12-aminododecanoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 June 1991 - 9 September 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

The study predates the last update of the guideline and used TA1538 instead of a cross-linking species

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

12-aminododecanoic acid

EC Number:

211-754-7

EC Name:

12-aminododecanoic acid

Cas Number:

693-57-2

Molecular formula:

C12H25NO2

IUPAC Name:

12-aminododecanoic acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Appearance: white powder
Storage: room temperature in the dark

Method

Target gene:

S. typhimurium: Histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535; TA1537; TA1538; TA98; TA100

Details on mammalian cell type (if applicable):

- Type and identity of media: top agar consisting of 0.6 % Difco agar, 0.5 % NaCl and 0.05 mM L-histidine. HCl/ 0.05 mM biotin. The agar was maintained at 46 ºC and 0.1 mL of a fully grown culture of the tester strain, 0.1 mL of the apropriate suspension of test material and 0.5 mL of the S-9 mix (if required) was added. the ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Toxicity test:
-0, 0.125, 1.25, 12.5, 125.0 and 1250.0 µg/plate

Mutagenicity assay:
-0, 154.3, 463.0, 1388.9, 4166.7 and 12500 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 2-nitrofluorene; 9-aminoacridine; 2-aminoanthracene

Details on test system and experimental conditions:

ORIGIN OF STRAINS
The genotypes of the Salmonella strains are given below.
-TA1537: His C3076, rfa, uvrB, -R-factor
-TA1538: His D3052, rfa, uvrB, -R-factor
-TA98: His D3052, rfa, uvrB, +R-factor
-TA1535: His G46, rfa, uvrB, -R-factor
-TA100: His G46, rfa, uvrB, +R-factor
rfa: this mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases the permeability to large molecules
uvrB: this mutation is a deletion of a gene coding for the DNA excision repair system, which results in greatly increased sensitivity in detecting many mutagens
R-factor: the R-factor strains contain the plasmid pKM 101, which increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA-repair system normally present in S. typhimurium. It carries an ampicillin resistance gene.

METABOLIC ACTIVATION SYSTEM
-Preparation of liver homogenate and S-9:
Male Wistar-outbred Hsd/Cpb:WU rats (body weights 202-216 g) were injected intraperitoneally with a single dose of Aroclor 1254 (500 mg/kg body weight) in soya bean oil (20 % w/v). The rats were provided with tap water and ITV stock diet ad libitum. Five days after the injection of Aroclor 1254 the rats were killed by CO2 gas. The livers were removed aseptically and immediately put into a cold, sterile 0.15 M KCl solution. After washing in the KCl solution, the livers were weighed, cut into pieces and homogenized in 3 volumes of 0.15 M KCl solution in a Potter-Elvehjem apparatus with a Teflon pestle. The homogenate was centrifuged for 10 minutes at 9000 x gravity. The supernatant, S-9, was collected and divided into small aliquots in sterile polypropylene vials. The vials were quickly frozen on dry ice and subsequently stored in a freezer at -80 °C. The S-9 was checked for sterility. The protein and cytochrome P-450 content of the S-9 fraction was determined according to the method published by Rutten et al. (1987); it contained 0.96 µmol cytochrome P-450 per gram protein.
-Preparation of S-9 mix
Immediately before use in the mutagenicity assay, an S-9 mix was prepared by mixing the thawed S-9 with a NADPH generating system. The final concentrations of the various ingredients in the S-9 mix were: MgCl2 8 mM; KCl 33 mM; G-6-P 5 mM; NADP 4 mM; sodium phosphate 100 mM (pH 7.4) and S-9 10 %. The S-9 mix was kept in ice before and during use.
-Activity of S-9
The ability of the S-9 to activate the metabolic system was tested with 4-amino-biphenyl and 2-aminoanthracene. Both compounds require metabolic activation to become mutagenic in S. typhimurium TA 98. The results of the experiments, carried out with different amounts of S-9 in the S-9 mix, show the bacteria exhibiting the expected sensitivity.

