Reaction mass of meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene-2 yl)phenoxy]methyl}oxirane and 2RS)-2-({4-[2-(4-{[(2RS)-oxiran-2-yl]methoxy}phenyl)-1,1-dichloroethylidene-2-yl]phenoxy}methyl)oxirane

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene -2- yl)phenoxy] methyl} oxirane

Molecular formula:

C20 H18 Cl2 O4

IUPAC Name:

meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene -2- yl)phenoxy] methyl} oxirane

Test material form:

solid

Details on test material:

Batch GRM193K01
Manufactured 19.07.2019
exp date : 03.09.2021
Density 1.32 g/cm3 at 23°C

Specific details on test material used for the study:

Name: Reaction mass of
meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene-2-yl)phenoxy]methyl}oxirane
and
(2RS)-2-({4-2-(4-{[(2RS)-oxiran-2-yl]methoxy}phenyl)-1,1-dichloroethylidene-2-yl]phenoxy}methyl)oxirane
Alternative Name: Bisphenol C Epoxy
CAS: 69415-01-6
Batch No.: GRM 193K01
Aggregate State at RT: viscous liquid
Colour: yellow
Storage Conditions: room temperature
Stability: stable at recommended storage conditions
Expiry Date: 03 September 2021

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

The test will be carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Controls
Controls will be set up in parallel to the test item cultures in order to confirm the validity of the test.
Negative Control
Distilled water (Aqua dest.)
Positive Control
8 N Potassium Hydroxide (KOH; CAS No.: 1310-58-3)

Dose Groups
1. Negative control 50 µL distilled water
2. Positive control 50 µL 8 N KOH
3. Test Item 50 µL (undiluted)

Duration of treatment / exposure:

3 minute and 60 minute experiment

Duration of post-treatment incubation (if applicable):

3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution will be aspirated. The wells will be refilled with PBS and the PBS will be aspirated. The rinsing will be repeated twice and the tissues will be dried. Then the inserts will be transferred into 12-well “extraction plates“. 2 mL of isopropanol will be pipetted into each insert, thus the insert will be covered from both sides. The extraction plates will be sealed in zip-bags to inhibit isopropanol evaporation. Extraction will be carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature.
After the extraction period the inserts will be pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts will be discarded and the extraction plates will be placed on a shaker for 15 min.
Per each tissue 3 x 200 µL aliquots of the extract will be transferred into a 96-well plate and OD will be measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

Number of replicates:

The test will be performed on a total of 4 tissues per dose group, 2 replicates for each treatment period (3 min and 60 min exposure time).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean at 3 min

Value:

99.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean at 60 min

Value:

92.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The potential of the test item to induce skin corrosion was analysed by using the three-dimensional human skin model EpiDerm, comprising a reconstructed epidermis with a functional stratum corneum.
In the present study Reaction mass of meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene-2-yl)phenoxy]methyl}oxirane and (2RS)-2-({4-2-(4-{[(2RS)-oxiran-2-yl]methoxy}phenyl)-1,1-dichloroethylidene-2-yl]phenoxy}methyl)oxirane was applied topically to the EpiDerm tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay.
Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues.
The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
The mixture of 25 mg test item per 300 µL Aqua dest. and per 300 µL isopropanol showed no colouring as compared to the solvent. Therefore, NSC equalled 0%.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.
The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was  50% (99.4%) after 3 min treatment and  15% (92.2%) after 60 min treatment.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no corrosive effects. The test item is classified as “non-corrosive“.

Executive summary:

In the present study the skin corrosivity potential of Reaction mass of meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene-2-yl)phenoxy]methyl}oxirane and (2RS)-2-({4-2-(4-{[(2RS)-oxiran-2-yl]methoxy}phenyl)-1,1-dichloroethylidene-2-yl]phenoxy}methyl)oxirane was analysed. Since corrosive chemicals are cytotoxic after a short time exposure to the stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDermÔ, a reconstituted three-dimensional human epidermis model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min and 60 min exposure period and compared to those of the concurrent negative controls.

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple.Therefore, NSMTT equalled 0%.

The mixture of 25 mg test item per 300 µL Aqua dest.and per 300 µL isopropanolshowed no colouring as compared to the solvent.Therefore, NSC equalled 0%.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was³ 50% (99.4%) after 3 min treatment and³ 15% (92.2%) after 60 min treatment.

In this study under the given conditions the test item showed no corrosive effects. The test item is classified as “non-corrosive“.