4-Amino-N-[3-(diethylamino)propyl]benzamid, hydrochloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-29 to 2017-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 640/2012, L 193, Part B.46. “In vitro Skin Irritation: Reconstructed Human Epidermis Test Method” 06-Jul-2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals No. 439: In Vitro Skin Irritation: Reconstructed human Epidermis Test Method, 28 July 2015

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: L’Oréal Standard Operating Procedure: “EpiSkin™ Skin Irritation Test Method“; - ECVAM Skin Irritation Validation Study - Validation of the EpiSkin™ Test Method15 min - 42 hours for the Prediction of Acute Skin Irritation of Chemicals.

Version / remarks:

Version 1.8, Feb-2009

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ECVAM DB-ALM Protocol No. 131 “EpiSkin™ Skin Irritation Test15 min – 42 hours. 09-Jun-2012

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-N-[3-(diethylamino)propyl]benzamide hydrochloride

EC Number:

814-672-5

Cas Number:

109342-99-6

Molecular formula:

C14H24N3OCl

IUPAC Name:

4-amino-N-[3-(diethylamino)propyl]benzamide hydrochloride

Test material form:

solid

Specific details on test material used for the study:

Name: 4-Amino-N-[3-(diethylamino)propyl]benzamid, hydrochloride
CAS No.: 109342-99-6
Batch No.: Betriebsprobe 3a + 3b
Molecular Weight: 285.82 g/mol
Physical State: paste
Colour: brown
Density: 1.1679 g/mL
Purity: 96.0%
Expiry Date: 23 May 2019
Storage Conditions: room temperature
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

In vitro test system

Test system:

human skin model

Source species:

human

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Details on test system:

Acute irritation is a local, reversible inflammatory response of normal living skin to direct injury caused by the application of an irritant substance for up to 4 hours. The potential to induce skin irritation is an important consideration included in procedures for the safe handling, packing and transports of chemicals.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

1. Negative control 10 µL Dulbecco's phosphate buffered saline
2. Positive control 10 µL 5% sodium dodecyl sulfate solution
3. Test Item 12 ± 2 mg
The test was performed on a total of 3 tissues per dose group.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 +/- 1 h

Number of replicates:

3

Test system

Details on study design:

The test was performed on EpiSkin, an organotypic reconstructed three-dimensional model of the human epidermis. 3 replicate tissues are dosed with the test item, the negative control (10µL DPBS) and the positive control (10µL 5% SDS), respectively. After 15 minutes treatment period at room temperature the test item and the controls are rinsed off with DPBS and the tissues are post-incubated for 42 +/- 1 h. Then the tissues are stained via MTT for 3 hours. The extracts are measured photometrically at 570 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

109

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Pre-Experiments

The mixture of 10 mg test item per 2 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 mixtures of 10 mg of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC_living equalled 0%.

 Result of the Test Item 4-Amino-N-[3-(diethylamino)propyl]benzamid, hydrochloride

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	0.720	0.700	0.619	0.080	0.068	0.074	0.684	0.798	0.734
	0.751	0.701	0.604	0.088	0.101	0.073	0.754	0.857	0.615
OD570 (Blank Corrected)	0.677	0.657	0.576	0.037	0.025	0.031	0.641	0.755	0.691
	0.708	0.658	0.561	0.045	0.058	0.030	0.711	0.814	0.572
Mean OD570 Of The Duplicates (Blank Corrected)	0.693	0.657	0.569	0.041	0.042	0.030	0.676	0.785	0.631
Total Mean OD570 Of 3 Replicate Tissues (Blank Corrected)	0.640*			0.038			0.697
SD OD570	0.064			0.006			0.079
Relative Tissue Viabilities [%]	108.3	102.8	88.9	6.3	6.5	4.8	105.7	122.7	98.7
Mean Relative Tissue Viability [%]	100.0			5.9**			109.0
SD Tissue Viability [%]^***	10.0			1.0			12.3
CV [% Viability]	10.0			16.6			11.3

^*              Corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

^**             Mean relative tissue viability of the three positive control tissues is ≤ 40%.

^***            Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: OECD 439

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 15 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study the skin irritant potential of 4-Amino-N-[3-(diethylamino)propyl]benzamid, hydrochloride was analysed. The EPISKIN-Standard Model™ (EPISKIN-SM^TM), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. 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 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (109.0%) after 15 min treatment and 42 h post-incubation.