2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropanoyl)phenoxy]phenyl]-2-methylpropan-1-one

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-14 - 2019-01-18 (experimental phase)

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:

OECD Guideline for the Testing of Chemicals, Version 439, adopted 28. July 2015, “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)

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending Regulation (EC) No. 761/2009, Annex III, EU method B.46 “IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST”, adopted 06. Jul. 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage: Fridge (2 – 8 °C); keep away from light; keep away from humidity
- Stability: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: multiple donors, not specified

Source strain:

other: not applicable

Details on animal used as source of test system:

Source: The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

Test System

Specification
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery: 15. Jan. 2018
Batch no.: 28679

Justification for test system used:

as stipulated under REACH and the respective Guidelines

Vehicle:

unchanged (no vehicle)

Remarks:

The tissues were wetted with 25 µL DPBS buffer before applying the test item.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava, EPI-200-SIT
- Tissue batch number(s): 28679
- Delivery date: 2018-01-15

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
- Observable damage in the tissue due to washing: none stated
- Modifications to validated SOP: none stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: plate spectrophotometer: 96-well-plate photometer, Anthos Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 replicates for each test item, positive and negative control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment, 3 replicates

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the viability after 1h exposure is greater than or equal to 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
One plate was used for treatment with the test item:
The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size.
The following amounts were applied to the tissues:
Tissue Amount
1 26.6 mg
2 23.9 mg
3 25.6 mg

NEGATIVE CONTROL
One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.

POSITIVE CONTROL
One plate was used as positive control; each tissue was treated with 30 µL 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

23 hours and 30 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

74.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 1

Value:

84.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 2

Value:

78.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 3

Value:

61.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction:no
- Colour interference with MTT: no

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

Interpretation of results:

other: EU-GHS criteria not met

Conclusions:

- mean value of relative tissue viability was reduced to 74.6 %
- test item is considered non-irritant to skin [since obtained value is above the threshold for skin irritation potential (50%)]

Executive summary:

A study was performed to assess the potential of the test substance to cause skin irritation in a reconstructed human epidermis (RhE) method according to OECD Guideline 439, in compliance with GLP. Three EpiDermTM human skin tissues were exposed for 60 minutes. The test substance was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control and 5% SDS solution as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability range of 0.8 ≤ mean OD ≤ 2.8. The positive control showed clear irritating effects. Mean relative tissue viability was reduced to 2.6% (required: ≤20%). The variation within the tissue replicates of negative control, positive control and test substance was acceptable (required: ≤ 18%). After treatment with the test substance, mean relative tissue viability was reduced to 74.6%. This is above the threshold for skin irritation (50%). Substances that induce values above the threshold are considered non-irritant. Therefore, under the study conditions, the test substance was concluded to be non-irritating to skin (Andres, 2019).

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Annex V, B4

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl:KBL (NZW)

Type of coverage:

semiocclusive

Vehicle:

other: Cottonseed oil

Duration of treatment / exposure:

4 h

Number of animals:

3

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

24/48/72 h

Score:

0

Other effects:

The dose of test subsatnce was 0,5 g per animal

Irritation parameter	Max. score	Remarks on result
erythema score	0	Max. duration: d; Max. value at end of observation period: 0 (related to all animals)
edema score	0	Max. duration: d; Max. value at end of observation period: 0 (related to all animals)

Interpretation of results:

other: not classified

Conclusions:

Under the test conditions, the test substance was concluded to be non-irritant.

Executive summary:

A study was performed to assess the potential of the test substance to cause skin irritation in rabbit according to Directive/ECC (67/548/EEC), Annex V, B4 “Acute Toxicity: Dermal Irritation / Corrosion”, comparable to OECD Guideline 404, in compliance with GLP. Three rabbits were treated with 0.5 g test substance via a semi-occlusive patch for 4 h. Observations were made at 24, 48 and 72 h post-exposure. No erythema or oedema were seen at any time point, with an average irritation score of 0. Under the study conditions, the test substance was concluded to be non-irritating to skin (Bioservice, 2005).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-10 - 2018-12-13 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

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:

OECD Guideline for the Testing of Chemicals, Part 492, adopted 25. Jun. 2018, “Re-constructed Human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye Irritation or serious eye damage”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was stored in the test facility in a closed vessel in the refrigerator (2 – 8 °C); protected from light and humidity.

