Benzamide, 3-amino-4-methoxy-N-methyl-

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

other: UN GHS (2003, last rev. 2015)

Principles of method if other than guideline:

Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-amino-4-methoxy-N-methylbenzamide

EC Number:

813-034-3

Cas Number:

878160-14-6

Molecular formula:

C9H12N2O2

IUPAC Name:

3-amino-4-methoxy-N-methylbenzamide

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg (~ 39 mg/cm2 according to guideline)

Duration of treatment / exposure:

60 minutes.

Number of replicates:

3

Test animals

Species:

other: reconstituted human epidermis model

Test system

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to each tissue, wetted with 25 µL DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS (MatTek) were used as negative control per tissue.


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% SLS solution in deionised water (MatTek) were used as positive control per tissue.

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: % relative absorbance

Run / experiment:

1

Value:

93.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

other: % relative absorbance

Run / experiment:

2

Value:

90.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

other: % relative absorbance

Run / experiment:

3

Value:

79

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

other: % relative absorbance

Run / experiment:

mean

Value:

87.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced irrelevantly to 87.8% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Any other information on results incl. tables

Results after treatment with 3-Amino-4-methoxy-N-methylbenzamide and the controls (exposure interval of 60 minutes):

Dose	Tissue	Absorbance	Absorbance	Absorbance	Mean	Mean
Group	No.	570 nm	570 nm	570 nm	Absorbance	Absorbance
		Well 1	Well 2	Well 3	of	of three wells
					3 Wells	blank
	-	-	-	-	-	corrected
Blank	-	0.037	0.038	0.038	0.037	0.000
Negative Control	1	1.873	1.874	1.815	1.854	1.817
Negative Control	2	2.080	2.059	2.045	2.062	2.024
Negative Control	3	1.924	1.847	1.775	1.849	1.811
Positive Control	1	0.100	0.099	0.099	0.099	0.062
Positive Control	2	0.099	0.108	0.109	0.105	0.068
Positive Control	3	0.108	0.110	0.107	0.108	0.071
Test Item	1	1.886	1.763	1.748	1.799	1.762
Test Item	2	1.807	1.733	1.712	1.751	1.713
Test Item	3	1.566	1.516	1.496	1.526	1.488

Dose	Tissue	Mean Absorbance of 3 tissues	Relative	Relative	Mean
Group	No.	after blank	Absorbance [%]	Standard	Relative Absorbance
		correction*	Tissue 1, 2, 3 ***	Deviation	[% of Negative
				[%]	Control] ***
Negative Control	1		96.4
Negative Control	2	1.884	107.4	6.4	100.0
Negative Control	3		96.1
Positive Control	1		3.3
Positive Control	2	0.067	3.6	6.8	3.5
Positive Control	3		3.8
Test Item	1		93.5
Test Item	2	1.654	90.9	8.8	87.8
Test Item	3		79.0

*         Mean of three replicate wells after blank correction

**       relative absorbance per tissue [rounded values]

***     relative absorbance per treatment group [rounded values]

Applicant's summary and conclusion

Interpretation of results:

other: not irritant

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, 3-Amino-4-methoxy-N-methylbenzamide is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of 3-Amino-4-methoxy-N-methylbenzamide by means of the Human Skin Model Test.

The test item passed the MTT- and the Colour Interference pre-tests.

Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissue.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following about 3 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.5% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 9% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced irrelevantly to 87.8% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.