N-(dodecylphenyl)naphthalen-1-amine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 July 1999 and 19 August 1999.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

-

EC Number:

437-450-6

EC Name:

-

Cas Number:

64654-05-3

Molecular formula:

Hill formula: C28 H37 N

IUPAC Name:

N-(dodecylphenyl)naphthalen-1-amine

Test material form:

liquid: viscous

Details on test material:

Sponsor's identification: APAN
Description: redbrown viscous liquid
Lot number: EL01010B01/KZ8911.5
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

No further details specified

Method

Target gene:

histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia col i strain WP2uvrA was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm
the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

The dose range was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate in the range-finding study. The experiment was repeated on a separate day using the same dose range as the range-finding study, fresh cultures of the bacterial strains and fresh test material formulations.

Vehicle / solvent:

The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes on the day of each experiment.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The dose range of the test material was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The study was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA),1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material and a vehicle control (dimethyl sulphoxide) were tested.
In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar was overlaid onto sterile Vogel-Bonner Minimal agar plates in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 μg/plate because of excessive precipitation.

Mutation Study - Experiment 1 (Range-finding Study)
Five concentrations of the test material (50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of 59-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 μg/plate because of excessive precipitation.

Mutation Study - Experiment 2 (Main Study)
The second experiment was performed using methodology as described for the range-finding study, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as the range-finding study (50 to 5000 μg/plate).

Rationale for test conditions:

Not specified

Evaluation criteria:

The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Study
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Study
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments of the main study was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation
Study.
No toxicity was exhibited to any of the strains of bacteria used. A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate, this did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

Preliminary Toxicity Study

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (μg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	109	109	104	111	104	110	118	131	112	127	91P
+	TA100	107	124	137	126	128	108	116	111	99	85	87P
-	WP2uvrA	21	26	19	26	24	21	25	24	21	23	26P
+	WP2uvrA	34	33	38	30	30	30	25	37	25	30	39P

P = precipitate

 

SPONTANEOUS MUTATION RATES

(CONCURRENT NEGATIVE CONTROLS)

 

RANGE-FINDING STUDY

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
91 93 87	(90)	16 14 10	(13)	27 26 22	(25)	21 13 16	(17)	9 3 6	(6)

MAIN STUDY

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
99 72 74	(82)	14 29 24	(22)	12 19 17	(16)	11 11 14	(12)	15 12 8	(12)
								12 12 13	(12)*

* Experimental procedure performed without S9-mix only.

 

RANGE-FINDING STUDY – WITHOUT METABOLIC ACTIVATION

With or without S9-mix	Test substance concentration (μg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA		TA98		TA1537
-	0	89 80 79	(83) 5.5#	25 31 21	(26) 5.0	35 32 21	(29) 7.4	17 22 32	(24) 7.6	10 15 10	(12) 2.9
-	50	74 85 76	(78) 5.9	19 20 19	(19) 0.6	22 30 18	(23) 6.1	18 17 16	(17) 1.0	10 12 10	(11) 1.2
-	150	95 95 82	(91) 7.5	19 21 25	(22) 3.1	10 25 C	(18) 10.6	20 13 18	(17) 3.6	7 10 6	(8) 2.1
-	1500	85 88 70	(81) 9.6	26 28 31	(28) 2.5	22 29 27	(26) 3.6	17 11 12	(13) 3.2	12 18 10	(13) 4.2
-	5000	88P 81P 73P	(81) 7.5	25P 19P 22P	(22) 3.0	12P 15P 18P	(15) 3.0	10P 20P 12P	(14) 5.3	11P 10P 11P	(11) 0.6
Positive controls	Name	ENNG		ENNG		ENNG		4NQO		9AA
S9-Mix	Concentration (μg/plate)	3		5		2		0.2		80
-	No. colonies per plate	342 329 367	(346) 19.3	219 179 139	(179) 40.0	834 635 629	(699) 116.7	153 150 130	(144) 12.5	1042 1000 689	(910) 192.8

ENNG     N-ethyl-N’-nitro-N-nitrsoguanidine                      #             Standard deviation

4NQO     4-Nitroquinolinw-1-oxide                                  C             Contaminated

9AA        9-Aminoacridine                                            P             Precipitate

 

RANGE-FINDING STUDY – WITH METABOLIC ACTIVATION

With or without S9-mix	Test substance concentration (μg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA		TA98		TA1537
+	0	94 124 121	(113) 16.5#	16 15 14	(15) 1.0	26 25 22	(24) 2.1	21 19 20	(20) 1.0	17 4 12	(11) 6.6
+	50	82 80 75	(79) 3.6	13 12 17	(14) 2.6	29 34 15	(26) 9.8	25 25 19	(23) 3.5	12 13 14	(13) 1.0
+	150	77 92 85	(85) 7.5	9 9 12	(10) 1.7	12 30 23	(22) 9.1	21 29 19	(20) 1.0	4 11 14	(10) 5.1
+	1500	90 86 80	(85) 5.0	14 18 11	(14) 3.5	25 20 19	(21) 3.2	34 13 20	(22) 10.7	14 13 11	(13) 1.5
+	5000	91P 95P 87[	(91) 4.0	13P 11P 10P	(11) 1.5	21P 30P 29P	(27) 4.9	17P 20P 24P	(20) 3.5	15P 12P 6P	(11) 4.6
Positive controls	Name	2AA		2AA		2AA		BP		2AA
S9-Mix	Concentration (μg/plate)	1		2		10		5		2
+	No. colonies per plate	1324 1214 1335	(1291) 66.9	155 318 216	(230) 82.4	1091 1124 1150	(1122) 29.6	550 603 532	(562) 36.9	295 328 X	(312) 23.3

