Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation/ corrosion:

The skin irritation potential of dibenzylbenzene, ar-methyl derivative, hydrogenated was examined in an in vitro test with the EPISKIN TM reconstructed human epidermis model. The test was performed according to the OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 28 July 2015) and the procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study. Exposure to the test substance lasted 15 minutes and was followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in the human epidermal cultures following topical exposure to the test item, with the MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The optical density was measured at 562 nm and MTT reduction in the test item treated tissues relative to negative control tissues was expressed as percentage viability. The relative mean viability of the test item treated tissues was 106.6% after the 15 -minute exposure period and hence, dibenzylbenzene, ar-methyl derivative, hydrogenated was classified as non-irritant.

Eye irritation:

The eye irritation potential of dibenzylbenzene, armethyl derivative, hydrogenated was assessed by means of the Human Cornea Model Test according to OECD Guideline No. 492 (adopted 28 July 2015) and in compliance with GLP. 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. 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 under the experimental conditions reported, dibenzylbenzene, ar-methyl derivative, hydrogenated does not possess any eye irritating potential.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate Good Laboratory Practice: The Department of Health of the Government ot the United Kingdom, Statement of compliance in accordance with Directive 2004/9/EC, date of inspection 05/07/2016
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 (unlimited shelf life)
- Storage condition of test material: room temperature in the dark
- Purity: 100%
Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, purchased from SkinEthic Laboratories (Lyon, France). The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes 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 a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Tissue batch number: 16-EKIN-040
- Production date: no details mentioned
- Delivery date: 04 October 2016
- Date of initiation of testing: 05 October 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
- Observable damage in the tissue due to washing: nothing mentioned
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: measured without reference filter
- Linear OD range of spectrophotometer: not mentioned

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- Classification of irritation potential is based upon relative mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period according to:
Relative mean tissue viability is <= 50%: irritant
Relative mean tissue viability is > 50%: non-irritant
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 μL
- Concentration: undiluted, used as supplied

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Details on study design:
Test for direct MTT reduction by the test material: 10 µL test material + 2 ml 0.3 mg/ml MTT solution incubated for 3 h at 37°C in the dark. Untreated MTT solution was used as control. If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

Assessment of Color Interference with the MTT endpoint: 10 μL of test item + 90 μL of sterile water, mixing for 15 minutes on a plate shaker, visual assessment of the color was made. A test item may interfere with the MTT endpoint if it is colored.

MAIN TEST
DAY 1-Application of test item and rinsing
Exposure: 15 min
Amount applied: 10 µl
Negative control: 10 µl DPBS
Positive control: 10 µl 5% w/v SDS
Washing: DPBS with Ca++ and Mg++ for 40 seconds
Post-exposure incubation: 42 h

DAY 3-MTT Loading/Formazan Extraction
Plate shaker: 15 min
MTT solution: 2 ml of 0.3 mg/ml per well
Incubation: 3 h
Biopsy of the epidermis was performed. The tissue was then transfered in micro tubes with 500 µl acidified isopropanol and refrigerated at 1-10°C till day 6.

DAY 6-Absorbance/Optical Density at 562 nm with Anthos 2001 microplate reader

DATA EVALUATION
Relative mean viability (%): (mean OD562 test material / mean OD562 negative control) *100

ACCEPTABILITY
Positive control: the relative mean tissue viability should be ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability should be ≤18%.
Negative Control: the mean OD562 negative control should be ≥0.6, and the SD value of the percentage viability ≤18%.
Test Item: the SD calculated from individual percentage tissue viabilities of the three identically treated tissues should be ≤18%.

Irritation / corrosion parameter:
% tissue viability
Value:
106.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. Quantitative correction of results was not necessary and a parallel determination of skin irritation potential on viable and waterkilled tissues was not performed.
- Colour interference with MTT: The solution containing the test item was a very pale green color. This color was attributed to the intrinsic color of the test item itself, it was therefore unnecessary to run color correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean OD562 for the negative control treated tissues was 0.782 and the standard deviation value of the viability was 12.7%
- Acceptance criteria met for positive control: yes, relative mean tissue viability for the positive control treated tissues was 7.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.8%
- Acceptance criteria met for variability between replicate measurements: yes, standard deviation calculated from individual tissue viabilities of test item treated tissues was 12.0%

Table 1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item 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

0.884

0.782

0.099

113.1

100*

12.7

0.775

99.1

0.686

87.8

Positive

Control Item

0.068

0.062

0.006

8.7

7.9

0.8

0.062

7.9

0.056

7.2

Test Item

0.904

0.833

0.094

115.7

106.6

12.0

0.869

111.2

0.727

93.0

 * = the mean viability of the negative control tissues is set as 100%

Interpretation of results:
GHS criteria not met
Conclusions:
The relative mean viability of the tissues exposed to the test material was 106.6%, and hence, the test material is not a skin irritant in this test.
Executive summary:

The skin irritation potential of dibenzylbenzene, ar-methyl derivative, hydrogenated was examined in this in vitro test with the EPISKIN TM reconstructed human epidermis model. The test was performed according to the OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 28 July 2015) and the procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study. Exposure to the test substance lasted 15 minutes and was followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in the human epidermal cultures following topical exposure to the test item, with the MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The optical density was measured at 562 nm and MTT reduction in the test item treated tissues relative to negative control tissues was expressed as percentage viability. The relative mean viability of the test item treated tissues was 106.6% after the 15 -minute exposure period and hence, dibenzylbenzene, ar-methyl derivative, hydrogenated was classified as non-irritant. The study is considered acceptable based on the quality criteria.

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:
September - October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
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
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%
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")
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).
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)

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    
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.

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

Additional information

Justification for classification or non-classification

Based on the available information on skin irritation/corrosion and eye irritation, Dibenzylbenzene, ar-methyl derivative, hydrogenated is neither irritating to the skin nor to the eyes and does not require classification according Regulation (EC) No 1272/2008.