Ethyl 4-methyl-2-oxocyclohexanecarboxylate

Endpoint summary

Description of key information

Skin corrosion (OECD TG 431): non-corrosive
Skin irritation (OECD TG 439): not irritating

Skin irritation in rabbit (OECD TG 404): mean scores of 24, 48 and 72 hrs were 1.3, 1.3 and 1.7 for erythema, 0.3, 0.6 and 1.3 for oedema for the three animals respectively. All findings recovered by day 7. -- no classification warratned under CLP or GHS

Eye irritation in BCOP (OECD TG 437): not requireing classification

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2017 - 12 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Stability at higher temperatures: maximum temperature: 50°C, maximum duration: 1440 minutes

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 minimise the need of in vivo testing. One of the validated in vitro skin corrosion test system is the EpiDerm Skin Model, 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: EpiDerm Skin Model (EPI-200)
- Lot no.: 25789
- Surface: 0.6 cm^2
- Date of analysis: 10 May 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during 3-minute treatment: room temperature
- Temperature used during 1-hour treatment: 36.0 - 37.0°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: once with phosphate buffered saline
- Observable damage in the tissue due to washing: no

The test item was checked for possible interference with the MTT endpoint before the start of the study.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Amount of MTT used: 300 μL
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.637 ±0.005 (within acceptance criteria)
- Barrier function: 7.9 hours (within acceptance criteria)
- Contamination: no contamination was detected

NUMBER OF REPLICATE TISSUES: 2 for the test item, the negative control and the positive control for each exposure period

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one experiment with a 3-minute exposure period and one experiment with a 1-hour exposure period.

DECISION CRITERIA (see table 1 in 'any other information on materials and methods')
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL

Duration of treatment / exposure:

3-minute and 1-hour

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2 for each exposure period (4 in total)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure period

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure period

Value:

97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- 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 (optical density at 570 nm) of the negative control tissues was within the acceptance limits (i.e. 1.556 for the 3-minute exposure and 1.742 for the 1-hour exposure)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following the 1-hour exposure to the positive control was 7.9%.
- Acceptance criteria met for variability between replicate measurements: yes, In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 30% (i.e. 26%) indicating that the test system functioned properly

Table 2 Individual OD measurements

	3-minute application (OD570)        A               B		1-hour application (OD570)        A               B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.6214	1.5705	1.6642	1.7940
	1.6185	1.5719	1.8605	1.7630
	1.6397	1.5603	1.8393	1.7822
FRET 13-0545 OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.3243	1.7427	1.7406	1.7500
	1.3004	1.7586	1.7239	1.7050
	1.3071	1.7672	1.7358	1.7465
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.1984	0.1724	0.1829	0.1708
	0.1912	0.1706	0.1801	0.1772
	0.1978	0.1694	0.1826	0.1783

OD = Optical density

Duplicate exposures are indicated by A and B.

Table 3 Historical data

	Negative control		Positive control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (% viability)	1-hour treatment (% viability)
Range	1.324 – 2.615	1.361 – 2.352	0.0172 – 0.56	0.046 – 0.339	6 – 25	3 – 13
Mean	1.84	1.85	0.19	0.14	11.03	7.45
SD	0.26	0.22	0.09	0.06	4.39	2.51
n	81	83	80	77	38	38

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016.

Interpretation of results:

study cannot be used for classification

Remarks:

Classification will be based on the combination of studies.

Conclusions:

Based on the results of an in vitro skin corrosion study, performed according to OECD guideline 431 and GLP principles, FRET 13-0545 is considered not corrosive (mean tissue viability of 96% and 97% after 3 minutes and 1 hour exposure, respectively).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 June 2017 - 26 June 2017

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:

28 July 2015

Deviations:

no

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

GLP compliance:

yes

Specific details on test material used for the study:

Stability at higher temperatures: maximum temperature: 50°C, maximum duration: 1440 minutes

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 minimise 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(TM)
- Tissue batch number(s): 17-EKIN-025
- Surface: 0.38 cm^2
- Expiration date: 26 June 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during test item treatment: room temperature
- Temperature used during other incubations: 36.3 - 37.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: once with phosphate buffered saline
- Observable damage in the tissue due to washing: no

The test item was checked for possible direct MTT reduction and color interference in an earlier performed skin corrosion test. The results showed that the test item did not interfere with the MTT endpoint.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 for the test item, the positive control and the negative control.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one single run with two individual OD measurements.

