4'-aminoacetanilide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In one disregarded study, the in vitro mutagenicity of the 4'-aminoacetanilide (4'-AA) was evaluated using the Ames assay (bacterial reverse mutation test) on Salmonella typhimurium TA102 and TA104 strains. 4'-AA exhibited a strong mutagenicity, in the presence of S9 and in particular with the S.typhimurium TA102 assay system, where 1022±25 revertant at the maximal dose tested of 250µg/plate was comparable to that of the positive control (2-aminoanthracene).

In a key study, the genotoxic properties of 4'-aminoacetanilide was investigated in the Ames test and by the human lymphocytes micronucleus test.

Given that 4'-AA is an actual human skin and hepatic metabolites of para-Phenylenediamine and represent the substance to which humans are systemically exposed, it was tested in the absence of metabolic activation. No increases in revertant numbers were observed for 4'-AA treatments that were statistically or biologically significant when tested at concentrations up to 5000µg/plate and neither did 4'-AA induce micronuclei in cultured human peripheral blood lymphocytes when tested up to 10mM concentrations in the absence of S-9. It was concluded that 4'AA is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Ames bacterial reverse mutation assay with Salmonella typhimurium TA 102 and TA 104 strains.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

4'-aminoacetanilide was purchased from Aldrich, Germany

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: histidine-requiring strain

Species / strain / cell type:

S. typhimurium, other: TA104

Additional strain / cell type characteristics:

other: histidine-requiring strain

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver

Test concentrations with justification for top dose:

10µg/plate, 50µg/plate and 250µg/plate

Vehicle / solvent:

not specified

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

ACTIVATION MIXTURE
The S9 microsome fraction was prepared from rats treated with Aroclor 1254 (Maron and Ames, 1983). The components of S9 mix were 1mL of salt solution; 0.25mL of 1M G6P; 2mL of 0.1M NADP; 25mL of 0.2M sodium phosphate buffer, pH 7.4; 7mL of S9 microsome fraction and 14.75mL H2O. The S9 mix was prepared freshly for each assay. Protein concentration of rat liver S9 was determined using protein BioRad assay (Bradford, 1976). It was found to be 12.3 mg/mL.

SALMONELLA-MICROSOME ASSAY
The mutagenicity assay with S. typhimurium was performed as described by Maron and Ames (1983). The experiments were performed with or without an exogenous metabolic system, the S9 fraction in S9 mix. 100µL of exponential-phase culture of bacteria, cultivated in oxoid nutrient broth medium supplemented with 10µL of 8mg/mL ampicilin in the presence of both S. typhimurium TA102 and TA104 strains, a supplementary 2µL of tetracycline 10mg/mL was added in the case of TA102 strain, and 500µL of sodium phosphate buffer (0.2 M, pH 7.4, for assay without S9) or 500µL of S9 mix was added to 2mL aliquots of top Agar (supplemented with 0.5mM l-histidine and 0.5mM d-biotine) containing 100µL of different concentrations of each tested compound. The resulting complete mixture was poured on minimal agar plates prepared as described by Maron and Ames (1983). The plates were incubated at 37°C for 48 h and the revertant bacterial colonies of each plate were counted. Negative and positive control cultures gave the numbers of revertants per plate that were within the normal limits found in the laboratory. The appropriate positive controls accepted for the Ames test were applied; these were selected according to the type of strain used, and the presence or absence of the S9 mix. Culture run without azo dye corresponds to negative response. Data were collected with a mean±standard deviation of three plates (n = 3).

Evaluation criteria:

According to Ames, a compound is classified as mutagen if it is able to increase at least twice the number of revertants compared to spontaneous revertant.

Statistics:

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

Species / strain:

S. typhimurium, other: T 104

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

4'-aminoacetanilide (4'-AA) showed an important mutagenic potential with or without the S9 preparation. Mutagenic activity of 4'-AA in the presence of S9 and in particular with the S.typhimurium TA102 assay system (1022±25 revertant at the maximal dose tested of 250µg/plate) was comparable to that of the positive control (2-aminoanthracene).

