Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion

Key: In vitro skin irritation (in vitro EpiDerm skin irritation assay, Andres 2018, OECD Guideline 439, GLP): skin irritating

Key: In vitro skin corrosion (in vitro EPISKIN skin corrosion assay, Buda 2018, OECD Guideline 431, GLP): not skin corrosive

Eye irritation/corrosion

Key: In vitro eye irritation (Reconstructed human Cornea-like Epithelium (RhCE), Geitlinger 2018, OECD Guideline 492, GLP): ocular irritating

Key:In vitro eye irritation/severe eye damage (in vitro isolated chicken eye test, Buda 2018, OECD Guideline 438, GLP): not ocular corrosive

Supporting: In vivo eye irritation (rabbit, Smyth 1969): positive, potential eye irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-15 to 2018-01-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz (8.4.2015)
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5°C) and kept under inert gas.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.
Details on animal used as source of test system:
not applicable
Justification for test system used:
This in vitro study was performed in order to evaluate the potential of hexanal to evoke skin irritation in a Reconstructed human Epidermis (RhE) test method.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm TM
- Tissue batch number(s): 25874
- Delivery date: 2018-01-16
- Date of initiation of testing: 2018-01-15 (pre-test)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C (35 min) room temperature (25 min)
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsing after exposure, after post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well plate. Then the inserts were transferred into the lower row of the 6-well plate

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Microtiter plate photometer Anthos Reader 2010 Flexi (Anthos Microsysteme GmbH)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.71 ± 0.09
- Barrier function: 5.28 h
- Morphology: presence of functional stratum corneum, a viable basal layer, and intermediate spinous and granular layers
- Contamination: sterile (no bacteria, yeast, other fungi, no HIV-1, Hepatitis B, Hepatitis C virus)

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 60 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than 50%
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): 100 %

VEHICLE
no vehicle used

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 100 %

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 %
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42.5 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
2.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
2.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
2.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
2.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
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:
Category 2 (irritant) based on GHS criteria
Remarks:
The OECD Guideline 439 addresses the human health endpoint skin irritation and does not allow a discrimination between skin irritation and skin corrosion.
Conclusions:
The test item hexanal is considered as at least irritant to skin. After the treatment, the mean value of relative tissue viability was reduced to 2.5 %. This value is well below the threshold for skin irritation (50 %). The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18 %). For these reasons, the result of the test is considered valid.
Executive summary:

One valid experiment according to OECD Guideline 439 following GLP with three tissues and two independent MTT measurements was performed. The tissues of the human skin model EpiDermTM were treated with hexanal for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). DPBS-buffer was used as negative control and 5 % SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 1.9 % (required: <= 20 %). The variation within the tissue replicates of negative, control, positive control and test item was acceptable (well below 18 %). Thus, confirming the validity and sensitivity of the test system. After the treatment with the test item, the mean value of relative tissue viability was only 2.5 % (reduction by 97.5 %). This value is well below the threshold for a skin irritation potential (50 % viability). Test items that induce viability values below the threshold of 50 % are considered to be at least irritant to skin. Therefore, hexanal is considered at least irritant to skin in the Reconstructed human Epidermis (RhE) Test Method. The OECD Guideline 439 addresses the human health endpoint skin irritation and does not allow a discrimination between skin irritation and skin corrosion.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-07 to 2018-03-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
OGYEI, Budapest (21.04.2016)
Test system:
human skin model
Source species:
human
Cell type:
other: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained.
Source strain:
other: Adult human-derived epidermal keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin(TM) Small Model (EpiSkin(TM)SM), EPISKIN SNC Lyon, France
- Tissue batch number(s): 18-EKIN-010
- Expiry date: 2018-03-12
- Date of initiation of testing: 2018-03-07

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL PBS, 1x

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT per well
- Incubation time: 3 h (± 15 min)
- Spectrophotometer: 96 well plate spectrophotometer (Thermo Scientific; Multiscan FC)
- Wavelength: 570 nm (±10nm; Read out range: 0-3.5 Abs)
- Linear OD range of spectrophotometer: 0.2136 – 3.1752

NUMBER OF REPLICATE TISSUES: 2 replicates of 12-well assay plate per item

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable):
– Placing untreated EpiOcular™ constructs (in a 24 well plate) in the –20°C freezer overnight.
– Thawing to room temperature.
– Refreezing (–20°C).
– Once frozen, the tissue may be stored indefinitely in the freezer.
– Before use, the killed tissues are de-frozen at room temperature.
– Further use of killed tissues is similar to living tissues.