DECISION CRITERIA
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
The in vitro skin irritation test is considered acceptable if it meets the following 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 ≤50% 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:

TEST MATERIAL
- Amount applied: 25 μL

NEGATIVE CONTROL
- Amount applied: 25 μL

POSITIVE CONTROL
- Amount applied: 25 μL, re-spread after 7 minutes of contact time

Duration of treatment / exposure:

15 ±0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours and 3 hours with MTT

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 3 replicates

Value:

78

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

tissue viability: 21%

Other effects / acceptance of results:

- 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 (i.e. 0.734 ± 0.043)
- Acceptance criteria met for positive control: yes, the positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 21%
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was ≤11%

For individual OD measurements, see table 1 in 'any other information on results'.

Table 1 Individual OD measurements

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	0.8361 0.8137	0.7391 0.7428	0.7753 0.7561
FRET 13-0545 OD570measurement 1 OD570measurement 2	0.5890 0.5869	0.5924 0.5859	0.6734 0.6515
Positive control OD570measurement 1 OD570measurement 2	0.1850 0.1777	0.2999 0.2842	0.1337 0.1233

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Table 2 Historical data for negative and positive control

	Negative control (absorption; OD570)	Positive control (absorption; OD570)	Positive control (viability; %)
Range	0.676 – 1.336	0.036 – 0.549	2.85 – 45.43
Mean	1.01	0.16	15.74
SD	0.16	0.10	9.22
n	155	154	163

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016.

Interpretation of results:

GHS criteria not met

Remarks:

The results of an in vitro skin corrosion study performed at the same test facility (study 517656) were taken into account to draw the conclusion as well.

Conclusions:

Based on the results of an in vitro skin irritation test, performed according to OECD guideline 439 and GLP principles, FRET 13-0545 is not classified according to GHS and Regulation (EC) No. 1272/2008 (relative tissue viability >50%).

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2017 - 05 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study is intended to provide information on the potential health hazards of the test item and data produced can be used for classification/labelling of the test item. This study should provide a rational basis for risk assessment in man.This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Appendix to Director General Notification, No. 12-Nousan-8147. Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan (JMAFF)

Version / remarks:

November 2000, including the most recent revisions

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

pH (1% in water, indicative range): 5.11 – 5.25

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: approx. 22-23 weeks old
- Weight at study initiation: 3653 to 3906 g
- Housing: individually in labeled cages with perforated floors.
- Diet: pelleted diet for rabbits (Global Diet 2030 Teklad®, Mucedola, Milanese, Italy), once daily and hay.
- Water: municipal tap-water, ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 71-83 (Deviations from the maximum level of daily mean target humidity occurred. This study plan deviation did not affect the integrity of the study because laboratory historical data do not indicate an effect of these deviations.)
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 September 2017 To: 05 October 2017

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Remarks:

re-clipped at least 3 hours before final observations

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

7 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 150 cm^2 (10x15 cm)
- % coverage: 100%
- Type of wrap if used: the test item was applied to a metalline patch which was mounted on Micropore tape. The tape was wrapped around the abdomen with Coban elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: using tap water
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
- Mortality/moribundity: twice daily for 7 days
- Clinical observations (postdose): once daily for 7 days
- Body weight: on day 1 and on the day of the final observation
- Irritation: 1, 24, 48 and 72 hours and 7 days after the removal of the dressings and test item.
- Histopathology: none

SCORING SYSTEM:
- Method of calculation: see table 1 in 'any other information on materials and methods'.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.6

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

All mean scores were below 1.5, except for mean erythema score in one animal, which was 1.7.

Other effects:

Scaliness was noted for all animals 72 hours after exposure.

