Nitrosodiphenylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Salmonella mutagenicity tests: IV Results from the testing of 300 chemicals

Author:

Zeiger E.

Year:

1988

Bibliographic source:

Environ. Molecular mutagenesis Vol 11, suppl 12: 1-158

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test chemical was tested for mutagenicity in salmonella typhimurium, using a preincubation protocol. All tests were performed in the absence of exogenoux metabolic activation, and in the presenceof liver S-9 from Aroclor-induced male Sprague Dawley rats and Syrian hamsters.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Nitrosodiphenylamine

EC Number:

201-663-0

EC Name:

Nitrosodiphenylamine

Cas Number:

86-30-6

Molecular formula:

C12H10N2O

IUPAC Name:

N-nitroso-N-phenylaniline

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% or 30% of liver rat or hamster S9

Test concentrations with justification for top dose:

0, 1000, 3300, 10000, 33000, 100000, 200000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (-S9, TA98)? 2-aminoanthracene (+S9, all strains)

Details on test system and experimental conditions:

Prepration of liver S9 fraction :
The S9 fractions of Aroclor 1254-induced, male SD rat ad male syrian hamster livers were prepared immediately prior to se and contained either 10% or 30% S9; occasionally, other levels were used. All chemicals were tested in the absence of metabolic activation, and with rat and hamster S9 fractions.

Test protocol :
The preincubation assay was performed as follow : the test chemical (0.05 ml), Salmonella culture (0.10 ml), and S9 mix or buffer (0.5 ml) were incubated at 37°C, without shaking for 20 min. The top agar was added and the contents of the tubes were mixed and poured onto the surfce of petri dishes containing medium. The histidine-independent (his +à colonies arising on these plates were counted following two days incubation at 37°C. Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the background agar. At the discretion of the investigators, plates with low numbers of colonies were counted by hand.
All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for mutagnicity assay. The toxicity assay was performed using TA100. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.
Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in the preliminary test. Chemicals that were not toxic were tested to a miximum dose of 10 mg/plate. Chemicals that were poorly soluble were tested up to doses defined by their solubilities. At least 5 doses of each chemical were tested in triplicate. Experiments were repeated at least one week following the initial trial. A maximum of 0.05 ml solvent was added to each plate.

Evaluation criteria:

Evaluationswere made at both the individual trial and overall chemical levels. Individual triales were judged as mutegnic or not depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response.

Statistics:

no

Results and discussion

Test results

Species / strain:

S. typhimurium, other: all strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see tables below

Any other information on results incl. tables

Tables of results (tables 207 : nitrosodiphenylamine)

Dose (µg/ plate)	TA100
	Without S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	82	6.2	95	9.7	86	24.7	209	1.3	95	2.3
1000	84	7.4
3300	90	9.0	91	3.5	97	6.0	200	8.1	91	6.1
10 000	84	6.2	114	5.0	96	10.2	203	17.3	89	2.0
33 000	86	4.7	101	7.5	92	5.5	206	1.7	96	10.9
100 000	61s	3.6	107	5.7	111	5.7	177	14.4	102	5.9
200 000			122s	5.0	76s	3.2	158s	2.1	90s	3.6
POS	429	7.6	260	14.2	363	16.5	424	17.9	323	16.4

HLI : hamster liver ; RLI : rat liver

Dose (µg/ plate)	TA1535
	Without S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	22	0.9	16	1.8	7	3.2	21	1.7	11	2.4
1000	23	1.0
3300	27	6.1	13	1.9	10	1.8	17	1.8	10	0.6
10 000	30	4.4	12	2.3	5	1.3	18	4.3	10	1.5
33 000	37	5.6	18	2.8	8	1.8	16	0.9	9	0.6
100 000	7s	2.0	12	1.2	12	1.7	18	2.0	14	1.8
200 000			15s	3.7	9s	3.2	17s	1.2	11s	0.3
POS	263	11.2	86	10.6	219	13.4	222	11.3	163	6.4

HLI : hamster liver ; RLI : rat liver

Dose (µg/ plate)	TA1537
	Without S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	8	2.2	9	2.0	6	0.7	16	3.2	12	1.5
1000	4	1.2
3300	5	2.3	9	1.8	8	2.3	12	2.3	12	1.3
10 000	7	0.7	11	1.5	7	0.6	14	1.7	9	1.9
33 000	7	0.9	10	0.9	8	0.0	14	0.6	8	1.5
100 000	4s	1.2	7	1.5	11	1.0	14	2.2	9	1.5
200 000			8s	2.4	10s	0.6	10s	0.9	9	2.8
POS	126	12.4	121	1.8	111	15.5	161	7.8	55	2.7

HLI : hamster liver ; RLI : rat liver

Dose (µg/ plate)	TA97
	Without S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	81	4.1	210	8.5	128	3.7	214	9.4	170	13.5
1000	76	9.7
3300	79	1.3	189	7.9	119	4.3	233	1.5	123	2.8
10 000	67	3.1	183	12.0	129	9.2	235	17.8	126	3.5
33 000	76	2.3	201	22.6	126	10.7	245	10.3	130	10.8
100 000	8s	1.5	220	4.8	132	3.9	265	12.5	124	4.4
200 000			141s	3.5	140	7.0	178s	7.8	130	5.5
POS	235	19.6	543	15.5	696	22.7	873	77.4	398	7.8

HLI : hamster liver ; RLI : rat liver

 

 

Dose (µg/ plate)	TA98
	Without S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	13	1.8	22	1.7	26	5.0	27	4.5	19	1.2
1000	11	1.8
3300	13	0.3	21	1.7	24	1.7	29	1.2	21	2.1
10 000	10	4.8	25	0.6	18	3.0	24	0.6	20	3.1
33 000	12	2.2	24	4.0	19	2.2	20	2.0	21	1.7
100 000	8s	3.6	25	2.8	27	1.5	21	1.0	20s	2.9
200 000			19s	1.0	25s	2.2	17s	1.3	22s	2.6
POS	157	12.3	193	6.7	164	24.1	158	8.1	152	16.4

HLI : hamster liver ; RLI : rat liver

 

Applicant's summary and conclusion

Conclusions:

Based on this assay, nitrosodiphenylamine showed negative results in the Ames test.

Executive summary:

Test chemical was tested for mutagenicity in Salmonella typhimurium, using a preincubation protocol. All tests were performed in the absence of exogenoux metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague Dawley rats and Syrian hamsters. Based on this assay, nitrosodiphenylamine showed negative results in the Ames test.