-Toxicity test
A preliminary test with 5 concentrations of the test material and the vehicle (DMSO), as negative control, was carried out to assess the toxicity of the test material to S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, both in the absence and in the presence of the S-9 mix.
Just before use, a stock suspension containing 12.5 mg of the test material per mL was prepared in DMSO. A suspension of the test material in DMSO was obtained after placing the stock solution in an Ultra Sonor waterbath (50°C) for 25 minutes. For the other dose levels a series of stepwise dilutions - by a factor of 10 - in DMSO were prepared from the stock suspension test material just before use.

To 2 mL molten top agar (containing 0.6 % Difco agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/ 0.05 mM biotin), maintained at 46 °C, were added subsequently: 0.1 mL of a fully grown culture of the appropriate tester strain, 0.1 mL of the appropriate suspension of the test material and 0.5 mL S-9 mix, if any. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % Bacto-Difco agar in Vogel and Bonner medium E with 2 % glucose). After incubation for three days at 37 °C, the background lawn of bacterial growth was examined microscopically and the his+ revertants were counted.
In view of the results, 1250 µg/plate was chosen as the highest concentration for the mutagenicity assays, both in the absence and in the presence of the S-9 mix.

-Mutagenicity assay
In both experiments the plate-incorporation method with the histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100 as indicator strains was applied. This assay has been described in detail by Ames et al. (1975) and by Maron and Ames (1983).
Appropriate suspensions of the test material, containing 0, 154.3, 463.0, 1388.9, 4166.7 and 12500 µg/ml, were prepared in DMSO just before use. The repeat experiment was carried out with the same dose levels. A suspension of the test material in DMSO was obtained after placing the stock suspension in an Ultra Sonor waterbath (50 °C) for 25 minutes.

The reference mutagens used as positive controls were:
-sodium azide: 1.0 µg per plate with the strains TA 1535 and TA 100, in the absence of the S-9 mix
-2-nitrofluorene; 2.0 µg per plate with the strains TA 1538 and TA 98, in the absence of the S-9 mix
-9-aminoacridine: 80.0 µg per plate with strain TA 1537, in the absence of the S-9 mix
-2-aminoanthracene: 2.0 µg per plate with the strains TA 1535, TA 1538, TA 98 and TA 100, 5.0 µg per plate with strain TA 1537, in the presence of the S-9 mix.

To 2 mL molten top agar, maintained at 46 °C, were added subsequently: 0.1 mL of the appropriate tester strain, 0.1 mL of the appropriate suspension of the test substance or the controls and 0.5 mL S-9 mix, if any. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates. All determinations were made in triplicate. The plates were incubated at 37 °C for three days. Then the his+ revertants were counted by an Artek Electronic Counter. The background lawn of bacterial growth was examined microscopically.

S-9 and S-9 mix were checked for sterility.

The viable count of each culture was made by plating appropriate dilutions of the cultures on nutrient broth agar plates. Each culture was examined for the number of spontaneous revertants, histidine requirement and sensitivity to ampicillin, crystal violet and UV radiation.

Evaluation criteria:

-Evaluation of test results
A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the vehicle, together with evidence of a dose-response.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA1535; TA1537; TA1538; TA98; TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Toxicity test:
-The results show that at all dose levels the test material was not toxic (both in the absence and in the presence of the S-9 mix) to all strains. No precipitation of the test material was observed. The number of spontaneous revertants was 29 (TA 1535), 11 (TA 1537), 38 (TA 1538), 46 (TA 98) and 181 (TA 100).