- Method of calculation used:
– Non-specific MTT reduction calculation (NSMTT):
NSMTT % = [(ODKT - ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD (mean)
ODKT: test item treated killed tissues OD (mean)
ODNC: negative control OD (mean)

If NSMTT % is > 5 0% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

– True MTT metabolic conversion (TODTT) is undertaken if NSMTT is ≤ 50 %:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues

– The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / ODNC] × 100
% RV 2= [TODTT2 / ODNC] × 100

– The mean value of the 2 individual relative viability % for test item is calculated
Mean Relative Viability % = (% RV 1 + % RV 2) / 2


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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL hexanal (undiluted)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL NaCl (9 g/L saline)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL glacial acetic acid
Duration of treatment / exposure:
4 hours (± 10 min)
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
replicate 1
Value:
67
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
replicate 2
Value:
84
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
75
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: OD values and cell viability percentages of the positive and negative control

Controls

Optical Density (OD)

Viability (%)

Δ%

Negative Control:
NaCl (9 g/L saline)

1

1.103

107

14.3

2

0.956

93

mean

1.029

100

 

Positive Control:
Glacial acetic acid

1

0.044

4

2.3

2

0.068

7

mean

0.056

5

 

 

 

Table 2: OD values and viability percentages of the test item (including corrected values):

Test Item

Optical Density (OD)

TODTT

Viability (%)

Relative Viability (%)

Δ%

Hexanal

1

0.712

0.687

69

67

17.2

2

0.890

0.864

86

84

mean

0.801

0.776

78

75

 

standard deviation (SD)

12.17

12.17

 

 

Table 3: OD values of additional controls for MTT-interacting test item:

Additional controls

Optical Density (OD)

Negative control killed tissues:
NaCl (9 g/L saline)

1

0.047

2

0.017

mean

0.032

Test item treated killed tissues:
Hexanal

1

0.056

2

0.059

mean

0.057

Δ%: The difference of viability between the two relating tissues

TODTT: true MTT metabolic conversion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
The OECD Guideline 431 only allows distinguishing corrosivity from non corrosivity
Conclusions:
The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the used testing conditions.
Executive summary:

A study according to OECD Guideline 431 and under GLP conditions was performed with the test item hexanal. Disks of EPISKIN (two units / incubation time) were treated with test item and incubated for 4 hours (±10 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1°C in an incubator with 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. The test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

For each treated tissue viability was expressed as a % relative to negative control.

The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not significantly reduce cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue relative viability was 75 % at 4 hours of exposure. The test item treated tissue viability was well above the threshold value of 35 % of the mean negative control value after 4 hours of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid. The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the used testing conditions. In conclusion, the test item hexanal can be classified as non-corrosive.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-01 to 2018-03-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
OGYEI, Budapest (21.04.2016)
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- indication of any existing defects or lesions in ocular tissue samples: the selected eyes were examined with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 µL
Duration of treatment / exposure:
10 seconds exposure
Duration of post- treatment incubation (in vitro):
up to 240 minutes after the post-treatment rinse
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES : The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS : At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0 % to 2 %) were observed in the eyes, finding considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES : test item: 3, positive control: 3, negative control: 1

NEGATIVE CONTROL USED : NaCl, 9 g/L saline

POSITIVE CONTROL USED: Acetic acid, 10 % (v/v)

