11-(heptylamino)undecanoic acid

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

Study period:

From November 1990 to March 1991

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

1984

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

11-(heptylamino)undecanoic acid

EC Number:

271-487-7

EC Name:

11-(heptylamino)undecanoic acid

Cas Number:

68564-88-5

Molecular formula:

C18H37NO2

IUPAC Name:

11-(heptylamino)undecanoic acid

Test material form:

solid

Details on test material:

CAS N°. 68564-88-5
Test item appearance : white solid
.Batch number : 091137

Method

Target gene:

hypoxanthine-phosphoribosyl-transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Established V79 cell line, clone 379-A, supplied by Flow Laboratories, France (Chinese hamster lung cells, fibroblast-like morphology). Cells were maintained in liquid nitrogen until needed. Each study was started with a frozen inoculum from a cell clone previously selected after exposure in a medium containing' hypoxanthine, aminopterin and thymidine, in order to obtain a Iow rate of spontaneous mutation frequency. At termination of experiments, the cell clone was discarded.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Toxicity study:
With and without S-9 mix: 5, 10, 50, 75, 100 and 150 Mutagenicity studies (first and second study):
With and without S-9 mix: 10, 50, 75, 100 and 150

Vehicle / solvent:

Pure Ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

no

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

Cel/s: Established V79 cell line, clone 379-A, supplied by Flow Laboratories, France (Chinese hamster lung cells, fibroblast-like morphology). Cells were maintained in liquid nitrogen until needed. Each study was started with a frozen inoculum from a cell clone previously selected after exposure in a medium containing' hypoxanthine, aminopterin and thymidine, in order to obtain a Iow rate of spontaneous mutation frequency. At termination of experiments, the cell clone was discarded.
Growth medûun: Eagle's minimal essential medium {MEM) with Earles' sait solution, penicillin (100 IU/ml), streptomycin (100 µg/ml), supplemented with non essential amino acid, and 9% foetal calf serum.
Treatment medi,un: same as growth medium but without foetal calf serum
Se/ective medium: growth medium supplemented with 5 µg/ml of 6-thioguanine.
Cel/ subcultures: trypsin solution (I/250).
Culture conditions: incubation at 37°C - 5% CO2 - 95-100% humidity.
Fixation and staining of clones: 0. l % met_hylene blue in 70% (V /V) ethanol.

Test and control compounds
2.4.1 Untreated and solveot controls
An untreated cell culture (treatment medium contrai, coded CC), and a solvent (pure ethanol) contral were included in each toxicity and· mutation assays. They were subjected to the same experimental conditions as cultures treated with NHAU.
2.4.2 Positive controls
The positive contrai used in the absence of S-9 mix was ethyl methane sulfonate (EMS) dissolved and diluted in DMSO, and tested at a final concentration of 6 mM.
In the presence of S-9 mix, Benzo(a)pyrene (B(a)P) was used, also dissolved and diluted in DMSO to obtain a final concentration of 20 µg/ml.
Both positive contrai cultures, whether with or without metabolic activation, were subjected to all the experimental manipulations that the treated cultures received.
2.4.3 Test compound
This assay was performed with NHAU, Batch 091137 (AT OCHEM filling number 1751/89); test article description and certificate of analysis are appended to this report.
NHAU was dissolved and diluted extemporaneously in pure ethanol.
The concentrations tested ranged between 0.6 mg/ml and 18 mg/ml (NHAU being not soluble at higher concentrations) and the final concentrations between 10 and 150 µg/ml.
Treatment was performed by adding 25 pl of each diluted concentration. Each concentration was run simple.

Preparation of S-9 fraction
A liver homogenate from rats injected with Aroclor 1254 was prepared following the technique of Ames et al (3).
Male Sprague Dawley rats were pretreated once by the intraperitoneal route with Aroclor 1254 at the dose level of 500 mg/kg. Five days later, animais were sacrificed and livers were removed, homogenized in a KCI 0.15 M solution, and centrifuged at 9000 x g for 10 minutes. The S-9 supernatant fraction was stored at -80°C. Two preparations were used and for each preparation total proteins and cytochrome P450/P448 were measured. The values obtained were: 31 mg/ml and 4.3 nmoles cytochrome P450/P448/mg microsomal proteins used for study 6TG056 and 42 mg/ml and 4.3 nmoles cytochrome P450/P448/mg microsomal proteins used for study 6TG058.
2.3 Preparation of S-9 mix
The S-9 mix metabolic activation system was reconstituted extemporaneously in ice: it consisted of 8 mM MgC12, 30 mM KCl, 5 mM glucose-6-phosphate, and 4 mM NADP, in 0.2M phosphate buffer, pH= 7.4. This preparation was immediately filter-sterilized by passage through a 0.45 µm Microflow filter before addition of the S-9 fraction at a concentration of 10%. The S-9 mix fraction was then added to the grawth medium at the final concentration of 10% just before treatment ( = 300 µl for 3 ml of final treatment medium).

Toxicity Test:
The highest concentration tested was 150 µg/ml (the compound was not soluble in pure ethanol at concentrations exceeding 18 mg/ml).

