Oct-7-enal

Endpoint summary

Description of key information

Skin corrosion

Oct-7-enal was found to be non-corrosive to the skin using the three-dimensional human skin model EPIDERM comprising a reconstructed epidermis with a functional stratum corneum. In a GLP, OECD 431 study, 50 uL of test article, negative or positive control were applied to EpiDerm tissue 3 and 60 minutes followed by a 42 hour post-incubation period and immediate determination of cytotoxic effects via the MTT reduction assay. Corrosivity potential of the test article was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with distilled water. The positive control, 8N potassium hydroxide viability was < 20% following a 60 minute exposure, thereby confirming that corrosivity could be detected in this test system. The test article showed no evidence of corrosivity effects during the short term (3 minute) and prolonged exposures (60 minutes). The mean relative tissue viability was > 50% (102%) after the 3 minute exposure and > 15% (22.6%) after the 60 minute treatment (+ 42 hour post-incubation). As the test article was not classified as skin corrosion in STEP 1, progression to STEP 2 was not required. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7 -enal has no obligatory labelling requirement for skin corrosion and is unclassified.

Skin irritation

Oct-7-enal was found to be irritant using the three-dimensional human skin model EPIDERM comprising a reconstructed epidermis with a functional stratum corneum. In a GLP, OECD 439 study, 30 uL of test article, negative or positive control were applied to EpiDerm tissue for a 35 minute exposure, followed by a 42 hour post-incubation period and immediate determination of cytotoxic effects via the MTT reduction assay. Irritant potential of the test article was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS. The positive control, sodium dodecyl sulfate viability was < 20%, thereby confirming that irritants could be detected in this test system. The test item showed irritant effects. The mean relative tissue viability (% vehicle control) was < 50% (2.6%) after 60 minute treatment and 42 hour post incubation. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal requires classification and labelling with skin irritation/corrosion category 2.

Eye Corrosion

Oct-7-enal showed irreversible effects on the eye using the BCOP permeability assay. In a GLP, OECD 437 study 750 uL of test article, negative or positive control were applied to isolated bovine cornea tissue for a 10 minute exposure, followed by a 2 hour post-incubation period. Irritant potential of the test article was predicted using an opacitometer to assess corneal opacity, with corneal permeability assessed with the sodium fluorescein solution, with optical density at 490 nm ready using a spectrophotometer. These values were corrected for background opacity and the negative control permeability. The mean opacity and permeability OD490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group. An IVIS score of 53.36 was obtained for the positive control (ethanol, 100%), thereby confirming that serious eye damage could be detected in this test system. The test item induced slight opacity, with an IVIS scored of 59.17 calculated after a 10 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal is classified as Category 1: Irreversible effects on the eye, H318: Causes serious eye damage.

Eye irritation

Oct-7-enal was found to be irritant using the three-dimensional human skin model EpiOcularTM comprising a reconstructed human cornea-like epithelium. In a GLP, OECD 492 study, 50 uL of test article, negative or positive control were applied to EpiOcular tissue for a 30 minute exposure, followed by a 2 hour post-incubation period and immediate determination of cytotoxic effects via the MTT reduction assay. Irritant potential of the test article was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with distilled water. The positive control, methyl acetate viability was < 50%, thereby confirming that irritants could be detected in this test system. The test item showed irritant effects. The mean relative tissue viability (% vehicle control) was < 60% (17.6%) after 30 minute treatment and 2 hour post incubation. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal requires classification and labelling with eye irritation/corrosion category 2.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-18 to 2017-04-19

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:

2016

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: Oct-7-enal (OEL)
- CAS no.: 21573-31-9
- EC-no.: not assigned
- Source and lot/batch No.of test material: Kuraray / 161110
- Expiration date of the lot/batch: not stated
- Molecular weight: 126.198 g/mol
- Purity: 94.2%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from a single donor

Source strain:

other: Normal human epidermal keratinocytes (NHEK), derived from neonatal-foreskin tissue

Details on animal used as source of test system:

TEST KIT
- Source: MatTex Corporation
- Type: Normal human epidermal keratinocytes (NHEK), derived from neonatal-foreskin tissue
- Lot no.: 25765

Cell culture media:
- Assay medium (MatTex Corporation)
- Lot no.: 041217CMHA
- Vehicle / postive controls: deionised water / 8N KOH (Wako Pure Chemical)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C, humidified incubator
- CO2 (%): 5%

IN-LIFE DATES: 18 April 2017 to 19 April 2017

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-experimental checks:
To check the non-specific MTT-reducing capability of the test article 50 µL of the test article was mixed with 1 mL MTT (1 mg MTT/mL) medium and incubated for 1 hour and observed visually after stirring.

