4-(Difluoro-(3,4,5-trifluoro-phenoxy)-methyl)-3,5-difluoro-benzaldehyde

Endpoint summary

Description of key information

Skin irritation in vitro

OECD 439: UN GHS Cat 2 or Cat 1

OECD 431: not corrosive

 

According to the result of the OECD Guideline study 439, the test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). To decide whether the test item is irritating or corrosive to skin, a further in vitro study according to OECD 431 was performed. Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin. Based on this weight of evidence approach, the test item is considered to be irritating to skin cat. 2.

 

Eye irritation in vitro

OECD 492: GHS 1 or GHS 2

OECD 437: no prediction can be made

Based on the result of the OECD Guideline study 492, the test item is considered to be UN GHS category 1 (cause serious eye damage) or category 2 (eye irritation). Since this study cannot resolve between UN GHS 2 or serious damage UN GHS 1, a further study was performed.

A study according to OECD 437 was performed to identify whether the test item induced serious eye damage (UN GHS 1) or not requiring classification for eye irritation or serious eye damage (No UN GHS category). The test item is not predicted to cause serious eye damage (UN GHS 1).

Based on the results of the weight of evidence approach, it can be concluded that the test item is irritating to eye (cat. 2).

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

Study period:

September 26, 2017 -November 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006. B.40.bis. In vitro skin corrosion: human skin model test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol SkinEthic Skin Corrosivity Test, April 2012.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

standard model

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used in this study; The test item was applied neat to the tissues.

Details on test system:

CELL CULTURE
- Supplier: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 17-RHE-108
- Expires: October 30, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF THE TEST MATERIAL AND CONTROL
At the end of the exposure periods, the test item, positive and negative control was removed immediately by gently rinsing with a minimum volume of 20 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg ± 3 mg of solid test material
- Concentration (if solution): n/a

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL ± 3 µl (deionised water )
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL ± 3 µl
- Concentration (if solution): An 8 N potassium hydroxide solution dissolved deionised water pure was used as positive control.

Duration of treatment / exposure:

3 min & 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1 (3 min)

Value:

98.6

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non-corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1 (1 hour)

Value:

99.6

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Non-corrosive

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The negative control OD values were 1.719, 1.745, 1.743 and 1.661 and, thus, in the range of ≥ 0.8 and ≤ 3.0.

Acceptability of the Positive and Negative Control Data:
After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.5 % and, thus, lower than the historically established threshold of 0.97 %.
After treatment with the negative control (deionised water) the mean ODs were 1.731 (3 minute exposure) and 1.703 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.590 and 1.468, respectively.

Negative Control, Positive Control and Test Substance Data Acceptance Criteria:
The range between identically treated tissues with the negative control was less than 30 % after 3 minutes exposure (1.4 %) and after 1 hour exposure (5.1 %).
The range between identically treated tissues with the positive control was higher than 30 % after 1 hour exposure (50.0 %), but the optical densities measured were <0.3.
The range between identically treated tissues with the test item was less than 30 % after 3 minutes exposure (0.3 %) and after 1 hour exposure (7.2 %).


The study met all acceptance criteria.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	3	1.731	100.0
Negative Control	60	1.703	100.0
Positive Control	60	0.009	0.5
Test Material	3	1.706	98.6
Test Material	60	1.696	99.6

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 431. Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 431. Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 17, 2017 - September 4, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending, for the purpose of its adaption to technical progress, Regulations (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

standard model

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used in this study; The test item was applied neat to the tissues.

