1-Piperazineacetamide, N-(2,6-dimethylphenyl)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2014 - 29 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 430. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-(2,6-dimethylphenyl)-2-(piperazin-1-yl)acetamide

EC Number:

610-916-8

Cas Number:

5294-61-1

Molecular formula:

C14H21N3O

IUPAC Name:

N-(2,6-dimethylphenyl)-2-(piperazin-1-yl)acetamide

Details on test material:

- Name of test material (as cited in study report): N-(2,6-dimethylphenyl)-1-piperazineacetamide
- Physical state: white solid
- Analytical purity: 99.8%
- Lot/batch No.: M13856C
- Expiration date of the lot/batch: December 2015
- Storage condition of test material: room temperature, i.e. 15-30ºC.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre for Experimental Medicine at the Medical University (Katowice)
- Age at study initiation: 23 days old
- Housing: in a plastic cage covered with a wire bar lid. Dimensions: 58 x 37 x 21 cm.
- Diet (e.g. ad libitum): ad libitum, "Murigran" standard granulated laboratory fodder.
- Water (e.g. ad libitum): ad libitum, drinking tap water.
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21 ºC
- Humidity (%): 44-63%
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 h light/ 12 h dark

Test system

Type of coverage:

other: Not applicable: in-vitro test

Preparation of test site:

other: Not applicable: in-vitro test

Vehicle:

water

Controls:

yes, concurrent vehicle

Amount / concentration applied:

VEHICLE
- Amount(s) applied (volume or weight with unit): 150 µL of distilled water.

Duration of treatment / exposure:

24

Number of animals:

2

Results and discussion

In vivo

Irritant / corrosive response data:

On the grounds of the study, it may be stated that the test item belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean disc Sulforhodamine B dye content of the treated skin discs was lower than the mean disc dye content of the concurrent 36% HCl positive control.

Any other information on results incl. tables

Table 1. Results of the control transcutaneous electrical resistance test (TER):

 

Animal number	Skin disc number	TER value (kΩ)
1	1	16.71
	2	15.38
2	1	13.23
	2	11.39

The skin disc resistance values were greater than 10 kΩ; therefore, the remainder of the animals' skin discs could have been used in the experiment.

Table 2. Results of the transcutaneous electrical resistance test (TER):

 

Animal number	Tested substance	Skin disc number	TER value (kΩ)	Mean TER value ± SD (kΩ)
1	Positive control – 36% HCl	1	0.85	0.90 ± 0.07
		2	0.98
		3	0.87
	Negative control – distilled water	1	17.61	17.83 ± 1.85
		2	16.11
		3	19.78
	Test item	1	3.45	3.44 ± 0.24
		2	3.21
		3	3.68
2	Positive control – 36% HCl	1	0.79	0.80 ± 0.04
		2	0.76
		3	0.84
	Negative control – distilled water	1	14.11	14.67 ± 1.08
		2	15.92
		3	13.98
	Test item	1	2.56	2.71 ± 0.30
		2	2.52
		3	3.06

 

The concurrent mean values for the positive and negative controls were within the acceptable ranges for the method:

Positive control: 0.5-1.0 kΩ

Negative control: 10 -25 kΩ

The mean TER results for the skin discs treated with the test item were equal to 3.44 kΩ (animal no. 1) and 2.71 kΩ (animal no. 2).

Table 3. Gross changes on the surface of the treated skin discs:

 

Animal number	Tested substance	Skin disc number	Gross changes
1	Positive control – 36% HCl	1	perforation
		2	perforation
		3	perforation
	Negative control – distilled water	1	no changes
		2	no changes
		3	no changes
	Test item	1	no changes
		2	no changes
		3	no changes
2	Positive control – 36% HCl	1	perforation
		2	perforation
		3	perforation
	Negative control – distilled water	1	no changes
		2	no changes
		3	no changes
	Test item	1	no changes
		2	no changes
		3	no changes

The gross examination showed that the positive control skin discs exhibited skin perforation, whereas the negative control skin discs and the ones treated with the test item did not reveal any changes.

Table 4. Results of the treated disc Sulforhodamine B dye content determination.

 

Animal number	Tested substance	Skin disc number	Optical density at 565 nm	Dye concentration (µg/disc)	Mean dye concentration ± SD (µg/disc)
1	Positive control – 36% HCl	1	0.586	95.60	79.94 ± 17.22
		2	0.507	82.71
		3	0.377	61.50
	Negative control – distilled water	1	0.139	22.68	21.70 ± 0.91
		2	0.129	20.88
		3	0.132	21.53
	Test item	1	0.145	23.66	24.64 ± 5.13
		2	0.184	30.18
		3	0.123	20.07
2	Positive control – 36% HCl	1	0.495	80.75	70.86 ± 20.62
		2	0.289	47.15
		3	0.519	84.67
	Negative control – distilled water	1	0.123	20.07	19.69 ± 0.34
		2	0.119	19.41
		3	0.120	19.58
	Test item	1	0.129	21.05	21.81 ± 0.73
		2	0.138	22.51
		3	0.134	21.86

 

The concurrent mean values for the positive and negative controls were within the acceptable ranges for the method:

Positive control: 40 – 100 µg/disc

Negative control: 15 – 35 µg/disc

The mean dye content rangesfor the skin discs treated with the test item were equal to 24.64µg/disc (animal no. 1) and 21.81 µg/disc (animal no. 2).

 

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test substance does not lead to skin corrosion/severe irritation. The mean disc Sulforhodamine B dye content of the treated skin discs was lower than the mean disc dye content of the concurrent 36% HCl positive control.

Executive summary:

The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD Guideline 430 and EU Method B.40 (GLP study). Skin discs used in the experiment were obtained from two 30 -day-old animals. The test item was uniformly applied to the epidermal surface of the skin disc placed inside a tube. Concurrent positive (36% hydrochloric acid) and negative (distilled water) controls were used. Three skin discs obtained from each animal were used for the test item and each control item. The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. Then, they were removed by washing with a jet of tap water. Prior to measuring the electrical resistance, the surface tension of the skin was reduced by adding a sufficient volume of 70% ethanol to cover. After removing the ethanol the tissue was hydrated by the addition of 3 mL of a solution of MgSO4 (154 mM). A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩ by placing the databridge electrodes on either side of the skin disc. The skin discs were subjected to a gross examination in order to reveal possible damage. The dye binding procedure was necessary in this case since all TER values for the test item were lower than 5 kΩ, and the skin discs did not exhibit any visible pathological changes. The mean treated disc dye contents were equal to 24.64 μg/disc (animal no. 1) and 21.81 μg/disc (animal no. 2). The concurrent positive and negative control values fell within the acceptable ranges for the method. On the grounds of the results, it may be stated that the test substance does not lead to skin corrosion/severe irritation, as the mean disc Sulforhodamine B dye content of the treated skin discs was lower than the mean disc dye content of the concurrent 36% HCl positive control.