APPLICATION DOSE AND EXPOSURE TIME : 30 µl, 10 seconds exposure

OBSERVATION PERIOD : 240 min

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 ml saline solution


SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: decision criteria as indicated in the TG were used.
Irritation parameter:
percent corneal swelling
Value:
>= 14 - <= 18
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
1.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: overall ICE Class
Remarks on result:
other: 1x I, 2x III

Test Item: Hexanal

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

14 %

III

Mean maximum corneal swelling at up to 240 min

18 %

II

Mean maximum corneal opacity

1.7

III

Mean fluorescein retention

0.5

I

Other Observations

None

Overall ICE Class

1xI, 2xIII

Interpretation of results:
other:
Remarks:
Not ocular corrosive (not Cat. 1). The assay does not allow a conclusion regarding ocular irritation (Cat. 2)
Conclusions:
In this ICET, hexanal did not cause ocular corrosion or severe irritation in the enucleated chicken eye. The overall ICE score was 1xI, 2xIII.
Positive and negative controls showed the expected results. The experiment was considered to be valid.


Executive summary:

The severe eye irritation potential of the test item hexanal was analysed in an Isolated Chicken Eye Test (ICET) according to OECD TG 438 under GLP conditions. Isolated chicken eyes were exposed to 30 µL of undiluted hexanal for 10 seconds, followed by a post-treatment observation period of up to 240 min. In this ICET, hexanal did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE score was 1xI, 2xIII. Positive and negative controls showed the expected results. The experiment was considered to be valid.

According to the OECD TG 438, the overall classification of hexanal is neither UN GHS Category I (an ocular corrosive or severe eye irritant) nor No Category.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-15 to 2018-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz (8.4.2014)
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel at room temperature (20±5°C), kept under inert gas.
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability : This test method is an in vitro procedure allowing the identification of chemicals (substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: 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. The tissues were sterile (no contamination with HIV-1, Hepatitis B and Hepatitis C virus, no bacteria, yeast and other fungi identified, long-term antibiotic and 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): 50 µL
- Concentration (if solution): 100 %

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): 100 %
- batch no.: 032817ISA

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): 100 %
- batch no.: 20170815
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
120 min
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used
- RhCE tissue construct used, including batch number : EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia. Designation of the kit: OCL-200-EIT, Day of delivery: 16. Jan. 2018, Batch no.: 27019
- Doses of test chemical and control substances used : 50 μL (83.3 μL/cm2)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min (37°C), 12 min (RT), 120 min (37°C)
- Description of any modifications to the test procedure : no modifications
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : colour development of test item and direct reduction of MTT by test item were assessed. No colour development of the test item could be observed after incubation. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.
- Number of tissue replicates used per test chemical and controls (positive control, negative control) : 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 520 nm
- Description of the method used to quantify MTT formazan : measurement of optical density at 520 nm in plate spectrometer
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : OD of negative control: > 0.8 - < 2.5, % mean relative viability of positive control: < 50 % of negative control, variation within replicates < 20 %, viability threshold for eye irritation potential (≤ 60%).
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : Values for negative control and for positive control were within the range of historical data of the test facility
- Complete supporting information for the specific RhCE tissue construct used : The specific tissue construct used passed the quality control
- Reference to historical data of the RhCE tissue construct : test with specific tissue construct lies within the acceptance criteria, which are based on 1996 QC Database
- Positive and negative control means and acceptance ranges based on historical data : negative control (OD): historical: 1.84, study: 2.01; positive control (viability, %): historical: 31.3, study: 30.5
- Acceptable variability between tissue replicates for positive and negative controls : < 20 %
- Acceptable variability between tissue replicates for the test chemical: < 20 %
Irritation parameter:
other: viability (%)
Run / experiment:
1
Value:
4.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: viability (%)
Run / experiment:
2
Value:
4.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: viability (%)
Run / experiment:
3
Value:
4.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
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: negative control (OD): 1.167 - 2.437; positive control (viability %): 12.4 - 57.2
Interpretation of results:
other: According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1).
Conclusions:
After treatment with the test item, the mean value of relative tissue viability was 4.1%. This value is well below the threshold for eye irritation potential (≤ 60%).
Executive summary:

