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Administrative data

Description of key information

In a BCOP assay conducted in accordance with OECD 437, 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) did not induce ocular irritation through opacity and permeability endpoints, resulting in a mean in vitro irritancy score (IVIS) of -0.7 after 10 minutes of treatment. In conclusion, since 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Under the experimental conditions of an in vitro skin corrosion test using the EpiDerm model (OECD 431), 1,1’-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) was observed to be a non-corrosive.

Under the experimental conditions of an in vitro skin irritaiton test using the EPISKIN model (OECD 439), 1,1’-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) was observed to be a non-irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 to 20 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Appearance: Clear colourless liquid
Batch: 170918339
Purity: 97.2%
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Source: SkinEthic Laboratories, Lyon, France
EPISKIN-SMTM, 0.38 cm2, Batch no.: 18 EKIN 033
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN TEST SYSTEM PREPARATION
- Procedure used: The liquid test item was applied undiluted (25 µL) was added into 12-well plates directly on top of the tissue

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 36.7 - 37.4°C)
- Temperature of post-treatment incubation: 37.0 ± 1.0°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: The tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 (The test was performed on a total of 3 tissues per test item together with negative and positive controls).

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- A test item is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 µL

NEGATIVE CONTROL Phosphate bufered saline
- Amount applied: 25 µL

POSITIVE CONTROL 5% (aq) Sodium dodecyl sulfate
- Amount applied: 25 µL
Duration of treatment / exposure:
Exposure period of 15 ± 0.5 minutes at room temperature
Duration of post-treatment incubation (if applicable):
42 hours at 37°C
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
91
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this in vitro skin irritation test using the EPISKIN model, 1,1’-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) is a non-irritant.
Executive summary:

Under the experimental conditions of this in vitro skin irritation test using the EPISKIN model, 1,1’-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) is a  non-irritant.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental start date was 25 Jun 2018, and the experimental completion date was 29 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
2008, B.40. bis. IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST
GLP compliance:
yes
Specific details on test material used for the study:
Purity: 97.2%
Batch: 170918339
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: All cells used are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions.
Justification for test system used:
Recommended test system in international guidelines (OECD and EC)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200, Lot no.: 28811, kit L and M)
- Tissue batch number(s): 28811

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.6 – 37.2°C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL
- Incubation time: 3 hours
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 4 tissues per test item

PREDICTION MODEL / DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability >= 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test item was applied undiluted (50 µL) directly on top of the tissue.
For the negative control 50 µL for both the 3-minute and 1-hour time points.
For the positive control 50 µL 8N KOH for both the 3-minute and 1-hour time points.
Duration of treatment / exposure:
3-minute and 1-hour exposure times.
Number of replicates:
Test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minutes exposure to the test item and two for a 1-hour exposure.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute application
Value:
94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour application
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
study cannot be used for classification
Remarks:
The results confirm the test item is not corrosive, but does not address skin irritation.
Conclusions:
The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report. Further work is required to determine the irritation potential of the test item.
Executive summary:

Based on this in vitro skin corrosion test according to OECD 431, the test item is not corrosive under the experiemental conditions described. Further work is required to determine the irritation potential of the test item.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
June 2018 to September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
2017
GLP compliance:
yes
Specific details on test material used for the study:
Clear colourless liquid
Purity: 97.2%
Species:
cattle
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch,The Netherlands). Eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Age at study initiation: 6 – 60 months of age

ENVIRONMENTAL CONDITIONS
- Eyes were collected and transported in physiological saline without antibiotics in a suitable container under cooled conditions.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test Material, Postive Control, Negative Control
- Amount applied: 750 µL
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.
Duration of post- treatment incubation (in vitro):
The corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.
Number of animals or in vitro replicates:
3 replicates/treatment
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

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

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

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

NEGATIVE CONTROL USED: Yes, physiological saline

POSITIVE CONTROL USED: Yes, ethanol

APPLICATION DOSE AND EXPOSURE TIME: The medium from the anterior compartment was removed and 750 µl of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

POST-INCUBATION PERIOD: yes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.
- POST-EXPOSURE INCUBATION: The corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated with the aid of a microplate reader (TECAN Infinite® M200 Pro Plate Reader). The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values.

SCORING SYSTEM:
- In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

DECISION CRITERIA: Yes, the IVIS cut-off values for identifying the test item as inducing serious eye damage were based on the UN GHS criteria: GHS Category 1 for serious eye damage; and the GHS No Category for not requiring classification for eye irritation/serious eye damage.

If: IVIS Range ≤ 3; UN GHS “No category”
If: IVIS Range > 3, ≤ 55: UN GHS “No prediction can be made”
If: IVIS Range >55; UN GHS “Category 1”
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
-0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
-0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The corneas treated with test item showed opacity values ranging from -1.3 to -0.3 and permeability values ranging from 0.000 to 0.006. The corneas were clear after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium.

The individual in vitro irritancy scores for the negative controls ranged from 2.0 to 2.6. The corneas treated with the negative control item were clear after the 10 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 29 to 31. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 30 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
Interpretation of results:
GHS criteria not met
Conclusions:
1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of -0.7 after 10 minutes of treatment. In conclusion, since 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of -0.7 after 10 minutes of treatment. In conclusion, since 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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

Additional information

Justification for classification or non-classification

In a BCOP assay conducted in accordance with OECD 437, 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) induced an IVIS ≤ 3, thus no classification is required for eye irritation or serious eye damage.

In an in vitro skin irritation test conducted in accordance with OECD 439, 1,1’-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) was determined to be a non-irritant, thus no classification is required for skin irritation.