Details on test system:

CELL CULTURE
- Supplier: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 17-RHE-085
- Expires: August 14, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Dulbecco`s Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5 % aqueous solution of sodium dodecyl sulfate in deionised water)

Duration of treatment / exposure:

42 min (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

6.8

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 1.754 to 1.943

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 1.827
Mean viability positive control < 40 % 1.3 %
SD of group-mean value ≤ 18 % 7.7 % (positive control)
5.5 % (negative control)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.455 1.827
Mean viability positive control ≤ 2.97 % 1.3 %

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18 % 2.9 %

The study met all acceptance criteria.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.827	100
Positive Control	42	0.024	1.3
Test Material	42	0.125	6.8

Interpretation of results:

other: UN GHS: Category 2 or Category 1

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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

Study period:

August 1, 2017 - October 17, 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:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaption to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL: 750 µL (i.e. 150 mg/750 µL)

NEGATIVE / VEHICLE CONTROL: 750 µL

Designation: 0.9 % sodium chloride solution
Supplier: B. Braun Melsungen AG, Germany
Batch: 16455011
Storage: 2 to 8 °C
Released until: October 2019


POSITIVE CONTROL: 750 µL

Synonym: Imidazole

Imidazole was dissolved with 0.9 % sodium chloride solution to a concentration of 20 % (w/v).

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

in vitro: triplicate design

Irritation parameter:

in vitro irritation score

Run / experiment:

Run 1 / Experiment 1

Value:

13.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.1 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.2).
- Acceptance criteria met for positive control: After treatment with the positive control (20% Imidazole) the calculated IVIS was 112.1 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 80.9 – 133.0).

Therefore, the study fulfilled the acceptance criteria.

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	SD
Negative control	0.9 % NaCl Solution	0.3	-0.005	0.225	0.1	0.6
		-0.4	-0.006	-0.490
		0.7	-0.005	0.625
Positive control	20 % Imidazole solution	77 .0	2.077	108.155	112.1	9.4
		65.0	2.689	105.335
		73.4	3.295	122.825
Test item		12.9	0.015	13.125	13.3	0.7
		12.6	0.009	12.735
		14.6	0.005	14.175

 

Interpretation of results:

other:

Remarks:

The IVIS obtained after treatment with the test item was 13.3 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

Conclusions:

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 437. The IVIS obtained after treatment with the test item was 13.3 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 27, 2017 - February 6, 2018

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)

Version / remarks:

October 9, 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT,

Version / remarks:

September 14, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation`s Reconstructed Human EpiOcular Model; MatTek Corporation

Version / remarks:

June 29, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL: 50 mg per tissue

NEGATIVE / VEHICLE CONTROL: 50 µL per tissue

Sterile deionized water was used as negative control.


POSITIVE CONTROL: 50 µL per tissue

Designation: Methyl acetate
Supplier: MatTek In Vitro Life Science Laboratories
Lot-No.: 032817ISA
Catalog #: TC-MA
Purity (GC): 99.7 %
Appearance: Colorless liquid
Expiration date: March 28, 2018
Storage: 15 to 30 °C

Duration of treatment / exposure:

6 hours

Number of animals or in vitro replicates:

in vitro: duplicate design

Irritation parameter:

other: Viability %

Run / experiment:

Run 1 / Experiment 1

Value:

7.5

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 observations

ACCEPTANCE OF RESULTS:
1. The negative control OD is > 0.8 and < 2.5 (1.403 and 1.615).
2. TThe mean relative viability of the positive control is below 50 % of the negative control viability (47.0 %).
3. The difference of viability between the two relating tissues of a single chemical is < 20 % (values between 0.3 % to 14.0 %) in the same run (for positive and negative control tissues and tissues of single chemicals).

The study met all acceptance criteria

 

	Mean OD	Mean Viability
Negative Control	1.509	100.0 %
Positive Control	0.709	47.0 %
Test Item	0.112	7.5 %

Interpretation of results:

other: UN GHS Category 1 or Category 2

Conclusions:

Under the conditions of the present study, the test item did show an eye hazard potential. The test item is labeled irritant (UN GHS Category 1 or Category 2).

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 492. Under the conditions of the present study, the test item did show an eye hazard potential. The test item is labeled irritant (UN GHS Category 1 or Category 2).

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 provided information there is evidence for classification for skin and eye irritation Cat 2 according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776.