4-[3-(1-naphthylamino)propyl]morpholine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study performed in accordance with GLP requirements (but not a GLP study).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

This is a screening non-GLP study. The current protocol is a streamlined version of OECD TG 439 test guideline.

Deviations:

no

GLP compliance:

no

Remarks:

The study was performed in accordance with GLP but was not reported under GLP due to lack of a GLP test lab for this assay

Test material

Test material information

Constituent 1

Reference substance name:

4-[3-(1-naphthylamino)propyl]morpholine

EC Number:

226-033-2

EC Name:

4-[3-(1-naphthylamino)propyl]morpholine

Cas Number:

5235-82-5

Molecular formula:

C17H22N2O

IUPAC Name:

N-[3-(morpholin-4-yl)propyl]naphthalen-1-amine

Test material form:

solid: granular

Details on test material:

Colour: Dark Brown to Black
GLP Characterized: January 21, 2016

Specific details on test material used for the study:

Test Material Name: 4-(3-(1-naphthylamino)propyl)morpholine
Chemical Name: N-1-Naphthalenyl-4-morpholinepropanamine
Lot/Reference/Batch Number: ZA01212016

Purity/Characterization (Method of Analysis and Reference):
The purity of the test material was determined to be 94.4% by liquid chromatography with identification by nuclear magnetic resonance spectroscopy and liquid chromatography mass spectrometry (Ferrer, 2016a).

Test Material Stability Under Storage Conditions:
4-(3-(1-Naphthylamino)propyl)morpholine, lot ZA01212016, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under U.S. EPA OPPTS Guideline 830.6313 (Ferrer, 2016b).

In vitro test system

Test system:

human skin model

Source species:

human

Cell source:

other: human-derived epidermal keratinocytes (NHEK)

Justification for test system used:

The EpiDerm three-dimensional human skin model has been extensively characterized (EC-ECVAM, 2009) and is approved (OECD TG 439, 2013) for identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/R38 or No label).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM:
The potential for chemically induced skin irritation and corrosion are important considerations in establishing procedures for the safe handling, packing, and transport of chemicals (UN, 2007). The EpiDerm three-dimensional human skin model has been extensively characterized (EC-ECVAM, 2009) and is approved (OECD TG 439, 2013) for identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/R38 or No label). The EpiDerm model uses human-derived epidermal keratinocytes (NHEK) as the cell source, which are cultured to form a multi-layered, highly differentiated model that closely mimics human epidermis at biochemical and physiological levels.
The test is based on the principle that chemicals with irritant potential can cause a cytotoxic response to the stratum cornea and the rate of cytotoxicity is proportional to irritation potency. In the assay, the EpiDerm tissue model was incubated with the test chemical for 60 minutes, followed by 42-hour incubation (recovery) under standard cell culture conditions. Following the post-treatment incubation period, cell viability was assessed using the MTT (3-[4,5- dimethylthiazol-2-yl] -2,5 –diphenyltetrazolium bromide) assay (Mosmann, 1983). Relative cell viability was calculated for each tissue as % of the mean of the negative control-treated tissues. A test chemical was interpreted as a potential skin irritant (UN GHS Cat 1/2) or non-irritant (UN GHS Cat NC), when the cell viability was ≤ or > 50%, respectively (OECD TG 439, 2013).

STUDY DESIGN:
The EpiDerm System (EPI-200) consists of normal, human-derived epidermal kerotinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues are cultured on polycarbonate membranes of cell culture inserts (MILLICELLs, 10 mm diameter, 0.6 cm² surface) and shipped as kit, containing 24 tissues mounted on agarose.

Supplier and Location:
MatTek Corporation; Ashland, MA.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Preparation of the Treatment Solution:
The chemical was tested at a concentration of 100% (neat/as provided), following supplier’s protocol (EpiDerm SOP) and OECD 439 test guideline. Thirty microliter of undiluted 431-NAPM was directly dispensed atop the tissue.

Route of Administration:
The test chemical was administered by topical application to the surface of the EpiDerm tissues.