One valid experiment according to OECD Guideline 492 was performed in accordance with GLP criteria. The test item hexanal was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 28 minutes. 50 μL of the liquid test were applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues 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 was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 2.0. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 30.5 % (< 50 %). Variation within tissue replicates was acceptable (well below 20 %). After treatment with the test item, the mean value of relative tissue viability was 4.1 %. This value is well below the threshold for eye irritation potential (≤ 60 %).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

in vitro skin irritation

The skin irritation potential of the test item hexanal was tested in Reconstructed human epidermis (RhE) (EpiDermTM) according to OECD Guideline 439 under GLP conditions. The tissues of the human skin model EpiDermTM were treated with hexanal for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). Test items that induce viability values below the threshold of 50 % are considered to be at least irritant to skin. The mean value of relative tissue viability was reduced to 2.5 %. Therefore hexanal is at least skin irritant.

The skin corrosion potential of the test item hexanal was tested in Reconstructed human epidermis (RhE) (EPISKINTM) according to OECD Guideline 431 under GLP conditions. The tissues of the human skin model EPISKINTM were treated with hexanal for 4 h. The test item was applied directly to each tissue. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not significantly reduce cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue relative viability was 75 % at 4 hours of exposure. Thus, hexanal is non corrosive to the skin.

in vivo skin irritation

Hexanal was tested positive in albino rabbits. It is considered as potential irritant in this study (Morris 1971).

in vitro eye irritation

The eye irritation potential of the test item hexanal was analysed in an Reconstructed human Cornea-like Epthelium (RhCE) assay according to OECD Guideline 492 under GLP conditions. The test item hexanal was applied to a three-dimensional human cornea tissue model for an exposure time of 28 minutes. After treatment with the test item, the mean value of relative tissue viability was 4.1 %. This value is well below the threshold for eye irritation potential (≤ 60 %).

The severe eye irritation potential of the test item hexanal was analysed in an Isolated Chicken Eye Test (ICET) according to OECD Guideline 438 under GLP conditions. Isolated chicken eyes were exposed to 30 µL of undiluted hexanal for 10 seconds, followed by a post-treatment observation period of up to 240 min. In this ICET, hexanal did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE score was 1 x I, 2 x III.

in vivo eye irritation

Exposure of rabbit eyes to hexanal showed positive results indicating effects of the test substance on the cornea of rabbit eyes (Smyth 1969). While in Smyth et al., 1969 the ocular toxicity potential of hexanal was graded as 5, a preceding publication of the same group graded hexanal as 2 (Smyth et al., 1954). However, this supports the eye irritating potential of the test substance hexanal in rabbit eyes.

Justification for classification or non-classification

Effects on the skin

In the in vitro EpiDerm skin irritation assay (OECD Guideline 439, GLP) the substance was tested positive for skin irritation. Thus, hexanal is at least skin irritating. This test does not allow a discrimination between skin irritation and skin corrosion (Andres 2018).

In the in vitro EPISKIN corrosion assay (OECD Guideline 431, GLP) the substance was tested negative for skin corrosion. Thus, hexanal is not skin corrosive (Buda 2018).

Effects on the eye

In a Reconstructed human cornea like Epithelium (RhCE) assay according to OECD Guideline 492 hexanal was evaluated to be at least eye irritant. This is also confirmed by in vivo studies.

Based on the findings in an in vitro Isolated Chicken Eye Test according to OECD Guideline 438 under GLP conditions, hexanal was interpreted as not corrosive to the eye (Buda, 2018).

As overall result for the damaging/irritation potential of hexanal to the eyes and to the skin, it is concluded that the substance is classified according to Regulation (EC) No 1272/2008 in Cat. 2 as irritating to eyes (Eye Irrit. 2, H319: Causes serious eye irritation) and in Cat. 2 as irritating to the skin (Skin Irrit. 2, H315: Causes skin irritation).