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcularTM Eye Irritation Test (EIT) predicts the acute eye hazard potential of chemi-cals by measurement of tissue damage caused by cytotoxic effects in the reconstructed human cornea-like tissue model. Within a testing strategy, the EpiOcularTM EIT can be used as a replacement of the in vivo Draize Eye Irritation Test.
It is utilized for the classification and labelling of chemicals concerning their eye hazard potential. The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B). For these purposes, further testing with other suitable test methods is required.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
Specification:
Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.
Origin:
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 11. Dec. 2018
Batch no.: 27085
The cells used to produce EpiOcular tissues are screened for potential biological contaminants. None of the following potential contaminants were detected:
HIV-1 virus (Oligonucleotide-directed amplification)
Hepatitis B virus (Oligonucleotide-directed amplification)
Hepatitis C virus (Oligonucleotide-directed amplification)
Bacteria, yeast, other fungi (long-term antibiotic, antimycotic free culture)

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): 52.6 and 50.8 mg / tissue
- Concentration (if solution): undiluted

VEHICLE
none

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

n/a

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number
Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 11. Dec. 2018
Batch no.: 27085
- Doses of test chemical and control substances used
Test chemical: 52.6 and 50.8 mg/tissue
Controls: 50 µL of the controls
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 29 minutes. After that, 50 µL of the controls and a defined amount of the test item (see table 7.2-a) were applied in duplicate in one- minute- intervals.
At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.
After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 17 hours and 45 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
After the post-treatment incubation, the MTT assay was performed.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
Spectrophotometer, 570 nm
- Description of the method used to quantify MTT formazan
MTT Assay and Extraction
A 24-well-plate was prepared with 300 µL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
Measurement
The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
OD value; > 60% of control
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
Parameter Optical Density Negative Control Relative Tissue Viability Positive Control
Demineralised H2O Methyl acetate
Exposure time 6 hours
Mean 1.631 34.8%
Standard deviation 0.271 7.3%
Range 1.047 - 2.340 21.1 - 53.9%
Current study 1.725 42.7%

- Complete supporting information for the specific RhCE tissue construct used
- Reference to historical data of the RhCE tissue construct
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
The validity of the EpiOcularTM test at the laboratory was demonstrated in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcularTM test was demonstrated.

- Positive and negative control means and acceptance ranges based on historical data
Yes
- Acceptable variability between tissue replicates for positive and negative controls
< 20%
- Acceptable variability between tissue replicates for the test chemical
< 20%

Irritation parameter:

other: % tissue viability

Run / experiment:

mean test item

Value:

77.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: % tissue viability

Run / experiment:

mean positive control

Value:

42.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none stated

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not different

Interpretation of results:

other: EU-GHS criteria not met

Conclusions:

Under the test conditions, the test substance was therefore concluded to be non-irritating to eyes.

Executive summary:

A study was performed to assess the potential of the test substance to cause eye irritation in a re-constructed human cornea-like epithelium (RhCE) method according to OECD Guideline 492, in compliance with GLP. The test substance was applied to a three-dimensional human cornea tissue model, in duplicate, for an exposure time of 6 hours. After treatment, the substance was rinsed from the tissue, then cell viability was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability being 42.7% (< 50%). The variation within tissue replicates of the controls and the test substance was acceptable (< 20%). After treatment with the test substance, mean relative tissue viability was 77.4%. This value is well above the threshold for eye irritation potential (≤ 60%). Under the test conditions, the test substance was therefore concluded to be non-irritating to eyes (Geitlinger, 2019).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

Annex V, B5

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl:KBL (NZW)

Amount / concentration applied:

100 mg

Number of animals or in vitro replicates:

3

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

24/48/72 h

Score:

1

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

24/48/72 h

Score:

0.33

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

(mean score 1)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

(mean score 2)

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

(mean score 3)

Time point:

24/48/72 h

Score:

0

Irritant / corrosive response data:

Reversibility of any observed effect: Changes fully reversible within 3 days

Other effects:

No Corrosion or irrevesible effects.

Irritation parameters	Max score	Remarks on result
conjunctivae score (redness)	1	Max. duration: h; Max. value at end of observation period: 0 (related to all animals)
chemosis score	0	Max. duration: h; Max. value at end of observation period: 0 (related to all animals)
cornea opacity score	0	Max. duration: h; Max. value at end of observation period: 0 (related to all animals)
iris score	0	Max. duration: 72 h; Max. value at end of observation period: 0 (related to all animals)

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

Under the test conditions, the test substance was concluded to be non-irritating to eyes (Bioservice, 2005).