Nylon mesh compatibility was also assessed, using the same methodology as outlined above. The nylon mesh was evaluated microscopically.

Procedure:
- Pre-incubation:
One 6-well plate was prepared for each 3 culture insert and the medium (0.9 mL) was added to each well of the plate and pre-incubated for 60 minutes in a CO2 incubator.

- Skin corrosion test:
3 and 60 minute exposures were conducted. 50 uL of the test article, vehicle (distilled water) and positive (8N KOH) controls were applied onto each tissue surface at 45 second intervals. Duplicate tissues were used for each dose level. After the exposure, a nylon mesh was placed on each tissue surface to spread the substances over the tissue surface. The 45 second interval allowed sufficient time for both application and washing procedures at the end of the exposure.

After the initial dosing of the 60 minute application, the plate was placed into the incubator for the remainder of the exposure period was reached.
At the end of the exposure period tissues were washed with PBS to remove any residual test article. Excess PBS was removed by blotting bottom with blotting paper. The inserts were placed into a new 24 well plate filled with 300 uL/well of fresh medium.

Cytotoxicity analysis (MTT):
Following the post-incubation period, the inserts were transferred in a prepared 24-well plate containing 300 µL pre-warmed MTT medium (1 mg/mL) and further incubated for 3 h, under the same conditions as previously stated.

After the 3 h MTT incubation period tissues, MTT medium was removed and 2 mL of 2-propanol was added to the tissue inserts to allow formazan extraction from the tissues. Plates were placed into a plastic bag and extraction was performed at room temperature for 2 h or more using a plate shaker.

The 200 uL of the extract were transferred into a 96-well plate and the optical density was measured at 570 nm without reference wavelength in a plate spectrophotometer. 2-propanol was used as a blank.

Cell viability was calculated as follows:
Cell viability (%) = (OD of each tissue / Mean OD of the negative control group) x 100

Tissue binding test was carried using the same procedure as described above, with the exception of medium without MTT was used. After the measuring of OD, the staining ratio was calculated by the following formula:
Staining ratio (%) = (OD of each tissue [without MTT] / Mean OD of the negative control group [without MTT]) x 100

Control samples:

yes, concurrent vehicle

Amount/concentration applied:

50 uL of test article

Duration of treatment / exposure:

single exposure / 3 and 60 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

2 replicates/dose

Species:

other:

Details on test animals or test system and environmental conditions:

in vitro test system used

Amount / concentration applied:

in vitro test system used

Duration of treatment / exposure:

in vitro test system used

Observation period:

in vitro test system used

Number of animals:

in vitro test system used

Details on study design:

in vitro test system used

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

ca. 102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

no indication of corrosivity

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

ca. 22.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

no indication of corrosivity

Other effects / acceptance of results:

refer to "Any other information on results incl. tables"

Formulation analysis:

None conducted.

 

Pre-experiment:

The mixture of test article and MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.

 

The mixture of the test article and aqua destain showed no colouring detectable by unaided eye-assessment

 

Skin corrosion:

The test article showed no evidence corrosivity effects during the short term (3 minute) and prolonged exposures (60 minutes). The mean relative tissue viability was > 50% (102%) after the 3 minute exposure and > 15% (22.6%) after the 60 minute treatment (+ 42 hour post-incubation). As the test article was not classified as skin corrosion in STEP 1, progression to STEP 2 was not required.

 

The positive control viability was < 15% (1.6%) after the 60 minute treatment, following a 42 hour post-incubation therefore confirming that corrosivity was detected with the test system.

 

The controls confirmed the validity of the study. The mean OD₅₅₀for the 3 minute and 60 minute negative control groups were 1.725 and 1.799, respectively.

 

Table CA 7.3.1/02-1: Summary of in vitro EPIDERM corrosivity result following application of Oct-7-enal

Parameter	Negative control	Oct-7-enal	Positive control
3 minute exposure
OD 570 nm	1.725	1.760	0.148
Mean relative tissue viability (%) ± CV	100 ± 6.5	102 ± 4.1	8.6 ± 12.3
60 minute exposure
OD 570 nm	1.799	0.406	0.028
Mean relative tissue viability (%) ±CV	100 ± 1.3	22.6 ± 2.62	1.6 ± 0.0

 

Deficiencies:

None.

