Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

Endpoint summary

Description of key information

SKIN IRRIRATION

The aim of this study was to evaluate the irritation potential of the test material Ocimene PQ - Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene EC Number 933-779-9 using reconstructed the human epidermis model EPISKIN^TM(SM). The study was performed according to the OECD 439 Guideline and under GLP conditions.

The EPISKIN^TM(SM) disks were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS).

The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a > 95% humidified atmosphere.

The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere.

PBS treated epidermis were used as negative control and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control.

The result of the study showed that, following exposure with Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,triene (Ocimene PQ), the mean cell viability was 33.5% compared to the negative control. This value is below the threshold of 50%, therefore the test item was considered irritant to skin. The validity criteria were met and the study was considered to be valid.

 

SKIN CORROSION

The aim of this study was to evaluate the corrosion to skin potential of the test material Ocimene P - Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene EC Number 933-779-9 using reconstructed the human epidermis model EPISKIN^TM(SM). The study was performed according to the OECD 431 Guideline and under GLP conditions.

Disks of EPISKIN^TM(SM) were treated with the test item and incubated 4 hours at room temperature.

Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS).

The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere.

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control)

The result of this study showed that, following exposure with Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,triene (Ocimene PQ), the mean cell viability was 76.0% compared to the negative control.

This value is above the threshold of 35%, therefore the test item was considered non- corrosive to skin. The validity criteria were met and the study was considered to be valid.

 

EYE IRRITATION

The irritation effects of the test item Ocimene PQ, Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,- triene Ocimene, EC number 933-779-9 were assessed using the Isolated Chicken Eye Test (ICET). The study was performed according to the OECD No. 438 guideline (09 October 2017) and under GLP conditions.

The selected eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 µL of the test item. The three positive control eyes were treated in a similar way with 30 µL Benzalkonium chloride solution 5% (w/v). The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

The negative control and positive control results were within the historical control data range in the experiment and the quality control standards were met. The experiment was considered to be valid.

The results showed no significant corneal swelling during the four-hour observation period on the test item treated eyes. No significant cornea opacity change was observed in three eyes. No significant fluorescein retention change was noted. No other corneal effect was observed.

In conclusion, the test item Ocimene PQ, Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,- triene Ocimene, EC number 933-779-9 is classified as non-irritant, UN GHS Classification: No Category.

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:

30 may 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)

Deviations:

yes

Remarks:

The deviations were considered not to adversely affect the results or integrity of the study

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8

Deviations:

yes

Remarks:

The deviations were considered not to adversely affect the results or integrity of the study

Qualifier:

according to guideline

Guideline:

other: EpiSkin™(SM) SOP, INVITTOX Protocol No. 118

Deviations:

yes

Remarks:

The deviations were considered not to adversely affect the results or integrity of the study

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

yes

Remarks:

The deviations were considered not to adversely affect the results or integrity of the study

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Test Item: Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene
- Public name: Ocimene PQ
- EC number: 933-779-9 (pre-registration 222-081-3)
- CAS number: n/a (pre-registration 3338-55-4)
- Batch/Lot number: A170524D
- Appearance: Colorless liquid
- Purity**: Considered as 100%
- Expiry date: 06 June 2019
- Storage conditions: Room temperature (15-25 °C, ≤ 70% relative humidity (RH)), under inert gas, protected from humidity (tightly closed container)
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

- Identification and Receipt: The test item of a suitable chemical purity was supplied by the Sponsor.
- Preparation of the Test Item: The test item was applied as supplied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKINTM(SM) (Manufacturer: SkinEthic, France) - Tree-dimensional human epidermis model.

Details on animal used as source of test system:

TEST SYSTEM: EPISKINTM(SM) (Manufacturer: SkinEthic, France) is a three-dimensional human epidermis model. Adult humanderived 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 after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN TM (SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-022, Expiry Date: 04 June 2018)
- Units: EPISKIN TM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm*2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plates
- Punch: EPISKIN TM (SM) biopsy punch for easy sampling of epidermis
- A flask of sterile “Assay Medium” (Batch No.: 18 ESSC 024; Exp. Date: 06 June 2018)
- Storage: The kits were kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.

