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

Endpoint summary

Description of key information

HPP 12879-1 is not corrosive but irritant to the skin in tests using reconstructed human epidermis (Wingenroth, 2016a+b). An in vitro test with HPP 12879-1 on a human corneal epithelium (HCE) cell model does not reveal irritant effects to the eyes (Wingenroth, 2016c).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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): liquid

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test system

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Tissue batch number(s): Cat.-No.CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 3 min- exposure and incubator (37 ± 2° C) for 60 min- exposure

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of 3 inserts was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- classification according to UN GHS: if the mean percent tissue viability after 3 min-exposure is less than (<) 50 % the test item is classified as corrosive (Category 1A); if the mean percent tissue viability after 3-min-exposure is greater than or equal (≥) to 50 % AND < 15% after 60-min exposure the test item is classified as corrosive (Category 1B/1C); if the mean percent tissue viability after 60-min-exposure is ≥ 50 % after 3-min exposure AND ≥ 15% after 60-min exposure the test item is considered as non-corrosive

The corrosive potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item.
All tests were performed in triplets for each time point. The test item was applied undiluted , i.e. 50 µl of the melted and cooled down test item per insert for 3 min. (room temperature) and 60 min. Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density of the negative control).

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 μl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 0.9% NaCl in water

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure period: 3 min.

Value:

ca. 104.58

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not specified

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure period: 60 min.

Value:

ca. 71.42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not specified

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes, within regular intervals in the lab
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Tabular Summary of the results

Sample No.	Test item	Expousre time [min]	OD mean*	StdDev	% Viability
1 -3	negative control, 0.9% NaCl	60	2.34	0.38	100
7 -9	HPP 12879 -1	60	1.67	0.22	71.42
10 -12	negative control, 0.9% NaCl	3	2.21	0.39	100
16 -18	HPP 12879 -1	3	2.31	0.20	104.58

*6 values

Interpretation of results:

other: negative

Executive summary:

A study was performed for the assessment of the skin corrosion of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 431 and EU Test Method B.40 bis. The mean value of cell viability was recorded to be 105 % (3 min. exposure) and 71% (60 min. exposure). The test item was thus shown to be not corrosive to reconstructed human skin in vitro.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September 2014

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)

GLP compliance:

yes (incl. QA statement)

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): viscous liquid (melted and cooled down test item)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test system

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Tissue batch number(s): Cat.-No.CS-100

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) values obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- According to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 μl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μl
- Concentration (if solution): 0.9% NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% SDS in physiological saline

Duration of treatment / exposure:

20 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

three

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

OTHER EFFECTS:

- Direct-MTT reduction: In the control on freeze killed tissue the test item did not lead to significant OD values. This demonstrated that no direct MTT reduction is occuring.

Table 1: Tabular Summary of the results

Sample No.	Test item	OD mean*	StdDev	% Viability
1 -3	negative control, 0.9% NaCl	2.03	0.15	100
7 -9	positive control, 5% SDS	0.03	0.01	1.66
10 -12	HPP 12879 -1	0.04	0.01	1.88

freezed killed tissue	Test item	OD mean*	StdDev
4 -6	negative control, 0.9% NaCl	0.03	0.00
13 -15	HPP 12879 -1	0.02	0.00

*6 values

Interpretation of results:

other: positive

Executive summary:

A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The mean value of cell viability was recorded to be 2 %. The test item was thus shown to be irritating to reconstructed human skin in vitro.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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

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.

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 %.

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

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

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.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was performed for the assessment of the skin corrosion of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 431 and EU Test Method B.40 bis. The mean value of cell viability was recorded to be 105 % (3 min. exposure) and 71% (60 min. exposure). The test item was thus shown to be not corrosive to reconstructed human skin in vitro (Wingenroth, 2016a).

A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The mean value of cell viability was recorded to be 2 %. The test item was thus shown to be irritating to reconstructed human skin in vitro (Wingenroth, 2016b).

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 (Wingenroth, 2016c).

.

Justification for classification or non-classification

Based on the available study results a classification with Skin Irritation Cat. 2 (H315: Causes skin irritation) according to Regulation (EC) No. 1272/2008 (CLP) is required.