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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September - October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted July 2015
Deviations:
yes
Remarks:
The post-incubation time was 9 minutes instead of 12± 2 minutes. This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Certificate Good Laboratory Practice, Statement of GLP Compliance according to §19b Abs. 1 Chemikaliengesetz, Date of Inspection: 13.-16. July 2015

Test material

Constituent 1
Reference substance name:
dibenzylbenzene, ar-methyl derivative, hydrogenated
EC Number:
943-342-4
Molecular formula:
C21H38
IUPAC Name:
dibenzylbenzene, ar-methyl derivative, hydrogenated
Test material form:
liquid
Specific details on test material used for the study:
- Lot/batch No.of test material: hydrogenius 005
- Expiration date of the lot/batch: > 3 years
- Storage condition of test material: room temperature
- Puritiy: 100%

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: Eye irritation potential is usually determined in vivo in the Draize rabbit eye irritation test as described in OECD guideline 405. In a prevalidation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.
The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.

- Description of the cell system used, incl. certificate of authenticity: 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). A certificate of the EpiOcular™ tissue used in this study is attached (see "Attached background material")

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 µl
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
9 minutes post-soak period + 120 minutes post-treatment incubation
Number of animals or in vitro replicates:
duplicate tissues were treated with test substance and controls
Details on study design:
RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM (RhCE) MODEL
- Model used: EpiOcular Kit, MatTek Corporation, Bratislava, Slovakia
- EpiOcular Kit Components: 12/24 inserts with EpiOcularTM tissues on agarose in sealed 24-well plates, serum-free test medium (DMEM-Medium), Methyl acetate (CAS# 79-20-9 as positive control), serum-free assay medium (DMEM-based medium)
- Lot number: 23737
- Pre-treatment of tissues: On day of receipt (04 October 2016), the EpiOcular™ tissues were equilibrated for 15 minutes at room temperature. An appropriate volume of EpiOcular™ assay medium was pre-warmed to 37 °C. 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 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 plate, 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 assay medium. After one hour, the assay medium was replaced by 1 mL of fresh assay medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions overnight (about 17.25 hours).
- Date of initiation of testing: 05 October 2016

PRE TESTS
- Assessment of Direct Test Item Reduction by MTT: 50 µl test material + 1 ml 1.0 mg/ml MTT solution in DMEM incubated for 3 h at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air, a control (50 µl deionised water + 1 ml MTT solution) was performed in parallel. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
- Assessment of Coloured or Staining Materials: 50 μL test item + 1.0 ml of water, + 2 ml isopropanol resp. The water mixture was incubated at 37 ± 1.5 °C in the dark in a humidified atmosphere of 5 ± 0.5% CO2 in air for 1 hour, the isopropanol mixture for 3 hours at room temperature. The test item itself was no coloured, but if it becomes coloured after contact with water of isopropanol, this could interfere with the quantitative photometric MTT measurement. Therefore the test item was checked for its colorant properties.

MAIN TEST
Pre-treatment of tissues: overnight EpiOcular tissues were pre-wetted with 20 µl Ca++Mg++ free DBPS and pre-incubated for 30 minutes at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH)
Test item exposure: 50 µl test item were applied topically on duplicate tissues and incubated for 30 min at standard culture conditions
Negative control: 50 µl deionised water, same treatment conditions as test item
Positive control: 50 µl Methyl acetate, same treatment conditions as test item
Washing: 3 times DPBS without Ca++ and Mg++
Post-soak period: 9 minutes in assay medium at room temperature to remove any substance absorbed into the tissue
Post-treatment incubation: 120 minutes at standard culture conditions in assay medium
MTT assay: Placement of each tissue into a 24-well plate with 0.3 ml MTT solution (1.0 mg/mL in DMEM), followed by incubation for 180 minutes at standard culture conditions
Extration of formazan crystals: Isopropanol (2.0 ml) was applied overnight at 2-8°C in the dark, then shaken for 2-3 hours at room temperature
Optical density (OD) measurement: at 570 nm with a plate reader (Versamax Molecular Devices, Germany, Software Softmax Pro, version 4.7.1), no reference wavelength measurement was performed

PREDICTION MODEL / DECISION CRITERIA
Test item-treated tissue viability relative to the negative control treated tissue viability > 60%: non-irritant.
Test item-treated tissue viability relative to negative control treated tissue viability ≤ 60%: irritant.