MTT SOLUTION
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solutions were stored in refrigerator (2-8 °C) protected from light. These were diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

ACIDIFIED ISOPROPANOL
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

CHEMICALS USED IN THE EXPERIMENT:
- MTT
- Isopropanol
- Hydrochloric acid
- Phosphate buffered saline (PBS)

PREDICTION MODEL / DECISION CRITERIA
General validity criteria
- The mean OD value of the two or three negative control tissues should be ≥ 0.6 and ≤ 1.5.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.
Specific criteria for corrosivity
- The difference of viability between the two tissue replicates should not exceed 30%.
- The acceptable mean percentage viability range for positive controls is ≤ 20%.

Control samples:

other: Positive controls: Glacial acetic Negative controls: Physiological saline (0.9% (w/v) NaCl solution)

Amount/concentration applied:

TEST MATERIAL
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath.

50 µL of test item was applied evenly to each of two test units and each additional control skin units.

NEGATIVE AND POSITIVE CONTROLS
50 µL of negative control (Physiological saline) or positive control (glacial acetic acid) were added to each skin unit

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 4 hours (± 10 min) at room temperature (23.0-24.2°C) in the corrosivity test.

Number of replicates:

TEST ITEM TEST AND CONTROLS:
- Two replicates per time point were used for test item
- Two negative controls and two positive controls
- Two additional test item-treated living tissues were used for the non-specific OD evaluation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

77

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of skin corrosion

Remarks:

Mean experiments 1,2: 76.0%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of skin corrosion

Remarks:

Mean experiments 1,2: 76.0%

Other effects / acceptance of results:

VALIDITY OF THE TEST
General validity criteria:
- After receipt, the two indicators of the delivered kits were in proper conditions.
- The mean OD value of the two negative control tissues in the corrosivity and irritation test was in the recommended range (0.954 and 0.723).
- The mean OD value of the blank samples (acidified isopropanol) was 0.047 in both cases.

Specific criteria for corrosivity testing:
- The two positive control treated tissues showed 0.5% viability demonstrating the proper performance of the assay.
- The difference of viability between the two test item-treated tissue samples in the MTT assay was 2.7%.
- The difference of viability between the two negative control tissue samples in the MTT assay was 1.3%.


All these parameters were within acceptable limits and therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

The resuts of this study indicated that test material Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene (Batch number: A170524D), is non-corrosive to the skin.

Executive summary:

The aim of this study was to evaluate the corrosion to skin potential of the test material Ocimene P - Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene EC Number 933-779-9 using reconstructed the human epidermis model EPISKINTM(SM). The study was performed according to the OECD 431 Guideline and under GLP conditions.

 

Disks of EPISKINTM(SM) were treated with the test item and incubated 4 hours at room temperature.

Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS).

 

The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere.

 

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control)

 

The result of this study showed that, following exposure with Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,triene (Ocimene PQ), the mean cell viability was 76.0% compared to the negative control.

This value is above the threshold of 35%, therefore the test item was considered non- corrosive to skin. The validity criteria were met and the study was considered to be valid.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 may 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)

Deviations:

yes

Remarks:

The deviations were considered not to adversely affect the results or integrity of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The deviations were considered not to adversely affect the results or integrity of the study

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8

Deviations:

yes

Remarks:

The deviations were considered not to adversely affect the results or integrity of the study

Qualifier:

according to guideline

Guideline:

other: EpiSkin™(SM) SOP, INVITTOX Protocol No. 118

Deviations:

yes

Remarks:

The deviations were considered not to adversely affect the results or integrity of the study

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Test Item: Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene
- Public name: Ocimene PQ
- EC number: 933-779-9 (pre-registration 222-081-3)
- CAS number: n/a (pre-registration 3338-55-4)
- Batch/Lot number: A170524D
- Appearance: Colorless liquid
- Purity**: Considered as 100%
- Expiry date: 06 June 2019
- Storage conditions: Room temperature (15-25 °C, ≤ 70% relative humidity (RH)), under inert gas, protected from humidity (tightly closed container)
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

- Identification and Receipt: The test item of a suitable chemical purity was supplied by the Sponsor.
- Preparation of the Test Item: The test item was applied as supplied.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKINTM(SM) (Manufacturer: SkinEthic, France) - Tree-dimensional human epidermis model.