Mutagenicity test:
Exposure conditions
V79 cell populations of controls (untreated, solvent, positive) and plates treated with NHAU were in exponential phase of growth at the time of exposure ; to this purpose, cells were seeded in growth medium in each of the plates prepared for both studies (6TG056 and 6TG058) at a density of 10 6 cells/plate for 24 hours before exposure to the compound (6TG056 study) and of 5 x 105 cells/plate for 48 hours before exposure to the compound (6TG058 study).
A culture per concentration was performed in treatment medium for at a least 3 hours. After treatment, cultures were washed twice with calcium- and magnesium-free Hanks balanced sait solution, and were trypsinized: trypsin solution was neutralized by an equal volume of culture medium.
V79 cells were counted in every cell suspension with a Model ZBI Coulter counter. An aliquot of these suspensions was sampled, then diluted and 200 cells were seeded per 6-cm petri dish in order to determine the cell viability immediately after treatment (CFE 1). Triplicate dishes were performed for each concentration, and after 4-5 days, colonies were fixed, stained with methylene blue, and counted using an electronic counter.
At the same time, a first subculture in 175 cm2 flasks was performed with the remaining cells at a highest density (5.105 cells/flask for 4 days or 106 cells/flask for 3 days).
Then two subcultures have been performed to allow phenotype expression.

Evaluation criteria:

Values obtained for mutation frequency in treated and control groups were then compared.
The first criterion for acceptance of a genotoxic effect is that the mutation frequency in the exposed culture must be significantly greater than in the control, the second criterion being the evidence of a dose-related pattern, and the third one being the reproducibility of the positive response.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 150 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity test
At the concentrations tested : 5, 10, 50, 75, 100 and 150 pg/ml, no cytotoxicity was observed whatever the concentration with or without S-9 mix. Cell morphology was in each case similar to that of control cultures. Further to these results (no decrease in CFEI values), the following concentrations were selected for the mutagenicity assays: IO, 50, 75, 100 and 150 µg/ml with and without S-9 mix.
M utagenicity test
First study (6TG054i)
Thereafter present group mean results obtained under the different experimental conditions with an without S9
ithout S-9 mix
The mutation frequency values in cultures treated with 10, 50, 75, 100 and 150 µg/ml NHAU were in the same range as control cultures.
Besides, given the heterogeneousness of the values obtained for untreated cultures and cultures treated with ethanol, the statistical analysis could not be performed.
With S-9 mix
No gene mutation was induced at the locus HPRT whatever the concentration tested.
The values of mutation frequencies in treated cultures were in the same range as control cultures.
The statistically significant value at 10 µg/ml had no biological relevance since the mutation freq uency was decreased compared with contrais.
A marked increase in the mutant cells was obtained in cultures treated with positive contrais EMS or B(a)P which indicates chat the cell lines were normally sensitive and that the metabolic activation system was potentially active.
Second study ( 6TG058)
The concentrations tested were identical to those of the first study.
Tables (3.2.2).1 and (3.2.2).2 present group mean results obtained (individual results are presented in section 5).
Without metabolic activation, no increase in the mutation frequencies was observed at concen­trations ranging from 10 to 75 µg/ml. At 100 µg/nù and at 150 µg/ml, a slight statistically but not biologically significant increase in the mutation frequency was noted ; indeed, the values of 5.56 and 13.47 6TGr mutants per 106 cells remains within the laboratory historical values which are ranging from 1.22 to 10.62 6TGr mutants per 1()6 cells obtained with cell control.s (26 last studies) and from 0.52 to 16.52 6TGr mutants per 106 cells obtained with DMSO controls (24 last studies).
With S9-mix, no gene mutation was induced at the locu_s HPR T whatever the concentratio11 tested.
In addition, the mutation frequency observed with the positive controls (EMS and B(a)P) was very high.
In view of the se two studies, it can therefore be conduded that NHA U has no genotoxic .activity whether in presence or absence of S-9 mix, whatever the concentration tested.

Applicant's summary and conclusion

Conclusions:

NHAU was not genotoxic in vitro in the HPRT/V79 mutagenicity assay.

Executive summary:

The aim of the study was to assess the genotoxic potential of N-Heptylamino:- ll-undecanoic acid (NHAU) in the gene mutation assay at the locus HPRT in Chinese hamster V79 fibroblasts, both in the presence and absence of rat Iiver preparations (S-9 mix) and co-factors required for mixed-function oxidase activity.
NHA U was dissolved and diluted in ethanol. Two independent studies were perf ormed. The concentrations tested ranged from 5 to 150 µg/ml. The highest concentration corresponds to the maximal solubility of NHAU.
With and without metabolic activation, NHAU did not show any cytotoxic effect at the highest concentration of 150 µg/ml and at lower concentrations.
Five concentrations were selected for the mutagenesis studies: 10, 50, 75, 100 and 150 µg/ml. Under these conditions, no significant increase in the mutation frequency was noted whatever the concentrations tested, with and without metabolic activation.
In conclusion, NHAU was not genotoxic in vitro in the HPRT/V79 mutagenicity assay.