1-[4-(4-chlorophenoxy)-2-(trifluoromethyl)phenyl]ethanone

Endpoint summary

Description of key information

Skin

In an in vitro skin corrosion study (OECD 431) a mean tissue viability of 118.3 % after 3 min exposure and 104.0 % after 1 h exposure was determined.

In an in vitro skin irritation study (OECD 439) a mean tissue viability of 76.9 % was determined.

Eye

In an in vitro eye irritation study (OECD 437) an In Vitro Irritancy score (IVIS) of 0.0 was determined.

In an in vitro Reconstructed Human Cornea like Epithelium (RhCE) eye irritation study a tissue viability of 83.0 % was observed.

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:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: L85-76

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™

PREDICTION MODEL / DECISION CRITERIA:
lrritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant', if the mean relative tissue viability with a test material is less than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

76.9

Vehicle controls validity:

not examined

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: Yes

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

Table 2: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test substance			Tissue 1	Tissue 2	Tissue 3	mean	SD	CV [%]
NC	viable tissues	mean OD570	2.809	2.714	2.843	2.789	0.067
		viability [% of NC]	100.7	97.3	101.9	100.0	2.4	2.4
	KC tissues	mean OD570	0.066	0.064	0.064	0.065	0.001
		viability [% of NC]	2.4	2.3	2.3	2.3	0.0	1.6
Test substance	viable tissues	mean OD570	1.787	2.257	2.426	2.157	0.331
		viability [% of NC]	64.1	80.9	87.0	77.3	11.9	15.4
	KC tissues	mean OD570	0.013	0.020	0.004	0.012	0.008
		viability [% of NC]	0.5	0.7	0.1	0.4	0.3	66.8
	Mean viability of tissues after KC correction [% of NC]					76.9
PC	viable tissues	mean OD570	0.101	0.089	0.115	0.101	0.013
		viability [% of NC]	3.6	3.2	4.1	3.6	0.5	12.8

NC = negative Control

PC = Positive Control

OD570 = Optical Density [wavelength 570 nm]

SD = Standard Deviation

CV = Coefficient of Variation

KC = Killed Control for MTT-reduction control

 

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (mean viability 0.4% of NC). Thus for the test substance the final mean viability is given after KC correction.

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: L85-76

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™

PREDICTION MODEL / DECISION CRITERIA: Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with de-ionized water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ~ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

118.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

For the test substance the final mean viability is given after KC correction.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

104

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

For the test substance the final mean viability is given after KC correction.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes

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

Table 2: 3 min exposure

	Test substance		Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	viable tissues	mean OD570	2.039	2.035	2.037	0.003
		viability [% of NC]	100.1	99.9	100.0	0.1	0.1
	KC tissues	mean OD570	0.102	0.121	0.112	0.013
		viability [% of NC]	5.0	5.9	5.5	0.6	11.7
Test substance	viable tissues	mean OD570	2.230	2.619	2.425	0.275
		viability [% of NC]	109.4	128.6	119.0	13.5	11.4
	KC* tissues	mean OD570 KC NC corrected	0.000	0.030	0.015	0.021
		viability [% of NC]	0.0	1.5	0.7	1.0	141.4
	Final mean viability after KC correction [% of NC]				118.3
PC	viable tissues	mean OD570	0.287	0.441	0.364	0.109
		viability [% of NC]	14.1	21.7	17.9	5.3	29.9

Table 3: 1 hour exposure

	Test substance		Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	viable tissues	mean OD570	2.038	2.166	2.102	0.090
		viability [% of NC]	97.0	103.0	100.0	4.3	4.3
	KC tissues	mean OD570	0.111	0.108	0.110	0.002
		viability [% of NC]	5.3	5.1	5.2	0.1	1.6
Test substance	viable tissues	mean OD570	2.146	2.293	2.219	0.104
		viability [% of NC]	102.1	109.1	105.6	4.9	4.7
	KC tissues	mean OD570 KC NC corrected	0.027	0.039	0.033	0.008
		Viability [% of NC]	1.3	1.8	1.6	0.4	25.9
	Final mean viability after KC correction [% of NC]				104.0
PC	viable tissues	mean OD570	0.121	0.143	0.132	0.016
		viability [% of NC]	5.8	6.8	6.3	0.7	11.8

* Negative values are set to zero for further calculation

NC = Negative Control

PC = Positive Control

OD570 = Optical Density [wavelength 570 nm]

SD = Standard Deviation

CV = Coefficient of Variation

KC = Killed Control for MTT-reduction control

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. The results of the tissues indicate an increased MTT reduction (mean viability 0.7% of NC). Thus for the test substance the final mean viability is given after KC correction.

