Hexa-1,5-diene

Endpoint summary

Description of key information

Two GLP OECD studies were performed to assess skin corrosion and irritation potential: EpiDerm OECD 431 and EpiSkin OECD 439, respectively. GLP BCOP anf EpiOcular assays were performed to assess ocular irritation potential.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

EpiDerm Human Reconstructed Epidermis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 8 January 2018 Experimental completion date: 12 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Version adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC B.40 BIS

Version / remarks:

EC Guideline No. 440/2008. 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

Specific details on test material used for the study:

1,5-hexadiene. 99.4% purity.

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BSC-433-4-0488-5
- Expiration date of the lot/batch:1 June 2018
- Purity test date: 26 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Analyses conducted to support the information cited in the certificate of analysis for the test item were not conducted in compliance with the GLP or GMP regulations. The characterization of the test item was conducted under a sponsor or sponsor subcontractor quality system.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Human donor skin

Vehicle:

unchanged (no vehicle)

Details on test system:

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis (provided by MatTek Corporation, Ashland MA, USA). It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.ç

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number(s): 27667
- Delivery date: 10 January 2018
- Date of initiation of testing: 8 January 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37ºC for the 1-hour exposure; room temperature for the 3-minute exposure
- Temperature of post-treatment incubation (if applicable): N/A

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 wash with PBS, no volume specified
- Observable damage in the tissue due to washing: N/A

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL undiluted test item.
50µL Milli-Q water for the negative control.
50 µL 8N KOH for the positive control.

Duration of treatment / exposure:

2 tissues exposed for 3 minutes.
2 tissues exposed for 1 hour.

Duration of post-treatment incubation (if applicable):

N/A

Number of replicates:

4 tissues per test item (2 tissues per exposure time) together with 1 negative and 1 positive control.

Irritation / corrosion parameter:

% tissue viability

Value:

> 90

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:

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 equal to or below 2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 4.7%. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was equal to or below 21%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with 1,5-hexadiene compared to the negative control tissues was 91% and 109% respectively.

Mean tissue viability in the in vitro skin corrosion test with 1,5 -hexadiene:

	3 -min application viability (% of control)	1 -hour application viability (% of control)
Negative control	100	100
1,5 -hexadiene	91	109
Positive control	6.7	4.7

Interpretation of results:

GHS criteria not met

Conclusions:

1,5-hexadiene is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate 1,5-hexadiene for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of 1,5-hexadiene was tested through topical application for 3 minutes and 1 hour. The study procedures were based on the OECD 431 guideline. Batch BSC-433-4-0488-5 of 1,5-hexadiene was a clear colourless liquid. 1,5-hexadiene was applied undiluted (50 ¿L) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 4.7% after the 1-hour exposure. 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 equal to or below 2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was equal to or below 21%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 91% and 109%, respectively. Because the mean relative tissue viability for 1,5-hexadiene was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment 1,5-hexadiene is considered to be not corrosive. In conclusion, 1,5-hexadiene is not corrosive in the in vitro skin corrosion test under the experimental conditions described in the study report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

EPISKIN Small model

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 06 March 2018. Experimental completion date: 12 March 2018.

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:

Version adopted on 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

1,5-hexadiene. 99.4% purity.

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BSC-433-4-0488-5
- Expiration date of the lot/batch: 01 June 2018
- Purity test date: 26 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light

Analyses conducted to support the information cited in the certificate of analysis for the test item were not conducted in compliance with the GLP or GMP regulations. The characterization of the test item was conducted under a sponsor or sponsor subcontractor quality system.

Test system:

human skin model

Remarks:

EPISKIN

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other:

Details on test system:

This model is a three-dimensional human epidermis model, which consists of adult human derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 µL undiluted test item
25 µL PBS for the negative control
25 µL 5% SDS for the positive control

Duration of treatment / exposure:

15 +/- 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37ºC

Number of replicates:

Three replicates

Irritation / corrosion parameter:

% tissue viability

Value:

>= 74

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:

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 3.3%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 8%, indicating that the test system functioned.
properly.

Mean tissue viability in the in vitro skin irritation test with 1,5 -hexadiene:

	Mean tissue viability (% of control)	Standard deviation (%)
Negative control	100	4.6
1,5 -hexadiene	74	7.9
Positive control	3.3	0.3

Interpretation of results:

GHS criteria not met

Conclusions:

1,5-hexadiene is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the CLP Regulation.

Executive summary:

The objective of this study was to evaluate 1,5-hexadiene for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKINSMTM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes.

The study procedures were based on OECD 439 guideline.

Batch BSC-433-4-0488-5 of 1,5-hexadiene was a clear colorless liquid. The test item was applied undiluted (25 ¿L) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 74%. Since the mean relative tissue viability for 1,5-hexadiene was above 50% after 15 ± 0.5 minutes treatment 1,5-hexadiene is considered to be non-irritant.

The positive control had a mean cell viability of 3.3% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 8%, indicating that the test system functioned properly.

In conclusion, 1,5-hexadiene was non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and does not require classification according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the CLP Regulation.