Details on animal used as source of test system:

TEST SYSTEM: EPISKINTM(SM) (Manufacturer: SkinEthic, France) is a three-dimensional human epidermis model. Adult humanderived 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 after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN TM (SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-022, Expiry Date: 04 June 2018)
- Units: EPISKIN TM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm*2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plates
- Punch: EPISKIN TM (SM) biopsy punch for easy sampling of epidermis
- A flask of sterile “Maintenance Medium” (Batch No.: 18 MAIN3 028; Exp. Date: 06 June 2018)
- Storage: The kits were kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.

MTT SOLUTION
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solutions were stored in refrigerator (2-8 °C) protected from light. These were diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

ACIDIFIED ISOPROPANOL
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

CHEMICALS USED IN THE EXPERIMENT:
- MTT
- Isopropanol
- Hydrochloric acid
- Phosphate buffered saline (PBS)

PREDICTION MODEL / DECISION CRITERIA
General validity criteria
- The mean OD value of the two or three negative control tissues should be ≥ 0.6 and ≤ 1.5.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.
Specific criteria for irritation test
- Standard deviation value (SD) of the negative control tissues and the identically treated replicates % viability should be ≤ 18.
- The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.

Control samples:

other: Positive controls: 5% (w/v) Sodium Dodecyl Sulphate solution (irritation test) Negative controls: Phosphate Buffered Saline (irritation test).

Amount/concentration applied:

TEST MATERIAL
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath.

10 µL of test item were applied evenly to each of three test units and each additional control skin units. The test item was spread gently on the skin surface with a pipette tip without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

NEGATIVE AND POSITIVE CONTROLS
50 µL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette in the irritation test

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature (23.1-23.6°C).

Duration of post-treatment incubation (if applicable):

After rinsing, the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 41 hours (+58 minutes) at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere.

Number of replicates:

TEST ITEM TEST AND CONTROLS:
- Three replicates per time point
- Three replicates per time point were used for test item
- Three negative controls and three positive controls
- Two additional test item-treated living tissues were used for the non-specific OD evaluation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

31.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Mean experiments 1,2,3: 33.5%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

34.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Mean experiments 1,2,3: 33.5%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

34.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Mean experiment 1,2,3: 33.5%

Other effects / acceptance of results:

VALIDITY OF THE TEST
General validity criteria:
- After receipt, the two indicators of the delivered kits were in proper conditions.
- The mean OD value of the two negative control tissues in the irritation test was in the recommended range (0.723).
- The mean OD value of the blank samples (acidified isopropanol) was 0.047 in both cases.

Specific criteria for irritation testing:
- Standard deviation of the viability results for negative control samples was 2.6%.
- The positive control treated tissues showed 4.5% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.0%.
- The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 1.7%.

All these parameters were within acceptable limits and therefore the study was considered to be valid.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The resuts of this study indicated that test material Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene (Batch number: A170524D), is irritant to the skin, UN GHS Classification: Category 2.

Executive summary:

The aim of this study was to evaluate the irritation potential of the test material Ocimene P - Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene EC Number 933-779-9 using reconstructed the human epidermis model EPISKINTM(SM). The study was performed according to the OECD 439 Guideline and under GLP conditions.

 

The EPISKINTM(SM) disks were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS).

The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a > 95% humidified atmosphere.

 

The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere.

 

PBS treated epidermis were used as negative control and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control.

 

The result of the study showed that, following exposure with Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,triene (Ocimene PQ), the mean cell viability was 33.5% compared to the negative control. This value is below the threshold of 50%, therefore the test item was considered irritant to skin. The validity criteria were met and the study was considered to be valid.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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. QA statement)

Specific details on test material used for the study:

Name: Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene
Public name: Ocimene PQ
EC number: 933-779-9 (pre-registration 222-081-3)
CAS number:n/a (pre-registration 3338-55-4)
Batch/Lot number: A170524D
Appearance: Colourless liquid
Purity:Considered as 100% Expiry date: 06 June 2019
Storage conditions: Room temperature (15-25°C, ≤ 70% relative humidity (RH)), under inert gas, protected from humidity (tightly closed container)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Animal: chicken
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129, Hungary)
- Age at study initiation : approximately 7 weeks old
- Weight at study initiation: approximately 2.45 kg

ENVIRONMENTAL CONDITIONS
- Transporation: ambient temperature at the earliest convenience

The heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Citoxlab Hungary Ltd. and processed approximately within 2 hours of collection.

Vehicle:

physiological saline

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL undiluted test item
- Concentration (if solution):

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl) solution.