Interpretation of results:

GHS criteria not met

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:

weight of evidence

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:

July 2013

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: L85-76

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

No data.

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 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

NUMBER OF REPLICATES: 3 corneas were used per treatment group.

NEGATIVE CONTROL USED: De-ionized water

POSITIVE CONTROL USED: 100% ethanol (PC1) and 100% dimethylformamide (DMF) (PC2)

POST-INCUBATION PERIOD: 2 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Cornea opacity is measured quantitatively as the amount of light transmission through the cornea.
- Corneal permeability: Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Evaluation of results were based on the decision criteria used in OECD guideline 437.

Irritation parameter:

in vitro irritation score

Value:

0

Vehicle controls validity:

not examined

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: Not observed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values:Mean ± SD (Mean-2SD - Mean+2SD)

Historic range of NC
Opacity = 1.8 ± (-1.6 - 5.1)
Permeability = 0.001 ± 0.003 (-0.006 - 0.008)

Historic range PC 1 (100% ethanol)
Opacity = 30.2 ± 5.5 (19.2 - 41.1)
Permeability = 1.761 ± 0.364 (1.032 - 2.490)
IVIS = 56.6 ± 10.1 (36.3 - 76.9)

Historic range PC 1 (100% DMF)
Opacity = 103.6 ± 9.3 (85.1 - 122.2)
Permeability = 1.179 ± 0.627 (-0.074 - 2.432)
IVIS = 121.3 ± 15.2 (90.9 - 151.7)

Table 2: Opacity score of test substance, NC and PC

Test substance	Cornea-No.	Initial opacity	Final opacity	Opacity Change	Corrected Opacity Change*	Mean	SD
Test substance	25	3.2	1.9	-1.3	0.0	0.0	0.0
	26	2.1	3.0	0.9	0.0
	27	1.8	1.9	0.1	0.0
NC	1	3.7	5.4	1.7	NA	2.0	0.7
	2	2.9	5.8	2.8	NA
	3	1.5	3.1	1.6	NA
PC 1 (ethanol)	4	4.2	37.8	33.6	31.6	35.1	3.8
	5	3.3	40.0	36.7	34.7
	6	3.7	44.8	41.2	39.1
PC 2 (DMF)	7	3.4	122.5	119.2	117.1	105.2	10.5
	8	0.7	99.9	99.2	97.2
	9	2.3	105.6	103.3	101.2

Table 3: Permeability score of the test substance, NC and PC

Test substance	Cornea No.	Mean OD490	Dilution Factor	Mean Corrected OD490*	Mean	SD
Test substance	25	0.003	1	0.000	0.001	0.001
	26	0.003	1	0.000
	27	0.007	1	0.003
NC	1	0.001	1	NA	0.005	0.004
	2	0.005	1	NA
	3	0.009	1	NA
PC 1 (ethanol)	4	0.280	5	1.394	1.509	0.415
	5	0.395	5	1.969
	6	0.234	5	1.164
PC 2 (DMF)	7	0.383	5	1.910	1.321	0.809
	8	0.081	5	0.399
	9	0.332	5	1.654

Table 4: In Vitro Irritation score (IVIS) of the test substance, NC and PC

	Cornea No.	Opacity per cornea	Permeability per cornea	IVIS
				Per cornea	mean	SD
Test substance	25	0.0	0.000	0.0	0.0	0.0
	26	0.0	0.000	0.0
	27	0.0	0.003	0.0
NC	1	1.7	0.001	1.7	2.1	0.7
	2	2.8	0.005	2.9
	3	1.6	0.009	1.7
PC 1 (ethanol)	4	31.6	1.394	52.5	57.8	6.0
	5	34.7	1.969	64.2
	6	39.1	1.164	56.6
PC 2 (DMF)	7	117.1	1.910	145.8	125.0	21.3
	8	97.2	0.399	103.2
	9	101.2	1.654	126.0

NA = not applicable; SD = standard deviation, OD490 = optical density at 490 nano meter

*Negative values are set to zero for further calculation

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same 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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: L85-76

Species:

human

Strain:

other: three-dimensional non-keratinized tissue construct

Details on test animals or tissues and environmental conditions:

No data.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: MTT reduction control (KC)

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

2

Details on study design:

METHOD: The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three dimensional human cornea model EpiOcular™. After application of the test material to the surface of the EpiOcular™ tissue the induced cytotoxicity (= lass of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-(4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanolextraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of test-substance treated tissues is compared to values from negative control tissues and expressed as relative tissue viability. Two EpiOcular™ tissue samples were incubated with 50 μL of the undiluted test substance for 30 minutes followed by a 2-hours post-incubation period.

