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Administrative data

Description of key information

In vitro skin corrosion (OECD 431): Negative
In vitro skin irritation (OECD 439): Negative
In vitro eye irritation (OECD 437): Negative

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
Study period:
Start of experimental phase: 29 June 2015 and End of experimental phase: 31 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: Commercial reconstructed human epidermis (RhE) model named EPISKIN™.
Strain:
other: Not applicable
Details on test animals or test system and environmental conditions:
- Supplier: SkinEthic Laboratories (4, A. Fleming – 69007 Lyon – France).
- Batch number: 15-EKIN-034 (alive tissue)
- Arrived at RTC: on 25 August 2015

Functional controls:
- Quality controls: histology scoring, magnitude of viability and barrier function (IC50 determination).
- Biological safety: absence of HIV1 and 2, Hepatitis B and C antigens, absence of bacteria, fungi and mycoplasma.
Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Remarks:
The epidermis surfaces were moistened with 10 microL. sterile water before application of the test item.
Controls:
other: Positive control (SDS: Sodium Dodecyl Sulphate) diluted at the final concentration of 5% (v/v) in sterile water for injection and negative control is D-PBS.
Amount / concentration applied:
20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2)
Duration of treatment / exposure:
- An exposure time of 15 +/- 0.5 minutes was allowed in a ventilated cabinet at room temperature for the test item.
- Negative and postive control treatments were carried out staggering samples of approximately 1 minute +/- 0.5 minute, while test item treatments were performed staggering samples of approximately 2 minutes +/- 0.5 minute.
Observation period:
- A 42 1 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.
Number of animals:
Not applicable
Details on study design:
TEST SITE
- % coverage:20 mg/epidermis unit, each measuring 0.38 cm2
- Replicate: 3 replicates for positive,3 for negative control and 3 for the test item.

REMOVAL OF TEST SUBSTANCE
At the end of the exposure, tissue samples were rinsed with approximately 25 mL of sterile D-PBS filling and empting the tissue insert; only one treated sample was rinsed twice and the test item was mechanically removed by using sterile tweezers.
The excess liquid was carefully removed and the samples transferred into new wells pre-filled with 2 mL/well of maintenance medium.

SCORING SYSTEM:
Irritation / corrosion parameter:
other: other: Mean cell viability
Value:
93.5
Remarks on result:
other:
Remarks:
Time point: 15 minutes exposure and 42 hours recovery period. Max. score: 100.0. Remarks: Mean cell viability in %. Acceptable intra-replicate variability was obtained (SD of % viability = 15.4% lower than 18.). (migrated information)
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study, using a commercial reconstructed
human epidermis (RhE) model named EPISKIN™. The negative and positive controls gave the expected results and the study was accepted as valid.
The mean cell viability of the test item treated tissues was 93.5%. Based on the results obtained, the test item is classified as not irritant to the skin.
Executive summary:

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 +/- 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied

by the Sponsor.

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se; a colourless suspension, with yellow precipitate, was observed at the end of the incubation period, indicating that the test item could not interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se; a colourless suspension, without precipitate but with the presence of two different phases, was observed, which indicates that the test item does not have a colouring ability. Based on these results, no additional controls were

added in the Main Assay.

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2). Positive and negative controls [a 5% (v/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 μL/epidermis unit.

The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability. The positive control caused the expected cell death (6.2% of cell viability when compared to the negative control) and variability (SD of % viability

equal to 4.3). Based on the stated criteria (mean viability <= 40% and SD of % viability <=1 8), the assay was regarded as valid.

Only the OD-blank background subtraction was performed for the evaluation of irritant properties of the test item. The test item did not induce cell death in any replicate with a mean cell viability of 93.5%, when compared to the negative control.

Based on the results obtained, the test item is classified as not irritant to the skin.

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:
Experimental starting date: 19 August 2015 and Experimental completion date: 19 August 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer. This can be seen as uncritical, because the opacity can be calculated from the absorbances. Deviation was signed and assessed by the S.D. on 20 August 2015
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer. This can be seen as uncritical, because the opacity can be calculated from the absorbances. Deviation was signed and assessed by the S.D. on 20 August 2015
GLP compliance:
yes (incl. QA statement)
Species:
other: Bos primigenius Taurus (fresh bovine cornea)
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 hour.
Vehicle:
unchanged (no vehicle)
Remarks:
The test item is a solid substance. It was tested directly, without dilution or preparation of a solution.
Controls:
yes
Amount / concentration applied:
Tissue 1: 469.7 mg of the test item
Tissue 2: 394,3 mg of the test item
Tissue 3: 455.7 mg of the test item
Duration of treatment / exposure:
4 hours
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Details on study design:
Conduct of the study:
Preparations:
After having carefully cleaned and sterilized the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM without phenol red was filled. The holders were then incubated for 1 h in the incubation chamber at 32 ± 1 °C.