Remarks on result:

other: Mutagenic potential defined by the author

Tested product	Dose (µg/plate)	TA 104		TA 102
		- S9	+S9	-S9	+S9
Positive control	-	1846 ± 46***	1400 ± 53***	1721 ± 24***	1031 ± 231***
Negative control	-	203 ± 11	273 ± 14	175 ± 10	217 ± 14
4'-AA	250	544 ± 17**	905 ± 21***	442 ± 13**	1022 ± 25***
4'-AA	50	322 ± 12*	800 ± 12***	329 ± 09**	795 ± 17***
4'-AA	10	289 ± 14	421 ± 11*	198 ± 10	675 ± 13**
* P < 0.01 ** P < 0.001 *** P < 0.0001

Conclusions:

4'-aminoacetanilide exhibited a strong mutagenicity towards S. typhimurium TA102 and TA104 strains.

Executive summary:

Mutagenicity of 4'-aminoacetanilide was evaluated using the Ames assay, in the presence of Salmonella typhimurium TA102 and TA104 strains.

Mutagenic activity of 4'-aminoacetanilide (4'AA) in the presence of S9 and in particular with the S.typhimurium TA102 assay system at the maximal dose tested was comparable to that of the positive control (2-aminoanthracene). It was concluded that 4'AA is a strong mutagen of S.typhimurium TA 102 and TA 104 strains.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Remarks:

GLP compliance according to the author

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

N-monacetylpara-phenylenediamine (MAPPD; CAS 122-80-5) was obtained from Lancaster Synthesis Ltd., Strasbourg, France. MAPPD was at least 95% pure according to the specifications of the manufacturer.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: histidine-requiring strains

Metabolic activation:

without

Metabolic activation system:

S9 mammalian liver post-mitochondrial fraction

Test concentrations with justification for top dose:

1st experiment: 1.6 to 5000 µg/plate
2nd experimzent: 156.3-5000 µg/plate

Vehicle / solvent:

The vehicle control was sterile water for injection (purified water).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control (quintuplicate)

True negative controls:

not specified

Positive controls:

yes

Remarks:

as appropriate per strain (triplicate)

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Details on test system and experimental conditions:

N-monoacetyl-para-phenylenediamine (MAPPD) was tested in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102. Two independent mutation experiments were performed with all five bacterial strains, using appropriate vehicle and positive controls. MAPPD was assayed only in the absence of S-9. Triplicate platings were performed for each bacterial strain at each dose level, and for positive controls. Quintuplicate platings were performed for vehicle controls in each strain. For the mutation experiments using a plate-incorporation method, molten soft agar (comprising 0.9% Difco Bacto agar and 0.5% sodium chloride, 2.5mLper tube) was dispensed into sterile tubes and held at 46±1°C. Bacterial culture (0.1 mL), control or test solution (0.1 mL) and buffer solution (0.5 mL) were added to each tube, the contents homogenised by rapid mixing, and poured onto Vogel-Bonner E agar plates (25 mL) supplied by Biotrace Fred Baker Ltd., UK. When the soft agar had set, all plates were inverted and incubated at 37±1°C for 3 days. Plating of these mixtures and incubation then proceeded as for the plate incorporation procedure. Plates were scored for revertant colony numbers using a Seescan Colony Counter (Seescan plc, UK) and the data processed using Colony2 software (York Electronic Research Ltd., York, UK).

Statistics:

Dunnett’s t-test was used to compare the colony counts at each dose with the vehicle control. The presence or otherwise of a dose response was checked by linear regression analysis. A positive result was indicated by a dose-related and reproducible response that was statistically significant at the 1% level.

Species / strain:

S. typhimurium, other: TA1535; 1537; 98; 100; 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Remarks:

The mean numbers of revertant colonies on negative control plates were all within acceptable ranges.

Positive controls validity:

valid

Remarks:

The mean number of revertant colonies in positive controls were significantly higher from the negative control.

