4'-aminoacetanilide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Data source

Reference

Reference Type:

publication

Title:

Acid violet 7 and its biodegradation products induce chromosome aberrations, lipid peroxidation, and cholinesterase inhibition in mouse bone marrow

Author:

H. B. Mansour, Y. Ayed-Ajmi, R. Mosrati, D. Corroler, K. Ghedira, D. Barillier, L. Chekir-Ghedira

Year:

2010

Bibliographic source:

Environ Sci Pollut Res (2010) 17:1371–1378

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Bone marrow cells were obtained according to the technique of Yosida and Amano 1965 (Autosomal polymorphism in laboratory bred and wild Norway rats, Rattus norvegicus. Chromosoma 16:658–667). Chromosomes were prepared as reported by Evans et al. 1960 (An air drying method for meiotic preparation from mammalian tests. Cytogenetics 3:613–616). See: details of tissue and slide preparation.

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4'-aminoacetanilide

EC Number:

204-576-6

EC Name:

4'-aminoacetanilide

Cas Number:

122-80-5

Molecular formula:

C8H10N2O

IUPAC Name:

4'-aminoacetanilide

Test material form:

solid

Details on test material:

Name: 4'-aminoacetanilide

Specific details on test material used for the study:

4’-aminoacetanilide (4’-AA) was obtained from Aldrich (St Louis, MO, USA) and zearalenone were purchased from Sigma–Aldrich (Steinheim, Germany).
Methanol and acetic acid were obtained from Prolabo (Paris, France).

Test animals

Species:

mouse

Strain:

Balb/c

Details on species / strain selection:

Mice of similar age and weight (20–25 g) were selected.

Sex:

female

Details on test animals or test system and environmental conditions:

Animals were kept for 1 week before the experiments for acclimatization and were maintained on food (conventional chow) and water ad libitum.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

not specified

Details on exposure:

Administration of the tested substance was by intraperitoneal (IP) injection of 200 μl of the solution. Twenty-four hours before sacrifice, animals were given 500 μl of the mixture yeast extract/glucose (at final concentrations 100 and 200 mg/ml, respectively) to accelerate mitosis of bone marrow cells. Vinblastin (200 μl; at final concentration 250 μg/ml) was injected into the animals 45 min before sacrifice, in order to block dividing cells in metaphase. Finally, animals were sacrificed by cervical dislocation.

Duration of treatment / exposure:

not specified

Frequency of treatment:

A single dose for the groups of negative and positive control. Increasing doses for the group 4'-AA.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.5 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

2.5 mg/kg bw (total dose)

No. of animals per sex per dose:

3

Control animals:

yes

Positive control(s):

A single dose of zearalenone (4 mg/kg)

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

BONE MARROW PREPARATION
Bone marrow cells were obtained according to the technique of Yosida and Amano (1965). Briefly, femur and tibia were removed immediately after animal sacrifice and bone marrow was flushed out with KCl solution (0.075 M, 37 C) using a syringe. The bone marrow cell suspension was incubated for 20 min at 37°C and centrifuged at 3,500 rpm for 10 min. The supernatant was discarded, the pellet was resuspended in 5 ml of a fixative solution (acetic acid/methanol, 1:3, v/v), centrifuged (3,500 rpm for 10 min), and the supernatant was discarded again. This step was repeated three times in order to clean the pellet. Finally, the pellet was resuspended in 1 ml of the above fixative solution and used for chromosome preparation.

CHROMOSOME PREPARATION
Chromosomes were prepared as reported by Evans et al. (1960). Cell suspensions were dropped on glass slides giving smears that were blazed on a flame for 5 s, then air-dried for conservation at room temperature and/or directly stained with Giemsa. Giemsa working solution was freshly prepared (4 ml in 100 ml) in phosphate buffer (0.15 M, pH7.2). Slides were left for 15 min in this staining solution, then rinsed with water and allowed to dry at room temperature.

SLIDE ANALYSIS
The slides were examined under 100× magnifications using an optical microscope (Carl Zeiss, Oberkochen, Germany). Three hundred well-spread metaphases were analyzed per group for abnormalities. Metaphases with chromosome breaks, gaps, ring, and centric fusion (Robertsonian translocation) were recorded and expressed as percentage of total metaphases per group.

Statistics:

Data are expressed as mean±standard deviation from three replicates. The statistical analysis was performed with STATISTICA edition 99 France. Duncan test was used to compare tested compounds vs. control. Difference was considered significant when P<0.05.

Results and discussion

Test results

Sex:

female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: Important genotoxic potential according to the authors

Additional information on results:

4’-aminoacetanilide showed an important genotoxic potential and the percentage of chromosome aberrations increased significantly (33% at the highest tested dose) which was comparable to the positive control (zearalenone at 20 mg/kg bw).

Any other information on results incl. tables

Tested compound	Dose (mg/kgbw)	Structural aberration (%)				Total aberration (%)
		Rings	Centric fusion	Chromosomal breaks	Gaps
Negative control	2.5	00±0	2.5±0.5	02.2±0.1	02.1±0.5	6±1.0
Positive control (zearalenone)	20	2.3±1.5	21.3±2.5	8.3±3.5	0.6±0.5	33.0±1.0**
4'-AA	2.5	6.5±0.5	15.3±0.6	10.5±1.2	01.50±0.5	34±2**
	1	04.2±1.4	9.5±0.5	6.3±1.1	00±00	19±1.5*
	0.5	2.5±1.3	5.5±0.5	3.5±0.5	1.5±0.5	13±2.5
*P<0.05 **P<0.01

Applicant's summary and conclusion

Conclusions:

4’-aminoacetanilide showed an important genotoxic potential in the in vivo mouse bone marrow test.

Executive summary:

Toxicity of standard 4’-aminoacetanilide (4’-AA) was evaluated in vivo, in mouse bone marrow, by assessing the percentage of cells bearing different chromosome aberrations.

4’-AA showed an important genotoxic potential, the percentage of chromosome aberrations increased significantly (33% at the highest tested dose) which was comparable to the positive control (zearalenone at 20 mg/kg bw).