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Skin irritation / corrosion

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Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August 2017 - 14 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd 3 November 2015
Test material information:
Composition 1
Specific details on test material used for the study:
No correction factor for purity required.
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 minimize 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-032
- Surface: 0.38 cm^2
- Expiration date: 14 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 36.6 - 37.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: tissues were washed with PBS (1 washing step)
- Observable damage in the tissue due to washing: no

- Before the start of the study, the test item was checked for possible direct MTT reduction and color interference. It was concluded that the test item did not interfere with the MTT endpoint.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE:
- Incubation time: 3 hours
- Measurement method: TECAN Infinite M200 Pro Plate Reader (570 nm)
- MTT concentration: 0.3 mg/mL

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

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

CELL VIABILITY MEASUREMENT: After incubation, tissues were dried and incubated with 2 mL MTT-solution for 3 hours. After incubation, epidermis was separated from the collagen matrix and both parts were extracted with 500 μL isopropanol. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

PREDICTION MODEL / DECISION CRITERIA (see table 1 in 'Any other information on materials and methods')
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
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 afer 7 minutes of contact time
Duration of treatment / exposure:
15 +/- 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours; + 3 hours with MTT
Number of replicates:
3 for the test item, the negative control and the positive control each.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 replicates
Value:
12
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
Mean tissue viability: 4.8%
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 and the SD of the % viability was 1.3%.
- Acceptance criteria met for positive control: yes, the mean relative tissue viability was 4.8% and the SD of the % viability was 2.3%
- Acceptance criteria met for variability between replicate measurements: yes, the standard deviation of the three tissue replicates treated with test substance was 6.3%.

- Since the mean relative tissue viability for the test item was below 50% the test item is considered to be irritant.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Classified as Category 2 based on Regulation (EC) 1272/2008.
Conclusions:
In an in vitro skin irritation test, performed according to OECD guideline 439 and under GLP principles, Florantone T showed to be irritant with a tissue viability of 12% after 15 minutes of treatment. Based on these results, the substance is classified as Category 2 (irritant) according to GHS and Regulation (EC) 1272/2008.
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Oct 2017 - 20 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008
Version / remarks:
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, 31 May 2008.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
Chemical name (IUPAC, synonym or trade name: 1-(5,6,7,8-tetrahydro-2-naphthyl)ethan-1-one
CAS number: 774-55-0
Test item storage: At room temperature protected from light container flushed with nitrogen
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns
Source strain:
other: cultured tissues
Justification for test system used:
Recommended test system in international guidelines (OECD and EC)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
human-derived epidermal keratinocytes

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- washed with phosphate buffered saline

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
300 μl MTT-medium
- Incubation time:
3 hours at 37°C
- Wavelength:
570 nm

NUMBER OF REPLICATE TISSUES:
triplicate

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50% 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%.

- The test substance is considered to be non-corrosive to skin if the relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%. In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 μl of undiluted test item applied directly
Duration of treatment / exposure:
3-minute exposure
1-hour exposure
Details on study design:
Calculation of Cell Viability: The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows: %Viability = (ODc/mean ODlt_u+MTT) * 100

number of replicates: triplicate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
107
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour
Value:
102
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
other: Mean Absorption
Run / experiment:
3-minutes
Value:
>= 2.059 - <= 2.263
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: Mean Absorption
Run / experiment:
1-hour
Value:
>= 2.247 - <= 2.377
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.7%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 8%, indicating that the test system functioned properly.
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, the test substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
02 August 2017 - 04 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
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:
28 July 2015
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
No correction factor for purity required.
Species:
human
Details on test animals or tissues and environmental conditions:
TEST SYSTEM:
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
- Justification: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 μL
- The test material was directly applied to the tissues which were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS.