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

Remarks:

BCOP Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 09 January 2018. Experimental completion date: 09 January 2018.

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

Adopted July 26, 2013

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

1,5-hexadiene. 99.4% purity.

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BSC-433-4-0488-5
- Expiration date of the lot/batch: 01 June 2018
- Purity test date: 26 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light

Analyses conducted to support the information cited in the certificate of analysis for the test item were not conducted in compliance with the GLP or GMP regulations. The characterization of the test item was conducted under a sponsor or sponsor subcontractor quality system.

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands)
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL

Duration of treatment / exposure:

10 +/- minutes at 32ºC

Duration of post- treatment incubation (in vitro):

120 +/- 10 minutes at 32ºC

Number of animals or in vitro replicates:

Three corneas for each treatment group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle¿s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1ºC. The corneas were incubated for the minimum of 1 hour at 32 +/- 1ºC.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Physiological saline (Eurovet Animal Health, Bladel, The Netherlands).

POSITIVE CONTROL USED
Ethanol.

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 µl of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1ºC.

POST-INCUBATION PERIOD: yes
After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle¿s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 +/- 10 minutes at 32 +/- 1ºC. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA:
IVIS <= 3: No category.
3 < IVIS <= 55: No prediction can be made
IVIS > 55: Category 1

Irritation parameter:

cornea opacity score

Value:

3.7

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Remarks:

Mean permeability

Value:

0.143

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Value:

5.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

The corneas were clear, however two corneas had a spot after the 10 minutes of treatment with 1,5-hexadiene. No pH effect of the test item was observed on the rinsing medium.

Other effects / acceptance of results:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 64 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The corneas were clear following exposure, however two corneas had a spot after the 10 minutes of treatment with 1,5-hexadiene. No pH effect of the test item was observed on the rinsing medium.

Summary of opacity, permeability and in vitro scores:

Treatment	Mean opacity	Mean permeability	Mean in vitro irritation score
Negative control	-0.1	0.007	0.0
Positive control	17	3.092	64
1,5 -hexadiene	3.7	0.143	5.8

Interpretation of results:

study cannot be used for classification

Conclusions:

Since 1,5-hexadiene induced an IVIS > 3 ¿ 55, no prediction on the classification can be made.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of 1,5-hexadiene as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of 1,5-hexadiene was tested through topical application for 10 minutes.

The study procedures described in this report were based on OECD guideline 437. Batch BSC-433-4-0488-5 of 1,5-hexadiene was a clear colourless liquid with a purity of 99.4%. The test item was applied as it is (750 ¿L) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 64 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 1,5-hexadiene induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 5.8 after 10 minutes of treatment. In conclusion, since 1,5-hexadiene induced an IVIS > 3 ¿ 55, no prediction on the classification can be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

EpiOcular OECD 492

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 12 March 2018. Experimental completion date 16 March 2018.

Reliability:

1 (reliable without restriction)

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:

28 July 2015

Deviations:

no

GLP compliance:

yes

Remarks:

The characterization of the test item was conducted under a Sponsor or Sponsor subcontrator quality system and not under GLP or GMP standards.

Specific details on test material used for the study:

Hexa-1,5-diene 99.4% purity

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BSC-433-4-0488-5
- Expiration date of the lot/batch: 01 June 2018
- Purity test date: 26 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability . 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).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live. EpiOcular¿ (OCL-200-EIT MatTek Corporation, Lot: 2742, kit A and B). The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The ¿tissue¿ is prepared in inserts with a porous membrane through which the nutrients pass to the 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. The cells used to produce EpiOcular tissue are screened for potential biological contaminants: HIV-1 virus, Hepatitis B virus, Hepatitis C virus, Bacteria, Yeast and other fungi. The product resulted in "no detection" for these biological contaminants. Tissue viability and barrier function tests are within the acceptable ranges and indicate appropriate formation of the mucosal barriar and a viable basal cell layer.
On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed Assay Medium. Assay Medium was supplied by MatTek Corporation, Ashland, USA. All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 73 - 86%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

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): Undiluted
No correction was made for the purity/composition of the test item. The liquid test item was applied undiluted directly on top of the tissue.

Duration of treatment / exposure:

30 +/- 2 minutes

Duration of post- treatment incubation (in vitro):

After the exposure period with 1,5-hexadiene the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a prelabeled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post- Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C.

Number of animals or in vitro replicates:

2 tissues per test item.