Strain	First test (−S-9)		Second test (−S-9)
	Concentration (µg/plate)	Mean±S.D.	Concentration (µg/plate)	Mean±S.D.
TA 1535	Vehicle	17 ± 4	Vehicle	16 ± 2
	200	16 ± 3	1250	16 ± 7
	1000	23 ± 5	2500	10 ± 6
	5000	18 ± 6	5000	16 ± 3
	NaN3	604 ± 21	NaN3	612 ± 17
TA 1537	Vehicle	23 ± 2	Vehicle	21 ± 3
	200	22 ± 4	1250	23 ± 6
	1000	24 ± 4	2500	17 ± 3
	5000	17 ± 3	5000	21 ± 5
	9-AA	173 ± 9	9-AA	154 ± 57
TA 98	Vehicle	28 ± 3	Vehicle	25 ± 6
	200	28 ± 9	1250	37 ± 8
	1000	30 ± 2	2500	31 ± 6
	5000	25 ± 7	5000	22 ± 8
	2-NF	1027 ± 123	2-NF	928 ± 61
TA 100	Vehicle	109 ± 5	Vehicle	102 ± 12
	200	108 ± 12	1250	92 ± 6
	1000	115 ± 5	2500	86 ± 13
	5000	86 ± 6	5000	80 ± 4
	NaN3	590 ± 32	NaN3	520 ± 41
TA 102	Vehicle	248 ± 20	Vehicle	211 ± 15
	200	248 ± 17	1250	188 ± 14
	1000	243 ± 6	2500	187 ± 22
	5000	217 ± 29	5000	186 ± 36
	MMC	685 ± 22	MMC	401 ± 32

Conclusions:

4’-aminoacetanilide did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of S. typhimurium when tested at concentrations up to 5000 µg/plate in the absence of any exogenous metabolic activation.

Executive summary:

Genotoxic properties of N-monoacetyl-para-phenylenediamine (MAPPD) were investigated in the Ames test. Given that MAPPD is an actual human skin and hepatic metabolite of PPD (para-Phenylenediamine) and represents the substance to which humans are systemically exposed, it was tested in the absence of metabolic activation. No increases in revertant numbers were observed for MAPPD treatments that were statistically or biologically significant. It was concluded that MAPPD did not induce mutation in the five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of S. typhimurium when tested at concentrations up to 5000µg/plate in the absence of any exogenous metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The in vitro micronucleus assay using human lymphocytes was performed according to the IWGT recommendations (M. Kirsch-Volders et al. 2003).

GLP compliance:

no

Remarks:

GLP compliance according to the author

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

N-monacetylpara-phenylenediamine (MAPPD; CAS 122-80-5) was obtained from Lancaster Synthesis Ltd., Strasbourg, France. MAPPD was at least 95% pure according to the specifications of the manufacturer.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Blood from two healthy, non-smoking female volunteers (under 35 years of age) were used. No donor was suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals. Blood was stored refrigerated and pooled prior to use. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4mL heparinised blood into 8.1mL Hepes-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 µg/mL gentamycin. Phytohaemagglutinin (PHA, reagent grade) was included at a concentration of approximately 2% of culture volume to stimulate the lymphocytes to divide. Blood cultures were incubated at 37°C and rocked continuously.

Metabolic activation:

without

Test concentrations with justification for top dose:

769, 961, and 1502 µg/mL.

Vehicle / solvent:

The vehicle control was sterile water for injection (purified water).

Details on test system and experimental conditions:

Blood from two healthy, non-smoking female volunteers (under 35 years of age) were used in this study. No donor was suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals. Blood was stored refrigerated and pooled prior to use. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4mL heparinised blood into 8.1mL Hepes-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 µg/mL gentamycin. Phytohaemagglutinin (PHA, reagent grade) was included at a concentration of approximately 2% of culture volume to stimulate the lymphocytes to divide. Blood cultures were incubated at 37°C and rocked continuously. Two independent experiments were performed.
Treatments were performed in the absence of S-9. Cells were treated with PHA for 20 h followed by a 28-h recovery period prior to harvest. Cytochalasin B (at a final concentration of 6 µg/mL) was added for the final 28 h (approximately) of incubation. At the defined sampling time, cultures were centrifuged, treated with pre-warmed hypotonic (0.075 M) KCl for 5 min and fixed with ice-cold methanol/glacial acetic acid (3:1, v/v).
Drops of fixed suspension were put onto clean glass microscope slides, dried and stained for 5 min with 4%(v/v) filtered Giemsa stain in Gurr’s pH 6.8 buffer. The slides were rinsed, dried and mounted with cover slips.

Evaluation criteria:

Slides were scored for proportions of mononucleate, binucleate and multinucleate cells and the replication index (RI) calculated based on the analysis of 500 cells per replicate (1000 per dose). The RI was determined using the following formula:
RI = (Number of binucleate cells + 2 x number of multinucleate cells)/Total number of cells

Statistics:

The top dose for analysis was to be one at which at least 60% (approximately) reduction in RI (compared to concurrent controls) occurred. The slides from the selected doses were scored for the presence of micronucleated binucleate (MNBN) cells. Where possible, one thousand binucleate cells from each culture (2000 per dose level) were analysed for micronuclei. The proportions of micronucleated cells in each replicate were used to establish acceptable homogeneity between replicates by means of a binomial dispersion test. The proportion of cells with micronuclei for each treatment condition were compared with the proportion in solvent controls by using Fisher’s exact test. Probability values of p≤0.05 were accepted as significant.