Name: Physiological saline (Salsol solution, 0.9% (w/v) NaCl)
Batch number:72522Y05-2
Manufacturer: B. Braun Pharmaceuticals SA
Expiry date: 31 May 2020
Storage condition:Room temperature


POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): The positive control eyes were treated with 30 μL of 5% (w/v) Benzalkonium chloride solution

Name:Benzalkonium chloride solution 50% in water
CAS Number: 63449-41-2
Batch number: STBH0549
Expiry date: 31 May 2022
Manufacturer: Sigma-Aldrich Co.
Storage condition:Room temperature

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

NA

Duration of post- treatment incubation (in vitro):

Controls and test eyes: 30, 75,120, 180 and 240 minutes after the post-treatment rinse

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluoresceintreated cornea was examined with a hand-held slit lamp, with the eye in the head, to ensure that the cornea was not damaged. Only the eyeball with a cornea in good condition was carefully removed from the orbit.
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a 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. 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 wet papers in a closed box to mantein the appropriate humidity.
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head), to avoide too much pressure on the eye by the clamp. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus.
The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the
chamber was closed except for manipulations and examinations, to maintain stable temperature and humidity.
The appropriate number of eyes was selected and carefully checked to ensure that they were in good condition. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was
measured, any eye with cornea thickness deviating more than 10% from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. The selected eyes were acclimatize for approximately 45 to 60 minutes at a controlled temperature (32±1.5°C))

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% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in the experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related
effect after treatment. All eyes were considered to be suitable for the assay.

TREATMENTS - APPLICATION DOSE - EXPOSURE TIME - REMOVAL OF TEST SUBSTANCE
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal 30 µL undiluted test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
The positive control eyes were treated in a similar way with 30 µL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl) solution. Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.
After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with at least 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.

OBSERVATION PERIOD
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. A Haag-Streit Bern 900 slit-lamp microscope,
with depth-measuring device no. 1 and slit-width setting at 9½, was used for the measurements.

NEGATIVE CONTROL USED
Physiological saline (Salsol solution, 0.9% (w/v) NaCl)

POSITIVE CONTROL USED
Benzalkonium chloride solution 50% in water

METHODS FOR MEASURED ENDPOINTS:
Provided in a separate document - see attachments to this robust study summary

SCORING SYSTEM:
Provided in a separate document - see attachments to this robust study summary

DECISION CRITERIA: According to the OECD guideline

Irritation parameter:

percent corneal swelling

Remarks:

up to 75 min

Run / experiment:

1

Value:

ca. 0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE Class I

Irritation parameter:

percent corneal swelling

Remarks:

up to 240 min

Run / experiment:

2

Value:

ca. 0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE Class I

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

ca. 0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE Class I

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE Class I

Other effects / acceptance of results:

In the positive control group, severe loosening of epithelium was observed in one eye at 75 minutes after the post-treatment rinse and in the other two eyes at 240 minutes after the post-treatment rinse. No other morphological effect was observed in the study.

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

According to the result of this study, the substance Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene (Ocimene PQ), EC number: 933-779-9, did not cause eye irritation.

Executive summary:

The irritation effects of the test item Ocimene PQ, Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,- triene Ocimene, EC number 933-779-9 were assessed using the Isolated Chicken Eye Test (ICET). The study was performed according to the OECD No. 438 guideline (09 October 2017) and under GLP conditions.

The selected eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 µL of the test item. The three positive control eyes were treated in a similar way with 30 µL Benzalkonium chloride solution 5% (w/v). The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

The negative control and positive control results were within the historical control data range in the experiment and the quality control standards were met. The experiment was considered to be valid.

The results showed no significant corneal swelling during the four-hour observation period on the test item treated eyes. No significant cornea opacity change was observed in three eyes. No significant fluorescein retention change was noted. No other corneal effect was observed.

In conclusion, the test item Ocimene PQ, Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,- triene Ocimene, EC number 933-779-9 is classified as non-irritant, UN GHS Classification: No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results observed in the eye and skin irritation/corrosion studies, 2-tert-butyl-1,4-dimethoxybenzene meets the criteria to be classified as a Category 2 skin irritant ( H315: Causes skin irritation), but do not meet the criteria to be classified as irritant to eyes under EU Regulation (EC) No 1272/2008 (CLP).