Reaction mass of 2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol and 4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Principles of method if other than guideline:

PRINCIPLE of TEST:
Assessment of ocular irritation potential of the test substance by determination of cytotoxic effects on a human corneal epithelium (HCE) cell model (similar to EpiOcular).

The HCE model is currently involved in the eye irritation validation conducted by COLIPA following ECVAM guidelines. Furthermore, it is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). This model is recognized as the model of choice and scientifically relevant as documented by several publications (e.g. Alepee et. al., Toxicology in Vitro 34 (2016) 55–70).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

Molecular formula:

C27H44N4O2

IUPAC Name:

4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

Constituent 2

Reference substance name:

2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}- 1-methylethyl)phenol

Molecular formula:

C24H37N3O2

IUPAC Name:

2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}- 1-methylethyl)phenol

Constituent 3

Reference substance name:

2-[(Dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol

Molecular formula:

C21H30N2O2

IUPAC Name:

2-[(Dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol

impurity 1

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

impurity 2

Reference substance name:

Xylene

EC Number:

215-535-7

EC Name:

Xylene

Cas Number:

1330-20-7

Molecular formula:

C8H10

IUPAC Name:

Benzene, dimethyl-

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

impurity 4

Reference substance name:

Sulphated ash

IUPAC Name:

Sulphated ash

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): HPP 12879-1

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item was melted in a hot cabinet at 90 °C


FORM AS APPLIED IN THE TEST (if different from that of starting material): melted and cooled down viscous liquid

Test animals / tissue source

Species:

other: human corneal epithelial cells

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

The experiment was carried out on a Human Corneal Epithelial (HCE) Model, which is standardized and commercially available (SkinEthicTM Human Corneal Epithelial Model (HCE), SkinEthic, France). Inserts were of 0.5 cm² size. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 µl per insert

Duration of treatment / exposure:

60 min at room temperature

Duration of post- treatment incubation (in vitro):

16 hours in the incubator (37°C, 5% CO2, maximum humidity)

Number of animals or in vitro replicates:

three

Details on study design:

The irritation potential of the test item is assessed by determination of its cytotoxic effect on an reconstructed human ocular epithelium. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item.
The test item is applied pure. Since the test item is a solution (ca. 40% in ethyl acetate) the solvent is tested in parallel to distinguish potential toxic effects of the test item from the effects of the solvent.
After the post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity) MTT reduction is performed. Cell viability is measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative or positive control substances or the test item.

A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50 %.

Results and discussion

In vitro

Results

Irritation parameter:

other: cell viability (%)

Value:

ca. 77

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
As the test item solidified again after application to the surface of the HCE, it was difficult to properly remove the substance during the subsequent washing procedure. Due to that, part of the tissue could be lost from one of the wells explaining the outlier triplicate value for one well (sample 7). Therefore one mean value is based on duplicate instead of triplicate measurements

Any other information on results incl. tables

Table 1: Tabular Summary of the results

Sample No.	Test item	OD mean*	StdDev	% Viability
1 -3	negative control, PBS	1.09	0.08	100.00
4 -6	positive control, 0.3% SDS	0.22	0.08	20.25
8 -9	HPP 12879 -1	0.84*	0.23	77.05

*6 values/*4 values

Applicant's summary and conclusion

Interpretation of results:

other: negative

Executive summary:

The reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted with the test item. The undiluted test item was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes followed by a 16 hours post-treatment incubation period the cell viability was measured to be about 77% by the MTT conversion assay. Thus the test item is predicted as non-irritant under the conditions of this test method.