 

Conclusion

Under the conditions of this study the Oct-7-enal showed no evidence of corrosive effects during the short term (3 minute) and prolonged exposures (60 minutes). The mean relative tissue viability was > 50% (102%) after the 3 minute exposure and > 15% (22.6%) after the 60 minute treatment (+ 42 hour post-incubation). As the test article was not classified as skin corrosion in STEP 1, progression to STEP 2 was not required. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7 -enal has no obligatory labelling requirement for skin corrosion and is unclassified.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study the Oct-7-enal showed no evidence of corrosive effects during the short term (3 minute) and prolonged exposures (60 minutes). The mean relative tissue viability was > 50% (102%) after the 3 minute exposure and > 15% (22.6%) after the 60 minute treatment (+ 42 hour post-incubation). As the test article was not classified as skin corrosion in STEP 1, progression to STEP 2 was not required. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7 -enal has no obligatory labelling requirement for skin corrosion and is unclassified.

Executive summary:

The potential of the test article to induce skin corrosion was analysed using the three-dimensional human skin model EPIDERM comprising a reconstructed epidermis with a functional stratum corneum. In the present study Oct-7-enal was applied topically to the EPIDERM tissue for 3 and 60 minutes followed by a 42 hour post-incubation period and immediate determination of cytotoxic effects via the MTT reduction assay. Corrosivity potential of the test article was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with distilled water. The positive control, 8N potassium hydroxide viability was < 20% following a 60 minute exposure, thereby confirming that corrosivity could be detected in this test system. The test article showed no evidence corrosivity effects during the short term (3 minute) and prolonged exposures (60 minutes). The mean relative tissue viability was > 50% (102%) after the 3 minute exposure and > 15% (22.6%) after the 60 minute treatment (+ 42 hour post-incubation). As the test article was not classified as skin corrosion in STEP 1, progression to STEP 2 was not required. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7 -enal has no obligatory labelling requirement for skin corrosion and is unclassified.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-12 to 2017-12-15

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:

2015

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: Oct-7-enal (OEL)
- CAS no.: 21573-31-9
- EC-no.: not assigned
- Source and lot/batch No.of test material: Kuraray / 161110
- Expiration date of the lot/batch: not stated
- Molecular weight: 126.198 g/mol
- Purity: 94.2%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from a single donor

Source strain:

other: Normal human epidermal keratinocytes (NHEK), derived from neonatal-foreskin tissue

Details on animal used as source of test system:

TEST KIT
- Source: MatTex Corporation
- Type: Normal human epidermal keratinocytes (NHEK), derived from neonatal-foreskin tissue
- Lot no.: 24947

Cell culture media:
- Assay medium (MatTex Corporation)
- Lot no.: 120716CMHB
- Vehicle / postive controls: Dulbecco's PBS / 5% sodium dodecyl sulfate KOH (MatTex Corporation)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C, humidified incubator
- CO2 (%): 5%

IN-LIFE DATES: 12 December 2016 to 15 December 2016

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-experimental checks:
To check the non-specific MTT-reducing capability of the test article 30 µL of the test article was mixed with 1 mL MTT (1 mg MTT/mL) medium and incubated for 1 hour and observed visually after stirring.

Procedure:
- Pre-incubation:
One 6-well plate was prepared for each treatment, 3 culture insert and the medium (0.9 mL) was added to each well of the plate and pre-incubated for 60 minutes in a CO2 incubator. After 60 minutes of pre-incubation the plates were taken out of the CO2 incubator and culture inserts were transferred to the lower wells of the 6-well plates. The plates were incubated for 18-19 h in CO2 incubator.

- Exposure to the test material and rinsing:
After pre-incubation tissues were treated with each dose group in triplicate, starting with the negative control. The test article (30 µL) was added to the inserts and mesh was plated over the tissue surface. The exposure time was 35 minutes in a CO2 incubator, at this time plate were removed from the incubator and left at room temperature until the exposure period reach 60 minutes.
One 6-well plate was prepared for each negative (phosphate buffered saline), positive (5% sodium dodecyl sulfate) and test article and 0.9 mL of medium was added to each upper well of the plate.
At the end of the exposure period tissues were washed with PBS to remove any residual test article. Excess PBS was removed by blotting bottom with blotting paper. The inserts were placed in a prepared 6-well plate containing 0.9 mL pre-warmed fresh maintenance medium and post-incubated in a CO2 incubator for 42 hours.