Experimental Procedure:
EpiDerm tissue cultures were used within 24 to 48 hours of receipt from the supplier. Upon receipt, the EpiDerm tissues were stored at 2-8ºC until used. On the day prior to testing, EpiDerm assay medium was warmed to room temperature and an aliquot of 0.9 mL was dispensed into each well of a 6-well plate. The EpiDerm tissues were transferred from a 24-well shipping plate to a 6-well plate containing the fresh, pre-warmed assay medium. Prior to transferring, each EpiDerm tissue was inspected for air bubbles between the agarose gel and MILLICELL insert. Tissues with air bubbles greater than 50% of the MILLICELL area were discarded. Any unused tissues were stored in an air tight bag (filled with 5% CO2/95% air) and stored at 2-8ºC for subsequent use. The EpiDerm tissues were incubated at approximately 37ºC in a humidified atmosphere of approximately 5% CO2 for 60 ± 5 min. At the end of the first pre-incubation period, the inserts were transferred into wells containing fresh warm assay medium and were incubated overnight (18 ± 3 hrs) to acclimate the tissue.
On the day of treatment, the EpiDerm tissues were exposed to the test chemical as described above (30 μL of test material or controls). After dosing, the tissue plates were transferred to a cell culture incubator and were incubated for 35 ± 1 minutes at standard culture conditions. Following incubation, the plates were transferred to the laminar flow hood and incubated at room temperature for 25 minutes. Following the total of 60 ± 1 minutes treatment period, the tissues were rinsed with sterile DPBS to remove test substance. The tissues were then transferred to new 6-well plates containing 0.9 mL/well fresh pre-warmed assay medium and incubated at standard culture conditions for an initial post-treatment incubation of 24 ± 2 hours. After the initial post-treatment incubation, the inserts were transferred into new 6-well plates pre-filled with 0.9 mL fresh pre-warmed assay medium and incubated for additional 18±2 hours (total post-treatment incubation period of 42 ± 2 hours).
Following post-treatment incubation, tissue inserts were evaluated for cell viability using the MTT assay. Each tissue insert was transferred to a well containing 300 μL MTT solution in a 24-well plate and tissues were incubated for 3 ± 0.1 hr at standard cell culture conditions. After incubation the tissues were washed with DPBS and formazan was extracted in 2 mL extractant solution with 2 hours shaking at room temperature. The amounts of extracted formazan from each tissue was measured spectrophotometrically at 570 nm (OD570) using a Microplate Reader (Molecular Devices).

Duration of treatment / exposure:

60 ± 1 minute treatment period

Duration of post-treatment incubation (if applicable):

Following the total of 60 ± 1 minutes treatment period, the tissues were rinsed with sterile DPBS to remove test substance. The tissues were then transferred to new 6-well plates containing 0.9 mL/well fresh pre-warmed assay medium and incubated at standard culture conditions for an initial post-treatment incubation of 24 ± 2 hours. After the initial post-treatment incubation, the inserts were transferred into new 6-well plates pre-filled with 0.9 mL fresh pre-warmed assay medium and incubated for additional 18±2 hours (total post-treatment incubation period of 42 ± 2 hours).

Number of replicates:

Three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: Mean Viability %

Run / experiment:

Mean Viability % for 3 replicates

Value:

105.5

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Assessment of Direct Test Material Reduction of MTT:
As some test chemicals may interfere with MTT assay readout by directly reducing MTT dye (turn MTT solution to blue/purple formazan in the absence of viable cells), 1-phenyl was evaluated for this property. In this experiment 431-NAPM (30 μL) was incubated with 1 mL of the MTT reagent in the dark at standard cell culture conditions (37°C) for 60 min. Untreated (no test material) MTT medium was used as the negative control. Results from this experiment suggested no direct reduction of MTT dye by 431-NAPM, as the test material did not turn MTT solution to blue/purple colour.

ACCEPTANCE CRITERIA:
The results for negative and positive controls met assay acceptance criteria, suggesting appropriate conduct of the study.
1) The corrected mean OD570 value of the negative control tissues (exposed for 60 minutes) was 2.611 (i.e. ≥ 1.00; criteria set by the tissue manufacturer).
2) The relative mean viability of positive control (5% SDS) was 2.7% (i.e. < 50% compared to negative control).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative tissue viability for EpiDerm tissues treated with 431-NAPM and positive control (5% SDS) were 105.5% and 2.7%, respectively. According to the EpiDerm skin irritancy prediction model, a test substance is considered irritant to skin if the mean tissue viability is ≤ 50%. Therefore, based on the present EpiDerm study results, 431-NAPM was interpreted to be a non- irritant to skin.

Executive summary:

4-(3-(1-naphthylamino)propyl)morpholine was evaluated for skin irritation potential in an in vitro EpiDerm skin irritation assay (MatTek Corporation; Ashland, MA). The EpiDerm tissue consists of normal, human-derived epidermal keratinocytes (NHEK) that are cultured to form a multilayered, differentiated model of human epidermis. In this assay, 4-(3-(1- naphthylamino)propyl)morpholine was topically applied to the EpiDerm tissue for 60- minutes, followed by a 42-hour post-exposure recovery. Following recovery, the cell viability was measured in treated and control tissues using MTT (3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide) assay and the data reported as a percentage of the mean of negative control. A test chemical was considered to possess skin irritation potential (UN GHS Cat 1/2) if the relative cell viability was less than or equal to 50%. In this study, Dulbecco Phosphate Buffered Saline (DPBS) and 5% SDS served as the negative and positive controls, respectively.

The mean relative cell viability of 4-(3-(1-naphthylamino)propyl)morpholine and positive control-treated tissues were 105.5% and 2.7% (i.e. ≤ 50%), respectively. Therefore, 4-(3-(1- naphthylamino)propyl)morpholine was interpreted as a non-irritant (UN GHS Cat NC) to skin in the EpiDerm irritation assay.