Executive summary:

A study was performed to assess the potential of the test substance to cause irritation in rabbit eyes according to Directive/ECC (67/548/EEC), Annex V, B5 “Acute Toxicity: Eye Irritation / Corrosion”, comparable to OECD Guideline 405, in compliance with GLP. 100 mg of test substance was placed in the right or left eye of three rabbits. Irritation parameters were then evaluated at 24, 48 and 72 h. Treated eyes did not produce corneal opacity, chemosis, iritis or conjunctival irritation during the 24-72 h test period, except in one animal for which conjunctival irritation occurred but was fully reversible within 3 days. Based on the scoring system used, the test substance was considered to be non-irritating to eyes (Bioservice, 2005).  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin    

 

In vitro    

 

A study was performed to assess the potential of the test substance to cause skin irritation in a reconstructed human epidermis (RhE) method according to OECD Guideline 439, in compliance with GLP. Three EpiDermTM human skin tissues were exposed for 60 minutes. The test substance was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control and 5% SDS solution as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability range of 0.8 ≤ mean OD ≤ 2.8. The positive control showed clear irritating effects. Mean relative tissue viability was reduced to 2.6% (required: ≤20%). The variation within the tissue replicates of negative control, positive control and test substance was acceptable (required: ≤ 18%). After treatment with the test substance, mean relative tissue viability was reduced to 74.6%. This is above the threshold for skin irritation (50%). Substances that induce values above the threshold are considered non-irritant. Therefore, under the study conditions, the test substance was concluded to be non-irritating to skin (Andres, 2019).   

 

In vivo   

  

A study was performed to assess the potential of the test substance to cause skin irritation in rabbit according to Directive/ECC (67/548/EEC), Annex V, B4 “Acute Toxicity: Dermal Irritation / Corrosion”, comparable to OECD Guideline 404, in compliance with GLP. Three rabbits were treated with 0.5 g test substance via a semi-occlusive patch for 4 h. Observations were made at 24, 48 and 72 h post-exposure. No erythema or oedema were seen at any time point, with an average irritation score of 0. Under the study conditions, the test substance was concluded to be non-irritating to skin (Bioservice, 2005).    

 

QSAR    

 

The skin irritation potential of the test substance was predicted using the model ‘Skin irritation exclusion rules by BfR v3.0’ in the OECD QSAR Toolbox v4.2. The prediction consists in exclusion of substances for skin irritation classification, without predicting the exact Draize scores on which a classification is based. The test substance was predicted to be not irritating or corrosive to skin (Gerner, 2020).   

 

Eye   

 

In vitro   

 

A study was performed to assess the potential of the test substance to cause eye irritation in a re-constructed human cornea-like epithelium (RhCE) method according to OECD Guideline 492, in compliance with GLP. The test substance was applied to a three-dimensional human cornea tissue model, in duplicate, for an exposure time of 6 hours. After treatment, the substance was rinsed from the tissue, then cell viability was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability being 42.7% (< 50%). The variation within tissue replicates of the controls and the test substance was acceptable (< 20%). After treatment with the test substance, mean relative tissue viability was 77.4%. This value is well above the threshold for eye irritation potential (≤ 60%). Under the test conditions, the test substance was therefore concluded to be non-irritating to eyes (Geitlinger, 2019).   

    

In vivo 

 

A study was performed to assess the potential of the test substance to cause irritation in rabbit eyes according to Directive/ECC (67/548/EEC), Annex V, B5 “Acute Toxicity: Eye Irritation / Corrosion”, comparable to OECD Guideline 405, in compliance with GLP. 100 mg of test substance was placed in the right or left eye of three rabbits. Irritation parameters were then evaluated at 24, 48 and 72 h. Treated eyes did not produce corneal opacity, chemosis, iritis or conjunctival irritation during the 24-72 h test period, except in one animal for which conjunctival irritation occurred but was fully reversible within 3 days. Based on the scoring system used, the test substance was considered to be non-irritating to eyes (Bioservice, 2005).  

Justification for classification or non-classification

Based on in vitro and in vivo data, the test substance is considered to be non-irritating to skin and eyes and does not require classification for these endpoints according to Regulation (EC) No. 1272/2008.