4'-aminoacetanilide

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

In vitro genotoxicity of para-phenylenediamine and its N-monoacetyl or N,N'-diacetyl metabolites

Author:

J-L. Garrigue, M. Ballantyne, T. Kumaravel, M. Lloyd, G. J. Nohyneka, D. Kirkland, H. Toutain

Year:

2006

Bibliographic source:

Mutation Research 608 (2006) 58–71

Materials and methods

Test guideline

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

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:

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.

Method

Species / strain

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).

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.