Cytotoxicity analysis (MTT):
Following the post-incubation period, the inserts were transferred in a prepared 24-well plate containing 300 µL pre-warmed MTT medium (1 mg/mL) and further incubated for 3 h, under the same conditions as previously stated.
After the 3 h MTT incubation period tissues a total biopsy of the epidermis by using the special biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 µL of acidic isopropanol were added in order to extract the formazan. Extraction was carried out protected from light at 2 - 8°C over the weekend or, alternatively for 4 hours at room temperature.
The extract were transferred into a 96-well plate and the optical density was measured at 570 nm without reference wavelength in a plate spectrophotometer.

Data analysis:
Irritant potential of the test article was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with PBS. Classification of the test article was conducted in accordance with regulation EC 1272/2008:
- The test article was considered irritant to skin if the tissue viability after 60 min. of exposure and 42 h of post-incubation was =50%.
- The test article was considered non-irritant to skin if tissue viability after 60 min. of exposure and post-treatment incubation was >50%.

Control samples:

yes, concurrent vehicle

Amount/concentration applied:

30 uL of test article

Duration of treatment / exposure:

single exposure / 35 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3 replicates/dose

Details on test animals or test system and environmental conditions:

in vitro test system used

Amount / concentration applied:

in vitro test system used

Duration of treatment / exposure:

in vitro test system used

Observation period:

in vitro test system used

Number of animals:

in vitro test system used

Details on study design:

in vitro test system used

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 2.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

refer to "Any other information on results incl. tables"

Formulation analysis:

None conducted.

 

Pre-experiment:

The mixture of test article and MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.

 

The mixture of the test article and aqua destain showed no colouring detectable by unaided eye-assessment.

 

Skin Irritation:

The test article showed irritant effects. The mean relative tissue viability was < 50% (2.6%) after 60 minute treatment (+ 42 hour post-incubation). The positive control viability was < 20%, therefore confirming that irritants were detected with the test system.

 

The controls confirmed the validity of the study. The mean OD₅₅₀ of the six blank values was 2.007. The mean absolute OD₅₅₀of the three negative control tissues was >= 0.8 and <= 2.8. The mean relative tissue viability (% negative control) of the positive control was < 20% (3.6%). The maximum standard deviation of viability of replicate tissues of all dose groups was < 18 (0.1 - 2.9).

 

Table CA 7.3.1/01-1:
Summary of in vitro EPIDERM result following application of Oct-7-enal

Parameter	Negative control	Oct-7-enal	Positive control
OD 570 nm	2.007 ± 0.059	0.053 ± 0.002	0.073 ± 0.002
Mean relative tissue viability (%) ± SD	100 ± 2.9	2.6 ± 0.1	3.6 ± 0.1

 

Deficiencies:

None.

 

Conclusion

Under the conditions of this study the Oct-7-enal showed irritant effects. The mean relative tissue viability was < 50% (2.6%) after a 60 minute exposure and 42 hour post-incubation. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal requires classification and labelling with skin irritation/corrosion category 2.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of this study the Oct-7-enal showed irritant effects. The mean relative tissue viability was < 50% (2.6%) after a 60 minute exposure (+ 42 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal requires classification and labelling with skin irritation/corrosion category 2.

Executive summary:

The potential of the test article to induce skin irritation was analysed using the three-dimensional human skin model EPIDERMTM comprising a reconstructed epidermis with a functional stratum corneum. In the present study Oct-7-enal was applied topically to the EPIDERMTM tissue for 60 minutes followed by a 42 hour post-incubation period and immediate determination of cytotoxic effects via the MTT reduction assay. Irritant potential of the test article was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS. The positive control, sodium dodecyl sulfate viability was < 20%, thereby confirming that irritants could be detected in this test system. The test item showed irritant effects. The mean relative tissue viability (% vehicle control) was < 50% (2.6%) after 60 minute treatment and 42 hour post incubation. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal requires classification and labelling with skin irritation/corrosion category 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: Oct-7-enal (OEL)
- CAS no.: 21573-31-9
- EC-no.: not assigned
- Source and lot/batch No.of test material: Kuraray / 82725
- Expiration date of the lot/batch: 24 April 2018
- Molecular weight: 126.198 g/mol
- Purity: 95.4%

