N-1-naphthylaniline

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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 1

Reference Type:

study report

Title:

Unnamed

Year:

1976

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1977

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Metabolic activation with non-induced mouse liver S9

Principles of method if other than guideline:

The procedure was a modification of that reported by Clive and Spector (1975). Briefly, rapidly growing cells were cleansed of spontaneous Tk-/- mutants by growing them in a medium containing thymidine hypoxanthine, methotrexate and glycine (THMG). Only cells producing the enzyme thymidine kinase can utilize the exogenous thymidine from the medium and grow. Study was performed before actual guideline was established.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-1-naphthylaniline

EC Number:

201-983-0

EC Name:

N-1-naphthylaniline

Cas Number:

90-30-2

Molecular formula:

C16H13N

IUPAC Name:

N-phenylnaphthalen-1-amine

Details on test material:

- Name of test material (as cited in study report): N-Phenyl-Alpha-Naphtylamine
- Substance type: red-brown pellets
- Physical state: solid
- Analytical purity: no data
- Other: Source: United States Air Force

Method

Target gene:

thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Non-induced mouse liver S9-mix

Test concentrations with justification for top dose:

-S9: 0.5, 5.0, 10.0, 25.0 µg/ml
+S9: 0.005, 0.01, 0.05, 0.1 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulfonate (-S9); dimethylnitrosamine (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 days

SELECTION AGENT (mutation assays): BUdR (5' bromodeoxyuridine)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: loss in growth potential, plating efficiency (cell survival)

Statistics:

Data analysis:
A mutation frequency was determined for each test dose by dividing the number of mutants/ml by the number of surviving cells/ml (adjusted to 10e-4) as indicated by plating efficiency.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentration showing toxic effects was chosen as highest conentration tested tested was chosen

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The solubility, toxicity and doses for the test chemical were determined prior to screening. The effect of the chemical on cell survival was determined by exposing the cells to a wide range of chemical concentrations in complete growth medium. Toxicity was measured as loss in growth potential of the cells induced by a five-hour exposure to the chemical. Four doses were selected from the range of concentrations by using the highest dose that showed no loss in growth potential as the penultimate dose and by bracketing this with one higher and two lower doses. Toxicity produced by chemical treatment was monitored during the experiment.

Any other information on results incl. tables

Mouse lymphoma mutagenicity assay:

	Day 1 count	Day 3 count	?GS	%GS	MC	VC	%CE	GF	MF(10e-4)
-S9
Control	1.5	11.1	9.6	100	89	191	100	100	0.5
EMS	1.6	2.3	0.7	7	288	7	4	0.3	41.1
PANA (µg/ml)
0.5	3.1	11.2	8.1	84	68	214	112	94	0.3
5.0	2.7	8.6	5.9	61	5	300	157	95	0.02
10.0	2.6	10.9	8.3	86	50	202	105	122	0.3
25.0	3.4	12.4	9.0	93	57	215	112	104	0.3
+S9
Control	1.1	7.2	6.1	100	58	229	100	100	0.3
DMN	1.5	2.7	0.2	3	184	8	3	0.9	2.3
PANA (µg/ml)
0.005	3.3	17.8	14.5	246	8	56	69	170	0.1
0.01	3.1	15.6	12.5	212	3	58	72	152	0.05
0.05	2.8	6.5	3.7	62	32	70	86	54	0.5
0.1	0.9	1.7	0.8	14	6	36	44	6	0.2

Day 1 and 3 counts: Expression day cell counts (x10e6)

?GS: Represents cell population growth during expression. The value is obtained by subtracting the Day 1 counts from the terminal day counts.

%GS: Percent suspension growth is obtained by expressing the?GS values for treated cells as a percent of the?GS for the negative controls.

MC: Mutant counts. The total number of colonies counted in the BUdR plates.

VC: Viable counts. The total number of colonies counted in the VC plates.

%CE: Cloning efficiency. Obtained by expressing the VC in treated cultures as a percent of the VC in negative controls.

GF: Growth factor. %GS x %CE / 100

MF (x10e-4): Mutation frequency. MC / VC x 10e-4