Registration Dossier

Administrative data

Description of key information

in vitro skin corrosion: Non-corrosive; OECD 431; Anon. (2017)

in vitro skin irritation: Not irritating; OECD 439; Anon. (2017)

in vitro eye damage: No prediction could be made; OECD 438; Anon. (2017)

in vitro eye irritation: Not irritating; OECD 492; Anon. (2017)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 October - 04 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 - 8ºC) in the dark and under nitrogen.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): applied as supplied.

OTHER SPECIFICS: n/a
Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed human epidermis
Cell source:
other: MatTek model kit - source not reported
Justification for test system used:
Guideline specific test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek
- Tissue batch number(s): 23372
- Production date: not reported
- Shipping date: not reported
- Delivery date: 01 November 2016
- Date of initiation of testing: not reported

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ºC
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS -Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: A comparison of laboratory historical data for negative and positive controls was made to verify the functioning of the test system.
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: Not reported
- Reproducibility: Coefficient of Variation between tissue replicates was ≤ 30%.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not reduce MTT.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): unchanged - applied as supplied

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8.0 N
Duration of treatment / exposure:
3 and 60 mins
Duration of post-treatment incubation (if applicable):
3 h MTT incubation followed by overnight isopropnaol extraction.
Number of replicates:
2 per test group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
90.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure
Value:
101.5
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:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.836 for the 3-Minute exposureperiod and 1.863 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 2.7% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: All values for the positive and negative control were within the historical ranges achieved by the testing facility in the previous twelve months, thus confirming the acceptable functioning of the test system.

Table 1       Relative mean viabilities for each treatment group

Exposure Period

Percentage Viability*

Negative Control§

Positive Control

Test Item

3 minutes

100

5.7

90.3

60 minutes

100

2.7

101.5

*mean of 2 replicates

§negative controls set to 100 %

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin under the conditions of the test.
Executive summary:

OECD 431 (2017) - The skin corrosivity potential of reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 562 nm of each well was measured using a Anthos 2001 microplate reader.

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 90.3 and 101.5 %, respectively. The quality criteria required for acceptance of the results was met.

Under the conditions of this study the test substance is not considered to be corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 - 21 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated ( 2 - 8 ºC) in the dark and under nitrogen.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: applied as supplied
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: n/a
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Episkin RHE model supplied by SkinEthic Laboratories, France.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 16-EKIN-046
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 15 November 2016
- Date of initiation of testing: 15 November 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ºC
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%. The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: Not reported
- Reproducibility: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- n/a, test item did not reduce MTT.
- Procedure used to prepare the killed tissues (if applicable): n/a
- N. of replicates : n/a
- Method of calculation used: n/a

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure is equal to or less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure is greater than or equal to 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): unchanged- applied as supplied.

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5 % w/v
Duration of treatment / exposure:
15 min test item exposure
Duration of post-treatment incubation (if applicable):
42 h followed by 3 h MTT incubation
Number of replicates:
3 per test group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 min exposure
Value:
67.7
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:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
- Acceptance criteria met for positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
- Acceptance criteria met for variability between replicate measurements: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.
- Range of historical values if different from the ones specified in the test guideline: Laboratory historical control data was performed to verify the functioning of the test system

Table 1       Mean optical density values and viabilities for the negative control, positive control and test item

Item

OD562 of tissues

Mean OD562 of triplicate tissues

± sd of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± sd of relative mean viability (%)

Negative control item

0.867

0.881

0.041

98.4

100*

4.6

0.927

105.2

0.849

96.4

Positive control item

0.103

0.083

0.018

11.7

9.4

2.1

0.069

7.8

0.076

8.6

Test item

0.599

0.596

0.040

68.0

67.7

4.6

0.635

72.1

0.555

63.0

* mean viability of negative controls was set to 100 %

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study the test item was classified as non-irritant (UN GHS classification not met).
Executive summary:

OECD 439 (2017) - The skin irritation potential of Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

 

Triplicate tissues were exposed to the test item for 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 562 nm of each well was measured using a Anthos 2001 microplate reader.

Mean viability of tissues exposed to the test substance after 15 minutes were 67.7 %. The quality criteria required for acceptance of the results was met.