Species:

cattle

Strain:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied (volume or weight with unit): 750 uL
- Concentration (if solution): as recieved

VEHICLE
- Amount applied (volume or weight with unit): 750 uL
- Concentration (if solution): 0.9% NaCl

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

10 minutes

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with ethanol 100%

Details on study design:

SELECTION AND PREPARATION OF CORNEAS

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES: 3 corneas/dose

NEGATIVE CONTROL USED: yes, physiological saline 0.9% NaCl

POSITIVE CONTROL USED: yes, ethanol 100%

APPLICATION DOSE AND EXPOSURE TIME: Single 750 uL application for 10 minutes at 32°C

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: washed three times with Minimum essential medium (MEM) with phenol red (used for the rinsing of test substances only). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).

- POST-EXPOSURE INCUBATION: 2 hours.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).
- Others (e.g, pertinent visual observations, histopathology): each cornea was observed visually and pertinent observations were recorded

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given below:
IVIS UN GHS
= 3 No Category
> 3; = 55 No prediction can be made
> 55 Category 1

An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made

Irritation parameter:

in vitro irritation score

Value:

ca. 59.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
- Acceptance criteria met for positive control:

Individual Data

 

Table CA 7.3.2/02-1:Opacity

Test item	Cornea no.	Initial opacity	Final opacity	Change of opacity value	Corrected opacity value
Negative control (saline, 0.9% NaCl)	1	1.65	1.08	-0.57	n/a
	2	1.65	1.54	-0.11
	3	1.58	1.04	-0.53
	Mean	1.63	1.22	-0.40
Test item (undiluted)	1	0.02	13.12	13.10	13.51
	2	1.15	13.24	12.09	12.49
	3	2.54	16.72	14.19	14.59
	Mean	1.23	14.36	13.13	13.53
Positive control (ethanol, 100%)	1	3.11	40.60	37.49	37.90
	2	2.88	30.21	27.34	27.74
	3	3.03	33.25	30.22	30.62
	Mean	3.01	34369	31.68	32.09

 

Table CA 7.3.2/02-2: Permeability

Test item	Cornea no.	OD490	Corrected OD490 value
Negative control (saline, 0.9% NaCl)	1	0.009	n/a
	2	0.013
	3	0.004
	Mean	0.009
Test item (undiluted)	1	2.645	2.636
	2	2.995	2.986
	3	3.515	3.506
	Mean	3.052	3.043
Positive control (ethanol, 100%)	1	1.1013	1.004
	2	1.147	1.138
	3	2.120	2.111
	Mean	1.427	1.418

 

Table CA 7.3.2/02-3: In Vitro Irritation Score

Test item	Cornea no.	Corrected opacity	Corrected OD490 value	IVIS
Negative control (saline, 0.9% NaCl)	1	-0.57	0.009	-0.27
	2	-0.11	0.013
	3	-0.53	0.004
	Mean	-0.40	0.009
Test item (undiluted)	1	13.51	2.636	59.17
	2	12.49	2.986
	3	14.59	3.506
	Mean	13.53	3.043
Positive control (ethanol, 100%)	1	37.90	1.004	53.36
	2	27.74	1.138
	3	30.62	2.111
	Mean	32.09	1.418

 

Table CA 7.3.2/02-4: Historical Mean In Vitro Irritation Score of the Positive Control

	IVIS positive control – ethanol 100%
Mean value:	48.47
Standard deviation (SD):	10.08
Mean value - 2 SD:	28.31
Mean value + 2 SD:	68.64
No. of replicates providing historical mean: 43

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the conditions of this study the Oct-7-enal showed irreversible effects on the eye using the BCOP permeability assay. The mean in vitro irritancy score (IVIS) was 59.17 after a 10 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal is classified as Category 1: Irreversible effects on the eye, H318: Causes serious eye damage.

