N-1-naphthylaniline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test article was not mutagenic and not clastogenic in various in vitro studies.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

thymidine kinase locus

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

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.

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.

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

No information if duplicate cultures were performed.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

-S9: 2.99 - 29.9 µg/ml
+S9: 1.49 - 19.9 µg/ml

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9: 1.0 and 5.0 µg/ml, respectively

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9: 50 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 8 hours (-S9); 2 hours (+S9)
- Expression time (cells in growth medium): 8 hours (exposure time; -S9); 8 hours (+S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 10 - 10.5 hours (-S9); 12 hours (+S9)


SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): 5 % Giemsa


NUMBER OF CELLS EVALUATED: 200 per dose


DETERMINATION OF CYTOTOXICITY
- Method: other: loss of confluency of cell monolayer


OTHER EXAMINATIONS:
- Determination of polyploidy: only when it seemed excessive
- Determination of endoreplication: only when it seemed excessive
- Other: categories of chromosomal aberration: simple (chromatid gap, break, fragment and deletion; chromosome gap, break, double minutes), complex (interstitial deletions, triradials, quadriradials, rings, dicentric chromosomes); other aberrations (pulverized chromosomes or cells with greater than 10 aberrations). Chromatid and chromosome gaps were recorded but were not used in the analysis, polyploidy or endoreduplication were not included in the totals or in the statistical analysis.

Evaluation criteria:

A positive response at a single dose was designated "+W", weak evidence for clastogenicity. If there was a strong trend as the result of a large increase in aberrations at a single dose only,the result was designated as "+W*". A test was designated "+" if at least two doses gave significantly increased responses. As a general rule, a test was repeated to confirm a positive result if there was one or more elevated points, if toxicity was too great, or if the controls did not give the expected responses. When combining the conclusions from 2 or more trials, more weight was given to the trials(s) that had a more appropriate experimental design, based on dose range and harvest time. Factors such as a reduced number of cells scored or a high solvent control usually reduced the weight given to a result. In general, in combining the conclusions of 2 trials, a chemical was designated as "+" if both trials were "+", or "+W" and "+" or "+" and "?". A "+W" and "?" were judged to be a "+W". A "+" response that was not repeatable gave rise to a "?", whereas a "+W" or "?" response that was not repeatable was considered "-".

Statistics:

A binomial sampling assumption as described by Margolin et al. (1983) was used to examine absolute increases in aberrations over solvent control levels at each dose. The P values were adjusted by Dunnett's method to take into account the multiple dose comparisons. Only the "total" percent cells with aberrations were analyzed, and a positive response was defined as one for which the adjusted P value was <0.05.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Highest dose was chosen to allow sufficient numbers of M2 cells for analysis at time of harvest.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Chromosome aberrations:

-S9 (harvest time: 10.5 h)		+S9 (harvest time: 20.0 h, 2nd trial)
Dose (µg/ml)	% cells with aberrations (total)	Dose (µg/ml)	% cells with aberrations (total)
0	3.0	0	1.5
2.99	6.0	9.95	5.0
9.95	3.0	14.9	3.5
29.9	5.0	19.9	4.0

(With metabolic activation, the trial with the higher dose levels was chosen here for presentation)

Reference 3

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:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Dose and identity of positive control substance not reported

Principles of method if other than guideline:

Experiment performed before actual guideline was established.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA97, TA98, TA100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat and hamster liver S-9

Test concentrations with justification for top dose:

without S-9: 0, 0.3, 1.0, 3.0, 10.0, 16.0 µg/plate (all strains);
with S-9: 0, 10.0, 33.0, 100.0, 333.0, 666.0 µg/plate (TA1535, TA1537 and TA97 + 10% HLI, 10% RLI, 30% HLI or 30% RLI; TA100 and TA98 + 10% HLIor 10% RLI); 0, 3.0, 10.0, 33.0, 100.0, 333.0 µg/plate (TA100 and TA98 + 30% HLI or 30% RLI)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA97, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation occurred at 666 µg/plate with 10% HLI or 10% RLI in strains TA100, TA1537, TA97, TA98

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Maximum number of revertants (mean ± SEM):

Strain	TA100	TA1535	TA1537	TA97	TA98
Negative control
-S9	91 ± 2.8	16 ± 3.5	8 ± 0.3	126 ± 9.5	14 ± 2.8
+ 10% HLI	83 ± 4.6	10 ± 1.5	12 ± 1.9	147 ± 9.8	23 ± 2.2
+ 30% HLI	166 ± 6.6	9 ± 2.3	11 ± 1.3	187 ± 2.4	27 ± 2.2
+ 10% RLI	108 ± 3.2	11 ± 2.2	7 ± 0.7	179 ± 8.9	24 ± 3.0
+ 30% RLI	146 ± 4.4	16 ± 2.0	10 ± 1.2	193 ± 5.4	20 ± 1.7
Test substance
-S9	90 ± 1.5 (3 µg)	16 ± 2.3 (1 µg)	8 ± 1.3 (0.3 µg)	151 ± 7.4 (3 µg)	16 ± 2.7 (1 µg)
+ 10% HLI	108 ± 5.6 (100 µg)	9 ± 2.0 (100 µg)	11 ± 4.0 (10 µg)	172 ± 16.9 (100 µg)	31 ± 0.9 (10 µg)
+ 30% HLI	150 ± 0.9 (10 µg)	12 ± 1.5 (10 µg)	12 ± 0.9 (10 µg)	201 ± 5.6 (10 µg)	27 ± 4.1 (33 µg)
+ 10% RLI	115 ± 11.6 (10 µg)	8 ± 1.0 (10 µg)	10 ± 1.2 (10 µg)	194 ± 6.4 (10 µg)	25 ± 0.6 (10 µg)
+ 30% RLI	124 ± 11.6 (3 µg)	15 ± 0.3 (10 µg)	13 ± 1.0 (10 µg)	219 ± 8.4 (100 µg)	25 ± 5.0 (33 µg)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test article was negative in an in vivo dominant lethal assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Principles of method if other than guideline:

Study performed before actual guideline was established.