Details on study design:

- Details of the test procedure used . EpiOcular OECD 492.
- RhCE tissue construct used, including batch number . EpiOcular. OCL-200-EIT MatTek Corporation, Lot 272. Non-keratinized epithelium prepared from normal human keratinocytes (MatTek).
- Doses of test chemical and control substances used . 50 µL.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) . Before the assay was started the entire tissues were pre-wetted with 20 ¿L of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Exposure: 30 +/- 2 minutes at 37 +/- 1 ºC. After the exposure period with 1,5-hexadiene, the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to
and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a prelabeled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C.
- Number of tissue replicates used per test chemical and controls: two.
- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) . The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Description of the method used to quantify MTT formazan . After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. Formazan was extracted with 2 mL isopropanol refrigerated for 18 ± 2 hours in the dark. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well from which it was taken. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- Description of evaluation criteria used . 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 postexposure incubation is less than or equal (¿) to 60%.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria . The absolute mean OD570 (optical density at 570 nm) of the negative and positive control tissues was within the laboratory historical control data range.
- Positive and negative control means and acceptance ranges based on historical data .Negative control mean absorption: 1.52. Negative control absorption range: 1.077 - 1.805. Positive control mean absorption: 0.42. Positive control absorption range: 2.11 - 48.25.
- Acceptable variability between tissue replicates for positive and negative controls . 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.
- Acceptable variability between tissue replicates for the test chemical. The difference between the % tissue viabilities of the two identically treated replicates should be <20.

Irritation parameter:

other: Tissue viability (%)

Value:

77

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

1,5-hexadiene was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0017 and -0.0002, respectively. Therefore it was concluded that the test item did not induce color interference.
In addition, because no color change was observed in the presence of MTT it was concluded that 1,5-hexadiene did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range and was > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 30 ± 2 minutes exposure of 33%.
- The difference between the percentages of viability of two tissues treated identically was less than 15%, indicating that the test system functioned properly.

Mean tissue viability in the EpiOcular test with 1,5 -hexadiene:

	Mean tissue viability (% of control)	Difference between two tissues (%)
Negative control	100	7.6
1,5 -hexadiene	77	14
Positive control	33	0.51

Mean absorption in the EpiOcular test with 1,5 -hexadiene:

	Mean OD570	SD
Negative control	1.613	0.086
1,5 -hexadiene	1.235	0.16
Positive control	0.539	0.078

Interpretation of results:

GHS criteria not met

Conclusions:

1,5-hexadiene is non-irritant in the EpiOcular¿ test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of 1,5-hexadiene. For this purpose 1,5-hexadiene was topically applied on the Reconstructed Human EpiOcular¿ Model.

The possible eye hazard potential of 1,5-hexadiene was tested through topical application for 30 minutes.

The study procedures described in this report were based on OECD guideline 492.

Batch BSC-433-4-0488-5 of 1,5-hexadiene was a clear colorless liquid with a purity of 99.4%. 1,5-hexadiene was applied undiluted (50 ¿L) directly on top of the tissue for 30 ± 2 minutes.

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 33% after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentages of viability of two tissues treated identically was less than 15%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with 1,5-hexadiene compared to the negative control tissues was 77%. Since the mean relative tissue viability for 1,5-hexadiene was above 60% after 30 ± 2 minutes treatment 1,5-hexadiene is considered to be non-irritant. In conclusion, 1,5-hexadiene is non-irritant in the EpiOcular¿ test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

An in vitro skin irritation study using the EPISKIN Model is available with the submission substance. The study was conducted according to GLP and OECD 439. The test material was applied to the skin tissue undiluted for 15 minutes, followed by a 42 hour post-incubation period. The relative mean tissue viability obtained after treatment with the test material compared to the negative control tissue was 74%. Since the mean relative tissue viability for 1,5-hexadiene was above 50% after 15±0.5 minutes treatment 1,5-hexadiene is considered to be non-irritant.

An in vitro skin corrosion study using the EpiDerm Model is available with the submission substance. The study was conducted according to GLP and OECD 431. The test item material was applied to the skin tissue undiluted for 3 minutes and 1 hour. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissue was 91% and 109%, respectively. Because the mean relative tissue viability for 1,5-hexadiene was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment 1,5-hexadiene is not considered to be corrosive.

 

Eye Irritation

An in vitro eye irritation BCOP test is available for the submission substance. The study was conducted according to GLP and OECD 437. The test material was applied undiluted to the corneal surface for 10 minutes, followed by a 120 minute incubation period. Corneal opacity and permeability were determined after incubation. The corneas treated with 1,5-hexadiene showed opacity values ranging from 0.9 to 5.9 and permeability values ranging from -0.003 to 0.264. The corneas were clear, however two corneas had a spot after the 10 minutes of treatment with 1,5-hexadiene. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 3.4 to 9.9 after 10 minutes of treatment with 1,5-hexadiene; the mean in vitro irritancy score was 5.8. Since the IVIS was > 3 and ¿55, no prediction on the classification can be made.

Following the result of the BCOP, the Reconstructed Human EpiOcular Model was used to determine eye irritation potential. The study was conducted to GLP and OECD 492. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with 1,5-hexadiene compared to the negative control tissues was 77%. Since the mean relative tissue viability for 1,5-hexadiene was above 60% after 30 ± 2 minutes treatment 1,5-hexadiene is considered to be non-irritant.

Justification for classification or non-classification

1,5-hexadiene was negative in the in vitro skin irritation and corrosion assays, therefore classification for skin irritation/corrosion according to CLP is not required. Based on the EpiOcular in vitro assay, classification for eye irritation is not required.