and 1xI.

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 the available information, the substance is not classified for skin irritation/corrosion in accordance with CLP, Regulation (EC) No. 1272/2008.

Based on the available information, the substance is not classified for serious eye damage/eye irritation according to CLP Regulation no. 1272/2008.