Dibutylamine

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

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)

GLP compliance:

yes

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:

Dibutylamine

EC Number:

203-921-8

EC Name:

Dibutylamine

Cas Number:

111-92-2

Molecular formula:

C8H19N

IUPAC Name:

N-butylbutan-1-amine

Details on test material:

Di-n-butylamine

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 media
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

Range finder: 0; 40.41; 80.81; 161.6; 323.3; 646.5; 1293 µg/ml (with and without S9 mix)
Experiment 1: 0, 50, 100, 150, 240, 280, 320, 360 µg/ml (without S9 mix)
0, 80, 160, 240, 280, 400, 450, 525 µg/ml (with S9 mix)
Experiment 2: 0, 50, 100, 150, 250, 300, 320, 340, 360, 400, 450 µg/ml (without S9mix)
0, 100, 200, 300, 350, 400, 450, 500, 525, 550,600 µg/ml (with S9 mix)

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see remarks

Remarks:

4-nitroquinoline 1-oxide 0.10/0.15 µg/ml without S9 mix; benzo[a]pyrene 2.00/3.00 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in medium

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1.The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤ 0.05)
2.There was a significant concentration relationship as indicated by the linear trend analysis (p≤ 0.05)
3.The effects described above were reproducible. Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was copared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 40.41 to 1293 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration tested (1293 µg/mL) was not plated due to excessive toxicity and complete toxicity (0% RS) was observed at the highest concentration plated in the absence and presence of S-9 (646.5 µg/mL). The highest concentration to provide >10% RS was 323.3 µg/mL in the absence and presence of S-9, which gave 12% and 37% RS, respectively.
No marked changes in osmolality were observed in the Range-Finder at the highest concentration tested (1293 µg/mL) as compared to the concurrent vehicle controls (individual data not reported). Marked changes in pH were observed at 1293 µg/mL as compared to the concurrent vehicle controls but no marked changes were observed up to 646.5 µg/mL (individual data not reported). As no concentration tested in the Mutation Experiments was greater than 600 µg/mL no further measurements were performed.

Any other information on results incl. tables

In Experiment 1 ten concentrations, ranging from 50 to 500 µg/mL in the absence of S‑9 and from 80 to 600 µg/mL in the presence of S‑9, were tested. Seven days after treatment, the highest two concentrations tested in the absence of S‑9 (400 and 500 µg/mL) and the highest concentration tested in the presence of S‑9 (600 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, concentrations of 200 µg/mL in the absence of S‑9 and 320 and 360 µg/mL in the presence of S‑9 were not selected as there were sufficient non-toxic concentrations. All other concentrations were selected in the absence and presence of S‑9. The highest concentrations plated were 360 µg/mL in the absence of S‑9 and 525 µg/mL in the presence of S‑9, which gave 18% and 22% RS in the absence and presence of S-9, respectively. Although no concentration tested in the presence of S-9 gave the desired 10 to 20% RS the toxicity level observed at 525 µg/mL (22% RS) was sufficiently close to 10 to 20% RS to be considered acceptable and the data are therefore considered valid.

In Experiment 2 ten concentrations, ranging from 50 to 450 µg/mL in the absence of S‑9 and from 100 to 600  µg/mL in the presence of S‑9, were tested. Seven days after treatment all concentrations in the absence and presence of S-9 were selected to determine viability and 6TG resistance. The highest concentrations tested, 450 µg/mL in the absence of S‑9 and 600 µg/mL in the presence of S‑9, gave 26% and 20% RS in the absence, respectively (seeTable8). It may be noted that in the absence of S‑9 a concentration of 400 µg/mL gave 20% RS and the data are therefore considered valid.