Under the conditions of this study the test substance is not considered to be irritant to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. 2 - 8ºC in the dark and under nitrogen
- Stability under test conditions: Asumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a - applied unchanged as supplied

OTHER SPECIFICS: No
Species:
chicken
Strain:
other: Ross 308 Broiler
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Baileys Turkeys Ltd., Cheshire, UK
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): 3 kg and 56 days old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported in boxes containing moistened paper towels (isotonic saline) at ambient temperature.
- Time interval prior to initiating testing: Not reported
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No
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): 0.03 mL
- Concentration (if solution): n/a

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a
Duration of treatment / exposure:
240 mins
Observation period (in vivo):
30, 75, 120, 180 and 240 mins (± 5 mins) after the eyes were decontaminated with isotonic saline (i.e. removal of test item).
Number of animals or in vitro replicates:
3 replicates per test group
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES

Eyes that had a high baseline fluorescein staining (> 0.5) or corneal opacity score (> 0.5) after the enucleation process were rejected. The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes. Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2 % (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5. Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner. Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ± 1.5 °C. Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea.
Eyes were replaced when:
(i) the fluorescein score was > 0.5
(ii) the corneal opacity score was > 0.5
(iii) there was any additional signs of damage
(iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.

EQUILIBRATION AND BASELINE RECORDINGS

After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES

The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

NEGATIVE CONTROL USED

0.9 % Sodium chloride solution

SOLVENT CONTROL USED (if applicable)

n/a

POSITIVE CONTROL USED

5 % benzalkonium chloride

APPLICATION DOSE AND EXPOSURE TIME

0.03 mL for 10 seconds.

OBSERVATION PERIOD

Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Treatments rinsed from the eye using 20 mL of isotonic saline.
- Indicate any deviation from test procedure in the Guideline: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
- Damage to epithelium based on fluorescein retention: The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item.
- Swelling: Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea. Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point
- Macroscopic morphological damage to the surface: Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.
- Others (e.g, histopathology): No

SCORING SYSTEM:
- Mean corneal swelling (%): ((corneal thickness at time (t) - corneal thickness at t0) / corneal thickness at t0) * 100
- Mean maximum opacity score: Scoring from 0 - 4
- Mean fluorescein retention score: Scoring from 0 - 3

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. Yes
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
0.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean maximal
Value:
6.05
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
A test was considered acceptable if the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non-Classified and GHS Category 1, respectively.
Other effects:
No morphological effects were noted in the test item or control item treated eyes. Sloughing was noted in all positive control treated eyes.

Table 1       Individual scores and mean scores for corneal effects - test item

Endpoint

Eye #

Time (after eye rinsng) in mins

0

30

75

120

180

240

Corneal opacity

3B

0.5

0.5

0.5

0.5

0.5

0.5

6B

0

0.5

1

2

2

2

8B

0.5

0.5

0.5

0.5

0.5

0.5

Mean

0.3

0.5

0.7

1.0

1.0

1.0

ICE Class

II

Fluorescein retention

3B

-

0

-

-

-

-

6B

-

0.5

-

-

-

-

8B

-

0

-

-

-

-

Mean

-

0.2

 

-

-

-

ICE Class

I

Corneal thickness

3B

0.72

0.70

0.72

0.76

0.74

0.76

6B

0.70

0.74

0.78

0.75

0.76

0.76

8B

0.73

0.70

0.70

0.70

0.72

0.76

Mean

0.72

0.71

0.73

0.74

0.74

0.76

Mean Corneal Swelling (%)

 

-0.47

2.33

2.79

3.26

6.05

ICE Class

II

ICE Classes Combined

1 x I, 2 x II

Classification

No prediction can be made

Table 2       Individual scores and mean scores for corneal effects - positive control

Endpoint

Eye #

Time (after eye rinsng) in mins

0

30

75

120

180

240

Corneal opacity

2B

0

3

4

4

4

4

5B

0.5

4

4

4

4

4

7B

0

.

.