Species / strain:

mammalian cell line, other: Human lymphocytes

Metabolic activation:

without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Remarks:

Negative (solvent) control cultures showed frequencies of MNBN that fell within historical solvent control ranges

Positive controls validity:

valid

Remarks:

All positive control cultures showed statistically significant increases in frequencies of MNBN

Additional information on results:

In the first experiment, the dose levels for micronucleus analysis were selected by evaluating the effect of 4'-aminoacetanilide on the replication index (RI). Micronuclei were analysed at three dose levels. The highest concentration chosen for analysis, 1502µg/mL (10 mM), was not toxic and induced approximately 4% reduction in RI. In the second experiment, micronuclei were also analysed at three dose levels. The highest concentration chosen for analysis, 1502 µg/mL (10 mM), was also not toxic (zero reduction in RI). Negative (solvent) control cultures showed frequencies of MNBN that fell within historical solvent control ranges. All positive control cultures showed statistically significant increases in frequencies of MNBN.
Treatment of cells with 4'-aminoacetanilide in the absence of S-9 resulted in frequencies of MNBN that were similar to those observed in concurrent vehicle control cultures for all concentrations analysed, were not significantly different and fell within the historical negative control ranges.

Remarks on result:

other: no micronuclei induction up to 10mM concentration

	Concentration (µg/mL)	Cytotoxicity (% reduction in RI)	Mean MNBN cell frequency (%)
Experiment 1 treatment (24 h PHA)
20+28h (-S-9)	Vehicle	-	0.35
	769	0	0.50
	961	5	0.70
	1502	4	0.55
	NQO	Nd	10.30***
	VIN	Nd	3.15***

Experiment 2 treatment (48 h PHA)
20+28h (-S-9)	Vehicle	-	0.45
	769	0	0.60
	961	0	0.40
	1502	0	0.20
	NQO	Nd	9.10***
	VIN	Nd	9.50***
Nd: not determined; MNBN: Micronucleated binucleate; RI: replication index. Positive controls: NQO: 4-nitroquinoline-1-oxide, 5 µg/mL; VIN: vinblastine, 0.08 µg/mL; Key to significance (Fisher’s exact test): *p< 0.05; **p< 0.01; ***p< 0.001.

Conclusions:

Treatment of cells with 4'-aminoacetanilide in the absence of S-9 resulted in frequencies of MNBN (Micronucleated binucleate) that were similar to those observed in concurrent vehicle control cultures for all concentrations analysed, were not significantly different and fell within the historical negative control ranges. It was concluded that 4'-aminoacetanilide did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to 10mM concentrations in the absence of S-9 under two different experimental conditions.

Executive summary:

Genotoxic properties of N-monoacetyl-para-phenylenediamine (MAPPD) were investigated in the micronucleus test (MNT) in human lymphocytes in two different experimental conditions in the absence of S-9.

MAPPD did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to 10mM concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In one in vivo study, the genotoxicity of standard 4’-aminoacetanilide (4’-AA) was evaluated in vivo, in mouse bone marrow, by assessing the percentage of cells bearing different chromosome aberrations, membrane lipid peroxidation (MDA) and acetylcholinesterasic (AChE) activity inhibition. 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), significant increase of MDA was induced, equivalent production to 12.5, 13, and 15.5 nM at concentrations of 0.5, 1, and 2.5 mg/kg bw, respectively and the AChE activity was decreased by 8.15%.

In another poorly documented study, where the genotoxicity of aniline derivatives was examined by a DNA repair test with rat hematocytes, it was found that 4'-aminoacetanilide did not induce DNA repair and thus it was concluded to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

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

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

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).

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.

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.

Dose / conc.:

0.5 mg/kg bw (total dose)

Dose / conc.:

1 mg/kg bw (total dose)

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)

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.

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).

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

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).

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The genotoxicity of 4'aminoacetalinide was examined by a DNA repair test with rat hematocytes.