Method Description
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.
The baseline opacity was measured by placing the holder with the cornea in a spectral photometer and recording the absorbance at 570 nm.
For each treatment group (negative control solution, test item and positive control solution), three replicates were used. After removal of the pre-incubation medium, 750 μL negative control solution resp. positive control solution were applied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:

Open Chamber Method
The “open chamber-method” is used for solids. In order to apply the test item, the nut was unscrewed to remove the glass disc. The test item could be applied directly on the cornea now.

The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item.
Exposition time on the corneas was 4 h at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded at once (again by measurement at 570 nm). The cMEM without phenol red was then removed from the front chamber, and 1 mL sodium fluorescein solution (concentration:
5 mg/mL) was added to the front chamber.
The chambers were then closed again and incubated for 90 min at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid at 490 nm.

SCORING SYSTEM: Classification scheme.

IVIS<=3: No cetgory
IVIS >3 and <=55: No prediction can be made
IVIS>55: Eye damage category 1
Irritation parameter:
in vitro irritation score
Value:
< 3
Remarks on result:
no indication of irritation

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

 TEST GROUP IVIS  MEAN IVIS  Relative standard deviation IVIS 
   1.84    
 Negative control 0.9% NaCl  1.51  1.43  31.6%
   0.95    
   -0.20    
 Test item  -0.92  -0.61  60.3%
   -0.70    
   73.79    
 Positive control imidazole 20%  56.25  62.88  15.1%
   58.60    

VALIDITY:

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean. The negative control has to show an IVIS ≤ 3.

The validity criteria and findings are given in the following table:

 Parameter  Criterion  Found  Assessment
 IVIS of negative control 0.9% NaCl  <=3  1.43  OK
 IVIS of positive control 20% imidazole solution  33.19 -135.95 62.88   OK

Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

COMPARISON WITH HISTORICAL DATA:

In the following table, the means of the negative control and positive control of all experiments which were performed with this positive control up to 16. Jun. 2015 are stated and compared with the values which were found in this study.

 Parameter  IVIS negative control  IVIS positive control
 Substance  0.9% sodium chloride solution  20% imidazole solution
 Mean  1.18 84.57 
 Standard deviation  0.93 25.69 
 Range (min-max)  0.13 -6.86 32.62 -161.29 
 Range (validity)  <=3 33.19 -135.95 
 Study 15061202G850  1.43 62.88 
Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
Under the conditions of this study, the test item 2,5-bis[(glycidyloxy)phenyl]octahydro-4,7- methano-5h-indene (TK30250) showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.61.
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
Executive summary:

This in vitro study was performed to assess corneal damage potential of 2,5- bis[(glycidyloxy)phenyl]octahydro-4,7-methano-5h-indene (TK30250) by quantitative measurements of changes in opacity and permeability in a bovine cornea.

The test item 2,5-bis[(glycidyloxy)phenyl]octahydro-4,7-methano-5h-indene (TK30250) was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 h and whose opacity had been determined.

The test item was incubated on the cornea for 4 h at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

The test item was tested pure.

Under the conditions of this test, the test item 2,5-bis[(glycidyloxy)phenyl]octahydro-4,7- methano-5h-indene (TK30250) showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.61.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

The negative control (physiological sodium chloride solution) and the positive control (20% imidazole solution) have met the validity criteria.

Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 1.43.

20% Imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 62.88.

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Two in vitro experiment on the skin were performed (OECD 431 and OECD 439).

TK 30250 was classified as non-corrosive to the skin for the OECD 431. 

TK 30250 was classified as non-irritant to the skin for the OECD 439.

In the BCOP experiment (OECD 437) performed with TK 30250, the conclusion was:

Under the conditions of this study, the test showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is -0.61.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.


Justification for selection of skin irritation / corrosion endpoint:
Results from both in vitro studies (in vitro skin corrosion (OECD 431) and in vitro skin irritation (OECD 439)) are negative. Both study are taken into consideration.

Justification for selection of eye irritation endpoint:
Only this study is available and study Klimisch 1.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No.1272/2008) and DSD (Directive 67/548/EEC).