Experiment 1 (3 hour treatment in the absence and presence of S-9)

Treatment (µg/mL)		-S-9			Treatment (µg/mL)		+S-9
		%RS	MF§				%RS	MF§
0		100	1.71		0		100	2.62
50		94	3.38	NS	80		78	1.18	NS
100		73	2.49	NS	160		74	1.75	NS
150		64	2.19	NS	240		59	2.60	NS
240		58	1.63	NS	280		52	1.90	NS
280		54	2.28	NS	400		44	2.49	NS
320		30	1.69	NS	450		32	1.79	NS
360		18	1.81	NS	525		22	1.78	NS
Linear trend			NS		Linear trend			NS
NQO					B[a]P
0.1		62	24.79		2		54	32.03
0.15		73	16.36		3		39	58.99

Experiment 2 (3 hour treatment in the absence and presence of S-9)

Treatment (µg/mL)		-S-9			Treatment (µg/mL)		+S-9
		%RS	MF§				%RS	MF§
0		100	2.19		0		100	1.18
50		97	1.55	NS	100		94	1.36	NS
100		90	1.55	NS	200		81	1.45	NS
150		82	2.76	NS	300		69	2.42	NS
250		51	3.65	NS	350		65	3.61	NS
300		54	2.46	NS	400		68	4.32	NS
320		47	5.65	NS	450		52	2.67	NS
340		58	0.60	NS	500		34	1.51	NS
360		45	4.60	NS	525		35	2.02	NS
400		20	3.06	NS	550		25	2.98	NS
450		26	2.54	NS	600		20	2.45	NS
Linear trend			NS		Linear trend			*
NQO					B[a]P
0.1		86	12.51		2		39	25.22
0.15		58	15.93		3		18	26.40

§  6-TG resistant mutants/10⁶viable cells 7 days after treatment

%RS  Percent relative survival adjusted by post treatment cell counts

NS    Not significant

*, **, ***       Test for linear trend: χ²(one-sided), significant at 5%, 1% and 0.1% level respectively

Applicant's summary and conclusion

Conclusions:

A GLP-compliant study according to OECD 476 was performed. It is concluded that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study.

Executive summary:

A GLP-compliant in vitro mammalian cell gene mutation test using the Hprt and xprt genes according to OECD 476 was performed. In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of rat S9 ranging from 40.41 to 1293 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration tested (1293 µg/mL) was not plated due to excessive toxicity and complete toxicity (0% RS) was observed at the highest concentration plated in the absence and presence of rat S9 (646.5 µg/mL). The highest concentration to provide >10% RS was 323.3 µg/mL in the absence and presence of rat S9, which gave 12% and 37% RS, respectively.

No marked changes in osmolality were observed in the Range-Finder at the highest concentration tested (1293 µg/mL) as compared to the concurrent vehicle controls (individual data not reported). Marked changes in pH were observed at 1293 µg/mL as compared to the concurrent vehicle controls but no marked changes were observed up to 646.5 µg/mL (individual data not reported). As no concentration tested in the Mutation Experiments was greater than 600 µg/mL no further measurements were performed.

In Experiment 1 ten concentrations, ranging from 50 to 500 µg/mL in the absence of rat S9 and from 80 to 600 µg/mL in the presence of rat S9, were tested. Seven days after treatment, the highest two concentrations tested in the absence of rat S9 (400 and 500 µg/mL) and the highest concentration tested in the presence of rat S9 (600 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, concentrations of 200 µg/mL in the absence of rat S9 and 320 and 360 µg/mL in the presence of rat S9 were not selected as there were sufficient non-toxic concentrations. All other concentrations were selected in the absence and presence of rat S9. The highest concentrations plated were 360 µg/mL in the absence of rat S9 and 525 µg/mL in the presence of rat S9, which gave 18% and 22% RS in the absence and presence of rat S9, respectively. Although no concentration tested in the presence of rat S9 gave the desired 10 to 20% RS the toxicity level observed at 525 µg/mL (22% RS) was sufficiently close to 10 to 20% RS to be considered acceptable and the data are therefore considered valid.

In Experiment 2 ten concentrations, ranging from 50 to 450 µg/mL in the absence of rat S9 and from 100 to 600  µg/mL in the presence of rat S9, were tested. Seven days after treatment all concentrations in the absence and presence of rat S9 were selected to determine viability and 6TG resistance. The highest concentrations tested, 450 µg/mL in the absence of rat S9 and 600 µg/mL in the presence of rat S9, gave 26% and 20% RS in the absence, respectively (seeTable8). It may be noted that in the absence of rat S9 a concentration of 400 µg/mL gave 20% RS and the data are therefore considered valid.

It is concluded that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9).