GLP compliance:

not specified

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

p-aminoacetanilide was purchased from Tokyo Kasei Chem. Co. (Tokyo, Japan) and dissolved in dimethylsulfoxide (Nakarai Chem. Co., Tokyo, Japan). As a positive control, N-2-fluorenylacetamide (Nakarai Chem.) was used in the assay.

Species:

rat

Details on species / strain selection:

ACI rats weighing 200-250 g.

Sex:

male

Details on test animals or test system and environmental conditions:

not specified

Route of administration:

other: Hepatocytes were isolated from the livers of male rats.

Vehicle:

dimethylsulfoxide

Details on exposure:

HEPATOCYTE / DNA REPAIR TEST
The test was performed basically in accordance with the method of Williams et al. 1982 (Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens. Mutation Res., 97, 359-370). Hepatocytes were isolated from the livers of male ACI rats weighing 200-250 g. The isolated hepatocytes were allowed to attach for 2 h on plastic coverslips in primary culture using Williams' Medium E. The cultures were then washed and exposed to the test chemicals and [Me-3H]thymidine (10 µCi/ml; 49 Ci/mmole; Amersham International, Amersham, U.K.) for 20 h. All chemicals were dissolved in dimethylsulfoxide (Nakarai Chem. Co., Tokyo, Japan). The substance was logarithmically diluted before addition to the cultures.
As a positive control, N-2-fluorenylacetamide (Nakarai Chem.) was used in the assay. At the end of incubation, the cultures were washed, and the coverslips were mounted on glass slides. The slides were dipped in Sakura NR-M2 photographic emulsion and exposed for 14 days. Autoradiographic grains were counted on a television screen (Olympus, type S) with a microscopic attachment. The data were expressed as the average net counts/nucleus for 3 coverslips + the standard deviation (50 cells/coverslip).
The test chemicals were considered positive when the mean net nuclear grain count was more than 5 grains above background and statistically greater than that of controls (unpaired t-test; P < 0.01). Two experiments were performed on every test compound for the assays with rat hepatocytes.

Duration of treatment / exposure:

20h

Frequency of treatment:

one application

Post exposure period:

none

Dose / conc.:

0.001 other: mol/L

Dose / conc.:

0 other: mol/L

Dose / conc.:

0 other: mol/L

Dose / conc.:

0 other: mol/L

No. of animals per sex per dose:

not specified

Control animals:

yes

Positive control(s):

N-2-fluorenylacetamide

Tissues and cell types examined:

rat liver hepatocytes

Details of tissue and slide preparation:

At the end of incubation, the cultures were washed, and the coverslips were mounted on glass slides. The slides were dipped in Sakura NR-M2 photographic emulsion and exposed for 14 days.
Autoradiographic grains were counted on a television screen (Olympus, type S) with a microscopic attachment.

Evaluation criteria:

The test chemicals were considered positive when the mean net nuclear grain count was more than 5 grains above background and statistically greater than that of controls (unpaired t-test; P < 0.01).

Statistics:

The data were expressed as the average net counts/nucleus for 3 coverslips + the standard deviation (50 cells/coverslip). The test chemicals were considered positive when the mean net nuclear grain count was more than 5 grains above background and statistically greater than that of controls (unpaired t-test; P < 0.01). Two experiments were performed on every test compound for the assays with rat hepatocytes.

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: Not genotoxic according to the author

Additional information on results:

Historical data of bacterial mutagenicity assays by Suzuki et al. 1986 (Mutagenicity of aniline derivatives and comutagenic effects of norharman on them (Part 3), Proc. Jpn. Cancer Assoc. 45th Annual Meeting, p. 62) indicated induced mutagenicity by p-aminoacetanilide. No correlation was seen between these results and the current p-aminoacetanilide DNA repair study.

Chemical	Dose (M)	UDS grains/ nucleus	% of UDS-positive cells	DNA repair
N-2-Fluorenylacetamide (positive control)	10 -5	60.8 + 8.4	100
Dimethylsulfoxide (solvent control)		0.6 5- 1.3	0
p-Aminoacetanilide	10 -3	2.5_+ 1.5	10	-
	10 -4	2.5 _+ 1.7	10
	10 -5	1.6_+ 1.5	4
	10 -6	1.5+ 1.4	2

Conclusions:

The genotoxicity of p-aminoacetanilide was examined by a DNA repair test with rat hematocytes among 18 substances. p-aminoacetanilide did not induce DNA repair, thus it was concluded to be non-genotoxic.