EVALUATION
- The mean OD value of the blank control wells (OD Blk) for each experiment were calculated.
- The mean OD Blk from each mean OD value of the same experiment (blank corrected values) were subtracted.
- The mean value of the two aliquots for each tissue (= corrected test item OD) were calculated.
- The mean value of the two relating tissues for each control and test item (= corrected mean OD) were calculated. For further calculations only the corrected mean negative control OD value was needed.
- The corrected OD value of the negative control corresponds to 100% viability:
- Corrected negative control OD = Negative Control OD – OD Blk = 100% Viability
- 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: Viability [%] = 100 x (corrected test item OD / corrected mean negative control OD)
- The difference of the viability between duplicate tissues was calculated.
- The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model.

ACCEPTANCE CRITERIA
- The negative control OD is > 0.8 and < 2.5,
- The mean relative viability of the positive control is below 50% of the negative control viability.
- The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items).

Results and discussion

In vitro

Results
Irritation parameter:
other: relative mean viability value in %
Value:
106
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
for details see table below
Other effects / acceptance of results:
OTHER EFFECTS:
Assessment of direct test item reduction of MTT: The MTT solution colour did not turn blue/purple, the test item was not presumed to be a MTT reducer. An additional test with freeze-killed tissues was not necessary.
Assessment of Coloured or Staining Materials: The test item was non-coloured and did not dye water or isopropanol, additional tests with viable tissues did not have to be performed.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, absorbance values within the required acceptabiliy criterion of mean OD > 0.8 and < 2.5
- Acceptance criteria met for positive control: yes, treatment with positive control induced a decrease below 50% compared with the negative control value in the relative absorbance
- Comparison with range of historical values: the values of the negative and positive controls were within the range of historical control data of the testing lab (data of 30 studies performed from July 2015 until November 2016)

Any other information on results incl. tables

Table 1: Absorbance values after treatment for 30 minutes

 Dose group  OD, well 1, tissue 1/2
 OD, well 2, tissue 1/2 Mean OD, tissue 1/2  Mean OD*, tissue 1 and 2 Mean OD of 2 tissues* Rel. absorbance, tissue 1 and 2** [%] Absolute value of the difference of the rel. absorbance, tissue 1 and 2 [%]  Mean rel. absorbance [% of neg. control]
 Blank  0.039  0.039  0.039  0.000        
Neg. control   2.110  2.057  2.084  2.045  2.023  101.1  2.2  100.0
   2.027  2.052  2.040  2.001    98.9    
 Pos. control  0.943 0.921   0.932  0.893  0.878  44.2  1.6  43.4
   0.908  0.892  0.900  0.862    42.6    
 Test item  2.243  2.147  2.195  2.156  2.144  106.6  1.2  106.0
   2.197  2.144  2.170  2.132    105.4    

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the study, dibenzylbenzene, ar-methyl derivative, hydrogenated does not possess any eye irritating potential.
Executive summary:

This in vitro study was performed to assess the eye irritation potential of dibenzylbenzene, armethyl derivative, hydrogenated by means of the Human Cornea Model Test according to OECD Guideline No. 492 (adopted 28 July 2015) and in compliance with GLP. The colourless test item did not prove to be an MTT reducer in the MTT pre-test and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues (without MTT addition) to gain a correction factor for calculation of the true viability in the main experiment did not have to be performed. Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues). Irritating effects were not observed following incubation with dibenzylbenzene, ar-methyl derivative, hydrogenated. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (106.0%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, dibenzylbenzene, ar-methyl derivative, hydrogenated does not possess any eye irritating potential.