Diethylammonium chloride

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diethylamine

EC Number:

203-716-3

EC Name:

Diethylamine

Cas Number:

109-89-7

Molecular formula:

C4H11N

IUPAC Name:

N-ethylethanamine

Details on test material:

- Name of test material (as cited in study report): N,N-diethylamine (DEA)
- Physical state: liquid
- Analytical purity: >= 99.5 %
- Lot/batch No.: DEJ30990H0

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

Range finder: 22.86; 45.71; 91.43; 182.9; 365.7; 731.4 µg/ml (with and without S-9 mix)
Experiment 1: 100, 200, 300, 350, 400, 450, 500, 550, 650, 731.4 (without S-9 mix)
100, 200, 300, 400, 500, 550, 600, 650, 700, 731.4 (with S-9 mix)
Experiment 2: 50, 100, 200, 300, 350, 400, 450, 500, 600, 731.4 (without S-9 mix)
100, 200, 300, 400, 450, 500, 550, 600, 650, 731.4 (with S-9 mix)

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 4-nitroquinoline 1-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

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

SELECTION AGENT (mutation assays): 6GT, 15 µg/mL

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:
The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
There was a significant concentration relationship as indicated by the linear trend analysis (p≤0.05)
The effects described above were reproducible.
Results that only partially satisfy the assessment criteria described above are considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly 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

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Accordingly, for Experiment 1 ten concentrations, ranging from 100 to 731.4 µg/mL, were tested in the absence and presence of S 9. Following the treatment incubation period, the highest two concentrations in the absence of S-9 and the highest three concentrations in the presence of S-9 (650 to 731.4 µg/mL in each case) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest two remaining concentrations in the absence of S-9 (500 and 550 µg/mL) and the highest remaining concentration in the presence of S 9 (600 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected. The highest concentrations selected were 450 µg/mL in the absence of S 9 and 550 µg/mL in the presence of S 9, which gave 10% and 7% relative survival (RS), respectively. In the presence of S-9, no concentration gave 10 and 20% RS. Cultures treated at 500 and 550 µg/mL gave 21% and 7% RS, respectively, therefore both concentrations were analysed.

In Experiment 2, ten concentrations, ranging from 50 to 731.4 µg/mL in the absence of S-9 and from 100 to 731.4 µg/mL in the presence of S 9, were tested. Following the treatment incubation period, the highest two concentrations in the absence of S-9 (600 and 731.4 µg/mL) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest remaining concentration in the absence of S 9 (500 µg/mL) and the highest four concentrations in the presence of S 9 (550 to 731.4 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected. However, the concentration of 300 µg/mL in the presence of S-9 was later rejected from analysis due to extreme heterogeneity for viability. Marked heterogeneity (also for viability) was observed at concentrations of 50 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9, but these were included for comparative purposes. The highest concentrations analysed were 450 µg/mL in the absence of S 9 and 500 µg/mL in the presence of S 9, which gave 10% and 13% RS, respectively.

Any other information on results incl. tables

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

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		% RS	MF§					% RS	MF§
0		100	4.80			0		100	3.57
100		72	3.25		NS	100		96	3.14		NS
200		74	4.91		NS	200		69	2.75		NS
300		67	2.03		NS	300		62	3.91		NS
350		43	5.24		NS	400		62	4.65		NS
400		29	7.07		NS	500		21	2.87		NS
450		10	6.85		NS	550		7	6.33		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		44	44.33			2		52	21.21
0.15		51	56.73			3		23	56.04

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

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

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

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		% RS		MF§				% RS		MF§
0		100		2.59		0		100		2.49
50	$$	80		1.85		100		127		3.84	NS
100		71		2.38	NS	200		76		3.36	NS
200		68		3.50	NS	400		45		2.70	NS
300		48		2.28	NS	450	$$	30		2.96
350		27		4.48	NS	500		13		5.28	NS
400		18		3.32	NS
450		10		10.10	*
Linear trend				*		Linear trend				NS
NQO						B[a]P
0.1		61		24.70		2		58		39.09
0.15		41		21.20		3		32		69.84

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

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

* : Comparison of each treatment with control: Dunnett#s test (one-sided), significant at 5% level

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

$$ : Treatment has marked heterogeneity for viability but is included for comparative purposes

Applicant's summary and conclusion

Conclusions:

It is concluded that Diethylamine, anhydrous 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 highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).