Irritation parameter:

other: tissue viability

Value:

83

Vehicle controls validity:

not examined

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

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

Table 2: Individual and mean OD570 values, ndividual and mean viability values and inter-tissue variability

Test substance		Tissue 1	Tissue 2	Mean KC	mean	Inter-tissue variability [%]
NC	Mean OD570	2.437	2.427	0.040	2.432
	Viability [% of NC]	100.2	99.8	-	100	0.4
Test substance	Mean OD570	1.911	2.118	0.056	2.014
	Viability [% of NC]	78.6	87.1	-	83	8.5
PC	Mean OD570	0.456	0.536	-	0.496
	Viability [% of NC]	18.7	22.0	-	20	3.3

Due to the ability of the test substance to reduce MTT directly, a KC (killed control) was applied in parallel. However, the result of the KC did not indicate an increased MTT reduction (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability calculation.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two in vitro test batteries were performed in a weight of evidence approach in order to evaluate if the test substance is irritating or corrosive to either skin or eye.

 

Skin

The objective was to assess the potential for corrosive activity and skin irritation of the test substance. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) according to OECD guideline 431and Skin Irritation Test (SIT) according to OECD guideline 439.

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 50 µL (corrosion test) or 30 µL (irritation test) of the undiluted test substance to the surface of a human reconstructed epidermis model (EpiDerm™).

For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.

Cell viability is measured by dehydrogenase conversion of the yellow, water-soluble MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), present in cell mitochondria, into a blue formazan salt that is measured quantitatively after isopropanol - extraction from the tissues. The optical density of the extracts of test substance treated tissues is compared to negative control values from tissues treated with de-ionized water or PBS and is expressed as relative tissue viability.

The EpiDerm™ skin corrosion/irritation test showed the following results:

 

The test substance is able to reduce MTT directly.

 

Corrosion test:

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 118.3% after 3 min exposure and 104.0% after an exposure period of 1 hour.

 

Irritation test:

The mean viability of the test substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 76.9 %.

 

Eye

The objective of the present study was the determination of a possible eye irritating potential of test substance. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) according to OECD guideline 437 and EpiOcular Eye Irritation Test according to OECD guideline 492.

 

BCOP

The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750μL of the undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hours post-incubation period.

In addition to the test substance a negative control (NC: de-ionized water) and two positive controls (PC1: 100% ethanol; PC2: 100% DMF) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.The following results were obtained in the BCOP Test:

 

 

	Mean Opacity Score	Mean Permeability Score	Mean In Vitro Irritation Score
Test substance	0.0	0.001	0.0
NC	2.0	0.005	2.1
PC1 (ethanol)	35.1	1.509	57.8
PC2 (DMF)	105.2	1.321	125.0

 

EpiOcular

The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™).
Two EpiOcular™ tissue samples were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period. After application of the test material to the surface of the EpiOcular™ tissue the induced cytotoxicity (= lass of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of test-substance treated tissues is compared to values from negative control tissues and expressed as relative tissue viability.
The EpiOcular™ eye irritation test showed the following results:

The test substance is able to reduce MTT dirctly. The mean viability of the test substance treated tissues was 83.0%.

The Summary of the individual test results of the in vitro eye irritation turnkey testing strategy:

 

Test method	Test result	Test Evaluation	Evaluation Test Strategy
BCOP test	The mean IVIS of the test substance treated corneas was 0.0	not identified as corrosive or serve irritant	Non-irritant
EpiOcular	The mean viability of the test-substance treated tissues was 83%	Non-irritant

 

Based on the results for BCOP and EpiOcular Test and considering the evaluation criteria, the test substance does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

 

Justification for classification or non-classification

Based on the study results of the in vitro test battery and taking into account the provisions laid down in Regulation (EC) No 1272/2008 as amended for the ninth time in Regulation (EU) No 2016/1179, the test substance does not need to be classified as irritating or corrosive to eye or skin.