BP           Benzo(a)pyrene                                               #             Standard deviation

2AA        2-Aminoanthracene                                           C             Contaminated

X             Poor lawn and colony growth                               P             Precipitate

 

MAIN STUDY – WITHOUT METABOLIC ACTIVATION

With or without S9-mix	Test substance concentration (μg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA		TA98		TA1537
-	0	73 74 94	(80) 11.8#	21 17 29	(22) 6.1	20 12 20	(17) 4.6	11 12 17	(13) 3.2	9 13 10	(11) 2.1
-	50	95 86 70	(84) 12.7	33 29 29	(30) 2.3	22 23 18	(21) 2.6	13 11 12	(12) 1.0	10 11 13	(11) 1.5
-	150	81 89 91	(87) 5.3	19 26 27	(24) 4.4	18 24 17	(20) 3.8	23 17 14	(18) 4.6	16 9 8	(11) 4.4
-	500	90 93 92	(92) 1.5	25 29 19	(24) 5.0	14 15 20	(16) 3.2	19 22 14	918) 4.0	14 9 11	(11) 2.5
-	1500	85 76 89	(83) 6.7	22 26 38	(29) 8.3	11 12 11	(11) 0.6	15 14 12	(14) 1.5	14 7 10	(10) 3.5
-	5000	76P 74P 87P	(79)]7.0	31P 21P 36P	(29) 7.6	22P 21P 11P	(18) 6.1	19P 19P 14P	(17) 2.9	5P 13P 12P	(10) 4.4
Positive controls	Name	ENNG		ENNG		ENNG		4NQO		9AA
S9-Mix	Concentration (μg/plate)	3		5		2		0.2		80
-	No. colonies per plate	796 799 684	(760) 65.5	317 256 379	(317) 61.5	973 887 1172	(1011) 146.2	210 188 177	(192) 16.8	979 1070 988	(1012) 50.1

ENNG     N-ethyl-N’-nitro-N-nitrsoguanidine                      #             Standard deviation

4NQO     4-Nitroquinolinw-1-oxide                                  P             Precipitate

9AA        9-Aminoacridine

 

MAIN STUDY – WITH METABOLIC ACTIVATION

With or without S9-mix	Test substance concentration (μg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA		TA98		TA1537
+	0	79 95 84	(86) 8.2#	9 15 17	(14) 4.2	19 26 24	(23) 3.6	33 27 15	(25) 9.2	11 11 14	(12) 1.7
+	50	69 84 111	(88) 21.3	15 13 16	(15) 1.5	21 17 21	(20) 2.3	28 27 28	(28) 0.6	14 11 14	(13) 1.7
+	150	90 85 105	(93) 10.4	10 21 23	(18) 7.0	20 15 15	(17) 2.9	26 34 28	(29) 4.2	18 17 13	(16) 2.6
+	500	97 111 83	(97) 14.0	16 14 9	(13) 3.6	15 23 29	(22) 7.0	33 29 20	(27) 6.7	17 5 12	(11) 6.0
+	1500	72 92 82	(82) 10.0	15 17 13	(15) 2.0	22 12 29	(21) 8.5	25 20 20	(22) 2.9	10 10 12	(11) 1.2
+	5000	84P 79P 86P	(83) 3.6	18P 20P 13P	(17) 3.6	22P 29P 16P	(22) 6.5	14P 14P 21P	(16) 4.0	15P 11P 14P	(13) 2.1
Positive controls	Name	2AA		2AA		2AA		BP		2AA
S9-Mix	Concentration (μg/plate)	1		2		10		5		2
+	No. colonies per plate	778 919 836	(844) 70.9	279 397 352	(342) 59.6	452 459 411	(441) 25.9	415 453 476	(448) 30.8	255 424 210	(296) 112.9

BP           Benzo(a)pyrene                                   #             Standard deviation

2AA        2-Aminoanthracene                               P             Precipitate

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requiretnents of the OECD Guidelines for Testing of Chemicals No. 471 "Reverse Mutation Study", ethod B 14 of Commission Directive 92/69/EEC and the USA, EPA (TSCA) OPPTS harmonised guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate in the range-finding study. The experiment was repeated on a separate day using the same dose range as the range-finding study, fresh cultures of the bacterial strains and fresh test material formulations.

 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.

 

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A creamy, opaque film with an associated oily precipitate was observed at 5000 μg/plate, this did not prevent the scoring of revertant colonies.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.