Executive summary:

The BCOP test method was used to identify if Oct-7-enal induced serious eye damage as defined by UN GHS, i.e. chemicals to be classified as UN GHS Category. In the present study Oct-7-enal was applied topically to the isolated bovine corneas for 10 minutes followed by a 2 hour post-incubation period. Negative (0.9% NaCl) and positive controls (ethanol, 100% were included). Irritant potential of the test article was predicted using an opacitometer to assess corneal opacity, with corneal permeability assessed with the sodium fluorescein solution, with optical density at 490 nm ready using a spectrophotometer. These values were corrected for background opacity and the negative control permeability. The mean opacity and permeability OD490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group. An IVIS score of 53.36 was obtained for the positive control (ethanol, 100%), thereby confirming that serious eye damage could be detected in this test system. The test item induced slight opacity, with an IVIS scored of 59.17 calculated after a 10 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal is classified as Category 1: Irreversible effects on the eye, H318: Causes serious eye damage.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-12 to 2017-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: Oct-7-enal (OEL)
- CAS no.: 21573-31-9
- EC-no.: not assigned
- Source and lot/batch No.of test material: Kuraray / 161110
- Expiration date of the lot/batch: not stated
- Molecular weight: 126.198 g/mol
- Purity: 94.2%

Species:

other: EpiOcular (reconstructed human cornea-like epithelium)

Strain:

other: supplied by MatTex Corporation

Details on test animals or tissues and environmental conditions:

TEST KIT
- Source: MatTex Corporation
- Type: Reconstructed human cornea-like epithelium
- Lot no.: 20973

Cell culture media:
- Assay medium (MatTex Corporation)
- Lot no.: 120516MWKC
- Vehicle / postive controls: deionised water / Methyl acetate (MatTex Corporation)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C, humidified incubator
- CO2 (%): 5%

IN-LIFE DATES: 12 December 2016 to 13 December 2016

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 uL
- Concentration (if solution): dosed as recieved (neat)

VEHICLE
- Amount(s) applied (volume or weight with unit): 50 uL
- Concentration (if solution): n/a
- Lot/batch no. (if required): municipal water purified using a water purifier to yield deionised water. Deionised water filter sterilised using a 0.22 um filter.
- Purity: sterile

Duration of treatment / exposure:

30 minutes / application to tissue surface

Observation period (in vivo):

in vitro method

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

2 replicates/treatment group

Details on study design:

Pre-experimental checks:
To check the non-specific MTT-reducing capability of the test article 50 µL of the test article was mixed with 1 mL MTT (1 mg MTT/mL) medium and incubated for 3 hours and observed visually after stirring. For comparison, only 1 mg/mL MTT solution was treated in a similar manner

Procedure:
- Pre-incubation:
One 6-well plate was prepared for each treatment, 2 culture inserts and the medium (1.0 mL) was added to each well of the plate and pre-incubated for 60 minutes in a CO2 incubator. After 60 minutes of pre-incubation the plates were taken out of the CO2 incubator and culture inserts were transferred to the lower wells of the 6-well plates. The plates were incubated for 18-19 h in CO2 incubator.

- Exposure to the test material and rinsing:
After pre-incubation tissues were treated with each dose group in duplicate, starting with the negative control. The test article (50 µL) was added to the surface of the tissues. When the test materials were not spread over the entire tissue surface, the culture inserts were gently tapped to penetrate the test material into the entire tissue. The exposure time was 30 minutes in a CO2 incubator.

At the end of the exposure period tissues were washed with PBS to remove any residual test article. Excess PBS was removed by blotting bottom with blotting paper. After completion of rinsing, the culture inserts were promptly transferred to a 12 well plate containing medium and left to stand for 12 minutes. At the end of the 12 minute period, the inserts were transferred to a 6-well plate containing medium and incubated for 2 h in a CO2 incubator.

Cytotoxicity analysis (MTT):
Following the post-incubation period, the inserts were transferred in a prepared 24-well plate containing 300 µL pre-warmed MTT medium (1 mg/mL) and further incubated for 3 h, under the same conditions as previously stated.

After the 3 h MTT incubation period, the inserts were transferred to a 24-well plate containing isopropanol (2 mL/well) in order to extract the formazan. Extraction was carried out protected from light at room temperature for 2 hours.

The extract were transferred into a 96-well plate and the optical density was measured at 570 nm without reference wavelength in a plate spectrophotometer.

Irritation parameter:

in vitro irritation score

Value:

ca. 17.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

refer to "Any other information on results incl. tables"

Formulation analysis:

None conducted.