Table 2 Individual skin irritation scores

Animal		292^*				297				300
Time after exposure		Erythema (0-4)	Oedema (0-4)	comments		Erythema (0-4)	Oedema (0-4)	comments		Erythema (0-4)	Oedema (0-4)	comments
1 hour		1	1	-		1	1	-		1	2	-
24 hours		2	1	-		1	1	-		2	2	-
48 hours		1	0	-		2	1	-		2	1	-
72 hours		1	0	s		1	0	s		1	1	s
7 days		0	0	-		0	0	-		0	0	-

* Sentinel; s: Scaliness

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) No. 1272/2008

Conclusions:

Based on the results of an in vivo skin irritation test, performed according to OECD guideline 404 and GLP principles, FRET 13-0545 is not classified according to GHS and 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:

06 April 2017

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:

26 July 2013

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Stability at higher temperatures: maximum temperature: 50°C, maximum duration: 1440 minutes

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 an 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
- Amount applied: 750 µL

NEGATIVE CONTROL: Physiological saline
- Amount applied: 750 µL

POSITIVE CONTROL: Ethanol
- 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 for the negative control, the positive control and the 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, the positive control or the test item was introduced onto the epithelium of the 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.

- 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 a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

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.

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

Irritation parameter:

in vitro irritation score

Remarks:

Mean of 3 replicates

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- The corneas treated with the test item showed opacity values between -1.4 and 0.6, permeability values were ranging from 0.001 to 0.0.26 and IVIS were -1.0, 0.4 and 0.7 (n=3).
- Since the mean IVIS was < 3 after 10 minutes of treatment, the test item is not classified according to GHS and Regulation (EC) No. 1272/2008.

OTHER EFFECTS:
- No 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 the historical range (IVIS ranging from -0.4 to 0.5)
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 38-59). Corneas were turbid after 10 minutes of treatment.

Table 2 Historical data for the BCOP studies

	Negative control			Positive control
	Opacity	Permeability	In vitroIrritancy Score	In vitro Irritancy Score
Range	-2.9 – 3.0	-0.016 – 0.042	-2.8 – 3.0	34.7 – 78.2
Mean	0.12	0.01	0.32	56.68
SD	1.07	0.01	1.17	12.64
n	72	65	66	47

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of February 2014 to February 2017.

Table 3 Individual permeability scores (corrected)

Treatment	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	6	0.352	0.354	0.350	0.352	2.112	1.992
	1	0.989	0.981	0.984	0.985	0.985
	6	0.484	0.475	0.480	0.480	2.878
Test item	1	0.025	0.026	0.026	0.026	0.026	0.011
	1	-0.001	0.005	0.000	0.001	0.001
	1	-0.002	0.008	0.008	0.005	0.005

¹    OD₄₉₀values corrected for the mean final negative control permeability (0.005).

²    Calculations are made without rounding off.

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) No. 1272/2008

Conclusions:

In the Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, FRET 13-0545 induced a mean IVIS of 0.0 after 10 minutes of treatment. Based on these results, the substance is not classified according to GHS and Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation/corrosive

Based on the results of an in vitro skin corrosion study using a human three dimensional epidermal model (EpiDerm (EPI-200)), performed according to OECD guideline 431 and GLP principles, FRET 13-0545 is considered not corrosive (mean tissue viability of 96% and 97% after 3 minutes and 1 hour exposure, respectively).

Based on the results of an in vitro skin irritation test using a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)), performed according to OECD guideline 439 and GLP principles, FRET 13-0545 is not classified according to GHS and Regulation (EC) No. 1272/2008 (relative tissue viability 78% after 15 minutes treatment).  

Based on the results of an in vivo skin irritation test conducted in rabbit with 4 hr semi-occlusive dressing, performed according to OECD guideline 404 and GLP principles, FRET 13-0545 is not classified according to GHS and Regulation (EC) No. 1272/2008 ( very slight to well-defined erythema and very slight to slight oedema in the treated skin areas of the rabbits, which had resolved within 7 days. Mean (24 – 72 hours) scores for erythema and oedema were below 1.5, except for mean erythema score in one animal, which was 1.7.).

Eye irritation

In the Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, FRET 13-0545 induced a mean IVIS of 0.0 after 10 minutes of treatment. Based on these results, the substance is not classified according to GHS and Regulation (EC) No. 1272/2008.

Justification for selection of skin adn eye irritation / corrosion endpoint:
The results of the studies are reliable and adequate for covering the endpoint.

Effects on skin irritation/corrosion: not corrovie or irriating, no need classification under CLP.

 

Effects on eye irritation/corrosion:no need classification under CLP.

Justification for classification or non-classification

Based on the results in the skin and eye irritation/corrosive tests the substance does not need classification according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.