Executive summary:

The genotoxicity of p-aminoacetanilide was examined by a DNA repair test with rat hematocytes aamong 18 substances. p-aminoacetanilide did not induce DNA repair, thus it was concluded to be non-genotoxic.

Reference 3

Endpoint:

genetic toxicity in vivo, other

Remarks:

Acetylcholinesterase inhibition assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

SERUM PREPARATION FOR STUDYING ACETYLCHOLINESTERASIC ACTIVITY INHIBITION
Blood was collected in heparin glass tubes (plastique Gosselin 59522, Hazebrouk, France); red blood cells were eliminated by centrifugation at 1,800 rpm for 10 min, and the plasma (supernatant) was then recovered. Mouse plasma was used immediately for studying acetylcholinesterase (AChE) activities or conserved at 2–8°C (stable for 7 days).

ACETYLCHOLINESTERASE INHIBITION ASSAY
AChE inhibiting activity was measured by the spectrophotometric method previously reported by Ellman et al. 1961(A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95), modified by Ortega et al. 2004(Anticholinesterase activity in an alkaloid extract of Huperzia saururus. Phytomedicine 11:539–543), and adapted according to the author’s experimental conditions. Before use, plasma was diluted (1/200) with 100 mM of phosphate buffer (pH8). Acetylcholine was used as substrate to assay AChE activity. In order to calculate the activity of the AChE activity, the following procedure was employed: 1 ml of phosphate buffer 100 mM; pH8, containing 0.26 mM of DTNB and 100 μl of plasma were placed in a microcuvette, which was incubated for 15 min at 30°C. The reaction (at 30°C) was started by the addition of acetylcholine (50 μl) and monitored by following the formation of yellow 5-thio-2-nitrobenzoate anions resulting from the reaction of DTNB with the thiocholine released by the enzymatic hydrolysis of acetylcholine. Absorbance was measured using an M350 double Beam UV–VIS spectrophotometer «Camespec» (TovaTech, South Orange, NJ, USA) at 405 nm, and the reading was repeated during 10 min at intervals of 30 s to verify the linearity of the reaction.
The enzymatic activity was calculated according to the relation:
Enzymatic activity (µm moles/ml/min)= Δ absorbance/min/1:36 x 10^4
The percentage (%) inhibition of AcChE activity was calculated as follows: (E−S)/E×100, where E is the activity of the enzyme in the absence of the test compound and S is the activity of enzyme in the presence of the test compound.
IC50 (concentrations of test compounds that inhibited the hydrolysis of substrate acetylcholine by 50%) values were calculated from dose–inhibition curves (Noor et al. 2007(L eufolins A and B, potent butyrylcholinesterase-inhibiting flavonoid glucosides from leucas urticifolia. Molecules 12:1447–1454)). All experiments were repeated three times.

GLP compliance:

no

Type of assay:

other: Acetylcholinesterase inhibition assay

Specific details on test material used for the study:

4’-aminoacetanilide (4’-AA) was obtained from Aldrich (St Louis, MO, USA).
Acetylcholine iodide and 5,5’-dithiobis[2-nitrobenzoic acid] (DTNB) were purchased from Quimica Clinica Aplicada S.A. (Amposta, Spain).

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.

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 control group, increasing doses fro 4'-AA group.

Dose / conc.:

0.5 mg/kg bw (total dose)

Dose / conc.:

1 mg/kg bw (total dose)

Dose / conc.:

2.5 mg/kg bw (total dose)

No. of animals per sex per dose:

3

Control animals:

yes

Positive control(s):

No positive control was used in this test.

Tissues and cell types examined:

Blood plasma

Details of tissue and slide preparation:

Serum preparation for studying acetylcholinesterasic activity inhibition
Blood was collected in heparin glass tubes (plastique Gosselin 59522, Hazebrouk, France); red blood cells were eliminated by centrifugation at 1,800 rpm for 10 min, and the plasma (supernatant) was then recovered. Mouse plasma was used immediately for studying acetylcholinesterase (AChE) and lipid peroxidation activities or conserved at 2–8°C (stable for 7 days).

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.

Sex:

female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: Not precised by the author

Remarks:

Insignificant rise in inhibition %

Additional information on results:

Acetylcholinesterase activity: 4’-aminoacetanilide at highest dose decreased the AChE activity by 8.15% (negative control was 5.21).