4

4

4

Mean

0.2

3.3

3.7

4.0

4.0

4.0

ICE Class

IV

Fluorescein retention

2B

-

3

-

-

-

-

5B

-

3

-

-

-

-

7B

-

3

-

-

-

-

Mean

 

3.0

 

-

-

-

ICE Class

IV

Corneal thickness

2B

0.72

0.94

0.90

0.92

0.84

1.08

5B

0.70

0.88

0.96

0.96

1.04

1.12

7B

0.70

0.82

0.84

0.90

0.98

1.06

Mean

0.71

0.88

0.90

0.93

0.95

1.09

Mean Corneal Swelling (%)

-

24.53

27.36

31.13

34.91

53.77

ICE Class

IV

ICE Classes Combined

3 x IV (sloughing in all eyes)

Classification

GHS Category 1

Table 3       Individual scores and mean scores for corneal effects - negative control

Endpoint

Eye #

Time (after eye rinsng) in mins

0

30

75

120

180

240

Corneal opacity

1B

0

0

0.5

0.5

0.5

0.5

4B

0

0

0.5

0.5

0.5

0.5

Mean

 

0

0

0.5

0.5

0.5

ICE Class

I

Fluorescein retention

1B

-

0

-

-

-

-

4B

-

0

-

-

-

-

Mean

-

0.0

 

-

-

-

ICE Class

I

Corneal thickness

1B

0.72

0.66

0.73

0.68

0.70

0.72

4B

0.70

0.70

0.72

0.70

0.74

0.70

Mean

0.71

0.68

0.73

0.69

0.72

0.71

Mean Corneal Swelling (%)

-

-4.23

2.11

-2.82

1.41

0.00

ICE Class

I

ICE Classes Combined

3 x I

Classification

No prediction can be made

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction for eye irritation can be made following assessment of the data for all endpoints.
Executive summary:

OECD 438 (2017) - The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye.

0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes were treated with saline for control purposes.

 

After 240 mins incubation (post rinsing) the mean corneal opacity of the chicken eyes was scored as 1.0 (ICE Class II), after 30 minutes the mean fluorescein retention scored as 0.2 (ICE Class I); and after 240 mins mean maximal corneal thickness scored as +6.05 % (ICE Class II).

 

The combination of the three endpoints resulted in an overall ICE score of 1 x I and 2 x II, thereby no classification for eye irritation could be made.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 March - 06 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. 2 - 8 ºC in the dark and under nitrogen.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: undiluted
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: no
Species:
human
Details on test animals or tissues and environmental conditions:
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ).

EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (29 November 2016) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.

Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (about 18 hours).
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

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a
Duration of treatment / exposure:
30 mins
Duration of post- treatment incubation (in vitro):
120 mins
Number of animals or in vitro replicates:
Each group tested in duplicate
Details on study design:
- Details of the test procedure used

Pre Test Assessments

The test items ability to directly reduce MTT was assessed. For this purpose approximately 50 µl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. The result indicated that the test item did not have MTT reducing effects. An additional test with freeze-killed tissues did not have to be performed.

The photometric properties of the test item after contact with water and isopropanol were assesed. For this purpose each 50 µL of the test item were added to 1.0 ml of water and to 2 ml isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least one hour, the isopropanol mixture or for 2 to 3 hours at room temperature. The result indicated that the test item did not dye the water or isopropanol and would therefore not interfere with the photometric assessment of MTT during the main test.

Main Test

After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.

After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.

At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).

After rinsing, the tissues were immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.

At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 ml of warm Assay Medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

After post-treatment incubation of 120 minutes the MTT assay was performed.

At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.

The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

- RhCE tissue construct used, including batch number: EpiOcular kit (Lot: 23574). Supplied by MatTek Coorp., Slovakia.

- Doses of test chemical and control substances used: 50 µL applied topically.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Tissues exposed for 30 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH. Tissues were immersed for 2 seconds during the rinsing process. Post exposue incubation was 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2.

- Justification for the use of a different negative control than ultrapure H2O (if applicable): N/A

- Justification for the use of a different positive control than neat methyl acetate (if applicable): N/A

- Description of any modifications to the test procedure: N/A

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): N/A

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per test group (test item, positive and negative control). 1 blank tissue prepared with no treatment.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm

- Description of the method used to quantify MTT formazan: See above.