 

Pre-experiment:

The mixture of test article and MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.

 

The mixture of the test article and aqua destain showed no colouring detectable by unaided eye-assessment.

 

Eye Irritation:

The test article showed irritant effects. The mean relative tissue viability was < 60% (17.6%) after 30 minute treatment (+2 hour post-incubation). The positive control viability was < 50%, therefore confirming that irritants were detected with the test system.

 

The controls confirmed the validity of the study. The mean OD₅₅₀of the vehicle control values was 1.279. The mean relative tissue viability of the positive control was < 50% (38%). The difference in viability of each dose group was < 20%.

 

Table CA 7.3.2/01-1:
Summary of in vitro EpiOcular result following application of oct-7-enal

Parameter	Negative control	Oct-7-enal	Positive control
Mean OD 570 (difference)	1.279 (0.097)	0.220 (0.016)	0.481 (0.112)
Mean relative tissue viability (%) (difference)	100 (7.6)	17.6 (1.2)	37.6 (8.8)

 

Deficiencies:

None.

 

Conclusion

Under the conditions of this study the Oct-7 -enal showed irritant effects. The mean relative tissue viability was < 60% (17.6%) after a 30 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal requires classification and labelling with eye irritation/corrosion category 2.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of this study the Oct-7-enal showed irritant effects. The mean relative tissue viability was < 60% (17.6%) after a 30 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal requires classification and labelling with eye irritation/corrosion category 2.

Executive summary:

The potential of the test article to induce ocular irritation was analysed using the three-dimensional human eye EpiOcular model comprising a reconstructed human cornea-like epithelium. In the present study Oct-7-enal was applied topically to the EpiOcular tissue for 30 minutes followed by a 2 hour post-incubation period and immediate determination of cytotoxic effects via the MTT reduction assay. Irritant potential of the test article was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with distilled water. The positive control, methyl acetate viability was < 50%, thereby confirming that irritants could be detected in this test system. The test item showed irritant effects. The mean relative tissue viability (% vehicle control) was < 60% (17.6%) after 30 minute treatment and 2 hour post incubation. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal requires classification and labelling with eye irritation/corrosion category 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No studies are available. In accordance with Annex I Regulation (EC) 1272/2008, Oct-7 -enal has no obligatory labelling requirement for skin corrosion and is unclassifed; therefore the following label "EUH071 – Corrosive to the respiratory tract" is not required.

Justification for classification or non-classification

Comparison with the CLP criteria

 

- Skin:

Oct-7-enal was found to be irritant using in the EpiOcular in vitro irritation model conducted under OECD 439.The mean relative tissue viability was <50% (2.6%) after 60 minute treatment (+42 hour post-incubation).

 

Oct-7-enal was deemed to be non-corrosive to the skin in the EpiDerm in vitro corrosivity model conducted under OECD 431.The mean relative tissue viability was >50% (102%) after the 3 minute exposure and >15% (22.6%) after the 60 minute treatment (+ 42 hour post-incubation).

 

Based on the results of these studies, Oct-7-enal was deemed to be irritant, but not corrosive to skin. Therefore, according to Annex I Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal is classified as Skin Irritant, Category 2.

 

- Eye:

Oct-7-enal was deemed to be an ocular irritant in the EpiDerm in vitro irritation model conducted under OECD 492 .However, this test guideline cannot resolve between GHS Categories 1 and 2, therefore assessment of skin corrosion potential is required to decide on the final classification.

 

Oct-7-enal showed irreversible effects on the eye using the BCOP permeability assay. The mean in vitro irritancy score (IVIS) was 59.17 after a 10 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal is classified as Category 1: Irreversible effects on the eye, H318: Causes serious eye damage.

 

- Respiratory tract:

No studies are available. In accordance with Annex I Regulation (EC) 1272/2008, Oct-7 -enal has no obligatory labelling requirement for skin corrosion and is unclassified; therefore the following label "EUH071 – Corrosive to the respiratory tract" is not required.

 

Overall conclusion:

Oct-7-enal was deemed to be irritant, but not corrosive to skin. Therefore, according to Annex I Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal is classified as Skin Irritant, Category 2.

 

Oct-7-enal was deemed to be cause irreversible effects on the eye. Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, Oct-7-enal is classified as Category 1: Irreversible effects on the eye, H318: Causes serious eye damage.