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Flow
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 30 ± 2.5 g
- Housing: 5/cage while treated, then after 2 days rest separately with two females for mating
- Diet (e.g. ad libitum): 4 % fat diet ad libitum
- Water (e.g. ad libitum): acidified according to approved laboratory animal health standards, ad libitum

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Duration of treatment / exposure:

5 days

Frequency of treatment:

daily

Post exposure period:

2 days

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

166 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Route of administration: intraperitoneal
- Doses / concentrations: 0.3 mg/kg bw in 0.85 % saline

Tissues and cell types examined:

number of dead, living, and total embryos in the uterus

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
1/10, 1/30 and 1/100 of LD50; but high dose of 500 mg/kg bw was selected by the contract monitor, other doses were then calculated in relation to this dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Males were treated for 5 consecutive days, after 2 days rest each male was mated with 2 virgin females from Monday through Friday. This sequence was repeated weekly with 2 new females each week for 8 weeks (period of spermatogenesis). Fourteen days from the midweek in which they were caged with the males (mid-pregnancy), females were sacrificed, dissected, and the number of dead, living, and total embryos in the uterus as well as the level of fertility recorded.

Evaluation criteria:

Dominant lethality is indicated by a high percentage of dead implants to total implants.

Statistics:

The number of dead, living, and total embryos in the uteri were statistically analyzed for indications of dominant lethality, and compared with control data for significance.

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Dominant lethality was not demonstrated by data from PANA administered to mice. In comparison, the positive control compound TEM demonstrated a clear dominant lethal effect in mice during weeks 1 through 3.

Dead implants / total implants:

Week	Negative control	50 mg/kg	166 mg/kg	500 mg/kg	Positive control
1	0/72 = 0.0	1/45 = 0.02	1/64 = 0.02	0/34 = 0.0	7/16 = 0.44*
2	3/118 = 0.03	3/126 = 0.02	5/77 = 0.06	5/135 = 0.04	39/50 = 0.78**
3	7/117 = 0.06	1/75 = 0.01	0/84 = 0.0	5/97 = 0.05	20/46 = 0.43**
4	4/138 = 0.03	5/81 = 0.05	5/61 = 0.08	1/106 = 0.01	8/160 = 0.05
5	4/50 = 0.07	5/106 = 0.06	1/44 = 0.02	3/60 = 0.05	0/12 = 0.0
6	4/204 = 0.02	3/143 = 0.02	9/148 = 0.06*	4/142 = 0.03	7/183 = 0.04
7	5/139 = 0.04	1/28 = 0.04	6/112 = 0.05	7/116 = 0.06	4/117 = 0.03
8	7/135 = 0.05	8/125 = 0.06	4/74 = 0.05	2/68 = 0.03	1/132 = 0.01

* P<0.05

**P<0.01

Isolated instances of apparent significant dominant lethality did occur among the experimental data. They were not associated with dose-related trends and had dead implant/total input ratios that fell within the range of all negative controls (intermediate dose, week 6).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro data

N-phenyl-1-naphthylamine was not mutagenic in bacterial tests conducted in the presence or absence of metabolic activation. These tests included bacterial Salmonella typhimurium (Zeiger et al., 1988; Rannug et al., 1984) or Salmonella typhimurium/E.coli WP2 (Baden et al., 1978; Brusick and Matheson, 1976, 1977) reverse mutation assays. A mitotic recombination assay in Saccharomyces cerevisiae yielded also negative results (Brusick and Matheson, 1976, 1977). In mammalian cells, no mutagenicity was observed in the mouse lymphoma (L5178Y cells) assay with and without metabolic activation (Brusick and Matheson, 1976, 1977). In the presence and absence of an external metabolising system, N-phenyl-1-naphthylamine did not lead to an increase in chromosomal aberrations in Chinese hamster ovary cells or in Chinese hamster lung fibroblasts (Loveday et al., 1990; Sofuni et al., 1990).

A sister chromatid exchange (SCE) assay in Chinese hamster ovary cells was marginally positive in the presence of an external metabolising system, but not without metabolic activation (Loveday et al., 1990). An unscheduled DNA synthesis (UDS) assay with human lung (WI-38) cells yielded ambiguous results (Brusick and Matheson, 1976, 1977). Repeatedly positive responses occurred in non-activated but not in activated (negative in the 1st trial, positive in the 2nd trial) test systems and the effects showed no dose-response relationship. The findings of the UDS assay were contradictory to the negative results in the chromosomal aberration assays as well as to the weakly positive result the SCE assay which was only positive in presence of an external metabolising system. With respect to the conflicting results and the lack of dose-response relationship, these findings should be considered as artefacts and not taken into account for assessment.  

In vivo data

A dominant lethal assay conducted in mice did not produce significant adverse effects on reproductive performance parameters (Brusick and Matheson, 1976, 1977). Male mice were intraperitoneally administered N-phenyl-1 -naphthylamine for 5 consecutive days followed by 2 days without exposure. Each male was then caged with two virgin females. The examination of the females showed no alterations in the number of dead embryos in the uteri compared to the control animals.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The in vitro and in vivo studies are negative for genetic toxicity. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.