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): N/A

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The percent viability of each of the two relating tissues for each control and test item relative to the negative control (100% control) were calculated. The difference of the viability between duplicate tissues was calculated. If the difference is >20% the test is considered as non-qualified. The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model. If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes

- Complete supporting information for the specific RhCE tissue construct used: Yes

- Reference to historical data of the RhCE tissue construct: Yes

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Yes

- Positive and negative control means and acceptance ranges based on historical data: Yes

- Acceptable variability between tissue replicates for positive and negative controls: Yes

- Acceptable variability between tissue replicates for the test chemical: Yes
Irritation parameter:
other: Spectrophotometric mean relative absorbance of MTT solution at 570 nm
Run / experiment:
Mean value
Value:
68.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD is > 0.8 and < 2.5 (1.922 and 2.068)
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (40.0%)
- Range of historical values if different from the ones specified in the test guideline: n/a

Table 1       Results after exposure to Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene for 30 minutes

Dose Group

Absorbance
Well 1
(Tissue 1/2)

Absorbance
Well 2 (Tissue 1/2)

Mean Absorbance* (Tissue 1/2)

Mean Absorbance* Tissue 1 and 2

Mean Absorbance of
2 Tissues*

Rel. Absorbance [%]
Tissue 1 and 2**

Absolute Value of the Difference of the Rel. Absorbances [%]
Tissue 1 and 2

Mean Rel. Absorbance

[% of Negative Control]**

Blank

0.038

0.039

0.038

0.000

 

Negative Control

1.928

2.068

1.998

1.960

1.927

101.7

3.3

100.0

1.922

1.946

1.934

1.895

98.3

Positive Control

0.780

0.849

0.815

0.776

0.771

40.3

0.6

40.0

0.802

0.805

0.803

0.765

39.7

Test Item

1.302

1.433

1.367

1.329

1.318

69.0

1.1

68.4

1.346

1.346

1.346

1.307

67.8

* mean of 2 replicates after blank correction

** relative absorbance = ((100 • test item absorbance) / mean absorbance of the negative control)

Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene does not possess an eye irritating potential.
Executive summary:

OECD 492 (2017) - This in vitro study was performed to assess the eye irritation potential of Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene by means of the Human Cornea Model Test.

Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol, and did not prove to be a MTT reducer.

Tissues of the human cornea model EpiOcularwere treated with the test item, the positive and the negative control for 30 minutes each in duplicate.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 40 %, thus the validity of the test system is ensured.

Relevant irritating effects were observed following 30 minutes incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 68.4 % compared with the value of the negative control (threshold for irritancy: ≤ 60%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene does not possess an eye irritating potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 431 (2017) - The skin corrosivity potential of reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 562 nm of each well was measured using a Anthos 2001 microplate reader. Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 90.3 and 101.5 %, respectively. The quality criteria required for acceptance of the results was met. Under the conditions of this study the test substance is not considered to be corrosive to the skin.

OECD 439 (2017) - The skin irritation potential of Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP. Triplicate tissues were exposed to the test item for 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 562 nm of each well was measured using a Anthos 2001 microplate reader. Mean viability of tissues exposed to the test substance after 15 minutes were 67.7 %. The quality criteria required for acceptance of the results was met. Under the conditions of this study the test substance is not considered to be irritant to the skin.

OECD 438 (2017) - The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes were treated with saline for control purposes. After 240 mins incubation (post rinsing) the mean corneal opacity of the chicken eyes was scored as 1.0 (ICE Class II), after 30 minutes the mean fluorescein retention scored as 0.2 (ICE Class I); and after 240 mins mean maximal corneal thickness scored as +6.05 % (ICE Class II). The combination of the three endpoints resulted in an overall ICE score of 1 x I and 2 x II, thereby no classification for eye irritation could be made.

OECD 492 (2017) - This in vitro study was performed to assess the eye irritation potential of Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene by means of the Human Cornea Model Test. Additional tests with viable or freeze-killed tissues were not performed, since the colourless test item did not dye water or isopropanol, and did not prove to be a MTT reducer. Tissues of the human cornea model EpiOcular™were treated with the test item, the positive and the negative control for 30 minutes each in duplicate. Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 40 %, thus the validity of the test system is ensured. Relevant irritating effects were observed following 30 minutes incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 68.4 % compared with the value of the negative control (threshold for irritancy: ≤ 60%). In conclusion, it can be stated that in this study and under the experimental conditions reported, Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene does not possess an eye irritating potential.

Justification for classification or non-classification

The substance does not meet the classification for skin or eye irritation/corrosion in accordance with GHS or Regulation (EC) No 1272/2008 (CLP).