POSITIVE CONTROL
- Amount applied: 50 μL Methyl Acetate

NEGATIVE CONTROL
- Amount applied: 50 μL Milli-Q water
Duration of treatment / exposure:
30 minutes ± 2 minutes
Duration of post- treatment incubation (in vitro):
Post-soak: 12 ± 2 minutes; incubation after the post-soak: 120 minutes ± 15 minutes
Number of animals or in vitro replicates:
2 tissues per test item and 2 tissues for the negative and the positive control each.
Details on study design:
- Details of the test procedure used:
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23491)
- Duration and temperature:
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS and incubated at standard culture conditions for 30 ± 2 minutes.
* exposure: 30 minutes ± 2 minutes at 37.0 ± 1.0°C
After the exposure period, the tissues were thoroughly rinsed with Ca2+Mg2+-free DPBS (brought to room temperature) to remove residual test item.
* post-soak immersion: 12 ± 2 minutes at room temperature in assay medium
* incubation: 120 minutes ± 15 minutes at 37°C in assay medium + 180 ± 10 minutes with MTT

- The test item was checked for possible color interference and possible direct MTT reduction before the study was started.

- Cell viability measurements:
After incubation, the tissues were incubated with 0.3 mL MTT-medium (1.0 mg/ml) for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were transferred to a 6-well plate containing 2 mL isopropanol. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

- Evaluation criteria:
* The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
* The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60%.

- Acceptability criteria:
* The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
* The mean relative tissue viability of the positive control should be <50% relative to the negative control.
* The difference between the % tissue viabilities of the two identically treated replicates should be <20%.
Irritation parameter:
other: Mean cell viability (%)
Value:
78
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
Mean tissue viability: 36%
Remarks on result:
other: SD: 6.6%
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: see 'any other information on results' for historical data. Results for the positive control were within the historical data range and therefore showing that the test system was functioning properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was between >0.8 and <2.5 (i.e., 2.005)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e., 36%)
- The difference between the % tissue viabilities of the two identically treated replicates was <20% (i.e., 2.9, 6.6 and 2.1 for the negative control, the treatment group and the positive control respectively)

Table 1 Historical data for EpiOcular studies

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Positive control

(viability; %)

Range

1.077 – 1.805

0.029 – 0.793

2.11 – 48.25

Mean

1.52

0.42

26.86

SD

0.21

0.23

14.11

n

16

16

16

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 January 2013 to May 2017.

Table 2 Individual OD570 measurements

 

A

(OD570)

B

(OD570)

Negative control

OD570 measurement 1

OD570 measurement 2

 

2.0133

1.9993

 

2.0902

2.0852

FLORANTONE T

OD570 measurement 1

OD570 measurement 2

 

1.7194

1.6748

 

1.5154

1.5050

Positive control

OD570 measurement 1

OD570 measurement 2

 

0.7296

0.7209

 

0.7955

0.7726

OD = Optical density

Duplicate exposures are indicated by A and B.

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) No. 1272/2008
Conclusions:
In an in vitro skin corrosion EpiOcular test, Florantone T was not irritant or corrosive to the eyes with a mean cell viability of 78% compared to the negative control. Based on these results, the test item is not classified according to GHS and according to Regulation (EC) No. 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin irritation/ corrosion

Two in vivo studies were available, one conducted in 1985 with 5% of the substance on Albino rabbits, and the other conducted in 1984 on Guinea pigs at up to 50% of the substance.

Neither study was considered sufficently reliable to conclude on the endpoint.

A first irritation test was conducted on Reconstituted Human Epidermis (OECD 439), the relative mean tissue viability was 12% Therefore, as it was not possible to conclude on the skin irritation or skin corrosion potential (cat. 2 or cat. 1), a second in vitro corrosion stuudy was conducted.

The corrosion test was conducted with EpiDerm (OECD 431), the 3 -min and the 1 -hr treatments gave respectively a relative mean viability of 107% and 102%.

Therefore, the test item is not a skin corrosive but it has to be claissified as a skin irritant cat. 2 (according to GHS & EU CLP).

Eye irritation/ corrosion

An in vitro irritation test was conducted with EpiOcular (OECD 492) and relative mean tissue viability was 78%.

It was concluded that the substance does not have to be classified as an eye irritant or corrosive.

Therefore for this endpoint it classified as "No category" (according to GHS & EU CLP).