Reaction mass of 1,4-bis(methoxymethyl)benzene, 1,6-dihydroxynaphtalene and Epichlorohydrin

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 June 2019 to 29 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,4-bis(methoxymethyl)benzene, 1,6-dihydroxynaphtalene and Epichlorohydrin

EC Number:

836-437-6

Cas Number:

577978-76-8

Molecular formula:

C40H38O8 to C112H104O20

IUPAC Name:

Reaction mass of 1,4-bis(methoxymethyl)benzene, 1,6-dihydroxynaphtalene and Epichlorohydrin

Test material form:

solid

Details on test material:

Name: CAS 577978-76-8
Chemical Name: Polyglycidyl ether of 1,6-naphthalenediol-pxylyleneglycol (or p-xylyleneglycol dimethylether) condensation polymer
CAS number: CAS 577978-76-8
Batch/Lot number: 0950985
Description: Brown solid
Purity: 100%
Expiry date: 08 February 2020 (as per updated CoA)
Storage conditions: Controlled room temperature (15-25oC, ≤70% relative humidity).
Safety precautions: Enhanced safety precautions above the routine safety precautions (lab coat, gloves, safety glasses, face mask) will be applied considering the supplied safety data sheet to assure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.

Cell source:

other: EPISKINTM(SM) (Manufacturer: SkinEthic, France,

Source strain:

not specified

Details on animal used as source of test system:

Not applicable for In-Vitro tests

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 19-EKIN-026, Expiry Date: 01 July 2019) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are 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 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin corrosivity and irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM(SM) Test Kits used in the present study)

Amount/concentration applied:

In case of the irritation testing, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of test item were applied evenly to each of three test units and each additional control skin units.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

In this assay, two replicates per time point were used for test item (in case of corrosivity part of the test) and three replicates per time point were used for test item (in case of irritation part of the test). Two negative controls and two positive controls were also run in corrosivity testing and three negative controls and three positive controls were also run in irritation testing. As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation in both cases.

Test animals

Species:

other: Not applicable

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Not applicable

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion testing

Value:

96.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Corrosivity testing:
Following exposure with CAS 577978-76-8, the mean cell viability was 96.2% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

The experiment met the validity criteria, therefore the study was considered to be valid.

Any other information on results incl. tables

Colour control for corrosivity testing:

Two additional test item-treated living tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.006, Non Specific Colour % was calculated as 0.7% . This value was below 5%, therefore additional data calculation was not necessary.

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues (Corrosivity test)

Additional control	Optical Density (OD)			NSC% (living)
		Measured	Blank corrected
Treated with CAS 577978-76-8	1	*0.049	*0.002	0.7
	2	0.057	0.010
	Mean	-	0.006

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3. *One sample was used for counting because another sample was excluded from the OD means calculation (negative value)

Corrosivity testing:

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in the table below. The OD values for the test item treated skin samples showed 96.2% relative viability compared to the negative control.

Optical Density (OD) and the calculated relative viability % of the samples (Corrosivity test)

Substance	Optical Density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative Control: Physiological saline (0.9% (w/v) NaCl)	1	0.851	0.804	91.5
	2	1.000	0953	108.5
	Mean	-	0.879	100.0
Positive Control: Glacial acetic acid	1	0.053	0.006	0.7
	2	0.059	0.012	1.4
	Mean	-	0.009	1.0
Test Item CAS 577978-76-8	1	0.915	0.868	98.7
	2	0.870	0.823	93.7
	Mean	-	0.846	96.2

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPISKINTM (SM) model test with CAS 577978-76-8, the results indicate that the test item is not corrosive and not irritant to the skin,

Executive summary:

An in vitro skin corrosivity of CAS 577978-76-8 was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosivity potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1) assay. The corrosivity potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines [1, 2].

Disks of EPISKINTM(SM) were treated with the powdered test item and incubated for 4 hours (two units for corrosivity testing) at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control). Two additional disks were used to provide in each case an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin.

Following exposure with CAS 577978-76-8, the mean cell viability was 96.2% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test with CAS 577978-76-8, the results indicate that the test item is not corrosive to the skin, UN GHS Classification: No Category.