Tested compound	Concentration (mg/kgbw)	Inhibition percentage	IC50(mg/kg)
Negative control	2.5	5.21	-
4’-AA	2.5	8.15	-
	1	5.30
	0.5	3.65

Conclusions:

4’-aminoacetanilide has no significant effect on acetylcholinesterase inhibition.

Executive summary:

Toxicity of standard 4’-aminoacetanilide (4’-AA) was evaluated in vivo, in mouse, by assessing the acetylcholinesterasic (AChE) activity inhibition. The AChE activity was decreased by 8.15%.

4'-aminoacetanilide was found not to have a significant effect on AChE inhibition.

Reference 4

Endpoint:

genetic toxicity in vivo, other

Remarks:

Lipid peroxidation assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

SERUM PREPARATION FOR STUDYING LIPID OXIDATION EFFECT
Blood was collected in heparin glass tubes (plastique Gosselin 59522, Hazebrouk, France); red blood cells were eliminated by centrifugation at 1,800 rpm for 10 min, and the plasma (supernatant) was then recovered. Mouse plasma was used immediately for studying lipid peroxidation activities or conserved at 2–8°C (stable for 7 days).

DETERMINATION OF LIPID PEROXIDATION PRODUCTS WITH THIOBARBITURIC ACID REACTIVE SUBSTANCES ASSAY
The well-known method termed thiobarbituric acid reactive species (TBARS) assay concerns the spectrophotometric measurement of the pink pigment produced through reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA) and other secondary lipid peroxidation products. TBARS were determined according to Ohkowa et al. 1979 (Assay for lipid peroxides in animals tissue by thiobarbituric acid reaction. Anal Biochem 95:351–358). In this method, 0.4 ml plasma from each mouse plasma was mixed with 1.5 ml of 20% acetic acid and 1.5 ml of 0.8% TBA. The mixture was brought to a final volume of 4.0 ml with distilled water and heated to 85°C for 120 min. After cooling for 10 min on ice, 5.0 ml of a mixture of n-butanol and pyridine (15:1 v/v) were added to each sample, and the mixture was shaken vigorously. After centrifugation (3,000 rpm, 20°C, 10 min), the supernatant fraction was isolated and the absorbance was measured at 532 nm. Induction of lipid peroxidation was expressed as equivalent of MDA. Data were reported as mean±SD for triplicate determinations.

GLP compliance:

no

Type of assay:

other: Lipid peroxidation assay

Specific details on test material used for the study:

4’-aminoacetanilide (4’-AA) was obtained from Aldrich (St Louis, MO, USA).
Acetic acid was obtained from Prolabo (Paris, France)

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.

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 control group, increasing doses for 4'-AA group.

Dose / conc.:

0.5 mg/kg bw (total dose)

Dose / conc.:

1 mg/kg bw (total dose)

Dose / conc.:

2.5 mg/kg bw (total dose)

No. of animals per sex per dose:

3

Control animals:

yes

Positive control(s):

No positive control was used in this test.

Tissues and cell types examined:

blood plasma

Details of tissue and slide preparation:

SERUM PREPARATION FOR STUDYING LIPID OXIDATION EFFECT
Blood was collected in heparin glass tubes (plastique Gosselin 59522, Hazebrouk, France); red blood cells were eliminated by centrifugation at 1,800 rpm for 10 min, and the plasma (supernatant) was then recovered. Mouse plasma was used immediately for studying lipid peroxidation activities or conserved at 2–8°C (stable for 7 days).

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.

Sex:

female

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: Significant increase in malondialdehyde

Additional information on results:

4’-amoniacetanilide induced a significant increase of malondialdehyde equivalent production to 12.5, 13, and 15.5 nM in the presence at concentrations of 0.5, 1, and 2.5 mg/kg bw, respectively (control values were 8nM).

Conclusions:

4’-aminoacetanilide has an important lipid peroxidation capacity according to the author.

Executive summary:

Toxicity of standard 4’-aminoacetanilide (4’-AA) was evaluated in vivo, in mouse, by assessing membrane lipid peroxidation (MDA).

4’-amoniacetanilide induced a significant increase of malondialdehyde equivalent production to 12.5, 13, and 15.5 nM in the presence at concentrations of 0.5, 1, and 2.5 mg/kg bw, respectively (control values were 8nM).