Mutagenicity assay:
-From the results obtained in both experiments it appeared that incubation of the test material with the bacteria did not increase the number of his+ revertants with S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, either in the absence or in the presence of the S-9 mix.
At all the dose levels tested, the test material appeared not to be toxic to all strains. No precipitation of the test material was observed.
The positive controls used in the present assays gave the expected strong increase in the number of his+ revertants, both in the absence and in the presence of the S-9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of the toxicity test in Salmonella typhimurium

Amount of test material per plate (µg)	S-9 mix (yes (Y) no (N))	Number of his+ revertants per plate
		TA 1535	TA 1537	TA 1538	TA 98	TA 100
0	N	30	12	20	55	206
0.125	N	30	16	39	43	143
1.25	N	36	5	31	52	173
12.5	N	26	13	24	45	134
125	N	29	13	25	43	132
1250	N	32	16	21	41	127
0	Y	37	17	33	92	169
0.125	Y	40	16	62	89	162
1.25	Y	38	25	48	90	179
12.5	Y	24	15	53	67	161
125	Y	19	20	52	73	178
1250	Y	26	16	48	115	158

Table 2: Results of the first experiment

Dose (µg/pl)	TA 1535		TA 1537		TA 1538		TA 98		TA 100
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	58	20	25	25	54	73	53	79	154	189
	56	29	21	33	41	74	50	64	151	185
	53	29	16	37	38	63	53	76	156	182
15.43	52	22	17	30	52	64	42	87	171	186
	56	23	14	27	62	75	43	57	165	180
	50	22	20	29	50	73	72	63	163	165
46.3	69	25	13	37	55	73	55	84	149	182
	42	24	15	28	49	61	33	69	158	167
	56	14	15	27	48	75	43	76	152	172
138.89	68	33	13	21	51	76	53	73	171	170
	68	28	18	28	43	66	43	86	134	154
	71	38	15	36	63	69	40	99	142	172
416.67	46	15	20	19	44	61	52	87	141	181
	40	23	20	26	44	72	55	68	140	145
	36	15	25	27	39	698	51	48	159	174
1250	59	28	18	33	49	69	54	87	148	163
	57	24	13	36	40	75	49	65	179	166
	54	22	14	30	44	53	53	79	188	178
Pos C.	379	361	1713	170	1306	7428	1057	1405	585	1571
	400	324	1412	199	1384	292	979	1584	463	1471
	417	412	1522	203	1789	349	988	1378	505	1250

Table 3: Results of the second experiment

Dose (µg/pl)	TA 1535		TA 1537		TA 1538		TA 98		TA 100
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	45	20	18	16	51	67	63	76	152	179
	51	31	14	25	52	72	45	74	159	162
	46	21	15	27	50	81	36	102	145	151
15.43	50	22	24	24	55	69	56	90	162	167
	47	34	15	8	42	68	69	78	138	174
	44	22	20	17	50	84	54	88	141	186
46.3	39	6	18	28	53	67	73	63	158	152
	50	25	19	18	52	72	69	81	156	183
	44	28	9	14	54	80	68	92	138	162
138.89	50	26	17	25	54	69	62	64	142	170
	48	38	13	17	45	78	56	78	141	170
	59	35	25	28	67	67	62	78	157	181
416.67	49	28	19	21	62	74	76	77	160	180
	33	24	18	20	38	67	62	73	145	189
	34	24	19	21	50	73	65	89	150	181
1250	38	17	6	15	45	61	65	62	151	158
	47	24	12	15	32	69	62	80	153	180
	47	28	13	17	43	55	66	84	156	182
Pos C.	338	305	2820	246	1595	1446	744	997	215	822
	293	267	2880	288	1462	1335	799	1158	315	828
	325	341	2900	317	1609	1359	721	1057	352	810

Pos C. = positive control

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not show mutagenic activity in Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 either in the absence or in the presence of the S-9 mix, under the conditions employed in this evaluation.

Executive summary:

The test material did not show mutagenic activity in Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 either in the absence or in the presence of the S-9 mix, under the conditions employed in this evaluation. The test was conducted in accordance with standardised guideline OECD guideline 471.