Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

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:

14 Apr - 18 May 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2015

Deviations:

no

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian cell gene mutation test

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

EC Number:

220-482-8

EC Name:

Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

Cas Number:

2781-11-5

Molecular formula:

C9H22NO5P

IUPAC Name:

diethyl {[bis(2-hydroxyethyl)amino]methyl}phosphonate

Details on test material:

Lot/batch No.: 1106C-1-3

Specific details on test material used for the study:

- Date received: 26 Mar 1981
- Color: amber
- Stability: 2 years
- Storage conditions: room temperature (~20°C), ambient humidity, in the dark

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: 3.7.2 clone of Fischer L5178Y cells
- Maintenance: Frozen stocks are maintained in liquid nitrogen; Laboratory cultures are maintained at a density less or equal to 1 x 10E6 cells/mL.

MEDIA USED
- Type and identity of media:
Growth medium: RPMI 1640 supplemented with 10% (v/v) horse serum, 2 mM glutamine, 22 mg/mL sodium
pyruvate, 50 mg/mL pluronic solution and penicillin-streptomycin
Treatment medium: growth media with reduced serum (5%)
Cloning medium: growth medium w/o pluronic solution and with noble agar (final concentration: 0.35%)
Selective medium: cloning medium with 4 µg/mL TFT
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

other: TK+/-

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 mix

Test concentrations with justification for top dose:

Experiment I
Without S9 mix: 31.3, 62.5, 125, 250, and 500 µL/L
With S9 mix: 250, 500, and 1000 µL/L

Experiment II
Without S9 mix: 200, 250, 300, 350, and 400 µL/L
With S9 mix: 1000, 1100, and 1200 µL/L

The highest chosen dose reduced the growth rate by 50-90% based on the results of the preliminary cytotoxicity test (for experiment I) and on the results of experiment I (for experiment II).

Vehicle / solvent:

- Solvent used: water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density: 6 x 10E5 cells/mL at the beginning of treatment; 3 x 10E6 cells cloned in soft agar for TFT resistant cells, 600 cells cloned in non-selective medium for viable counts.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated for determination of the cloning efficiency and the mutation frequency on agar plates containing TFT selective medium. The agar plates were incubated for 10-12 days.

SELECTION AGENT: Trifluorothymidine (TFT)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

Evaluation criteria:

The substance is considered mutagenic if it produces a dose-dependent increase in mutation frequency over three doses to a level at least 2.5 times the solvent control. If this increase is observed for the highest dose in a reproducible manner, than the substance is considered mutagenic even without an observed dose response.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at ≥ 0.3 µL/mL without activation, at ≥ 1.1 µL/mL with activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

solvent control

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: None reported

RANGE-FINDING/SCREENING STUDIES:
- Doses were selected based on the results of a range-finding assay from a range producing 10-100% relative suspension growth.

STUDY RESULTS: see Tables below

HISTORICAL CONTROL DATA: not reported

Any other information on results incl. tables

Table 1: Experiment I - with and without metabolic activation

		w/o S9 mix		with S9 mix
Observed parameter		Relative cloning efficiency	Mutant frequency x 10E6	Relative cloning efficiency	Mutant frequency x 10E6
Negative control		76	63	161	108
		88	67	75	152
Solvent control		100	54	100	129
		100	70	100	130
Positive control (EMS 0.5 µL/mL w/o S9 mix, DMN 0.3 µL/mL with S9 mix)		99	522	19	1058
Test material	0.0313	147	36	-	-
(µL/mL)	0.0625	98	60	-	-
	0.125	115	55	-	-
	0.250	93	79	135	119
	0.500	74	241	166	161
	1.000	-	-	67	300

Table 2: Experiment II - with and without metabolic activation

		w/o S9 mix		with S9 mix
Observed parameter		Relative cloning efficiency	Mutant frequency x 10E6	Relative cloning efficiency	Mutant frequency x 10E6
Negative control		57	92	87	123
		83	54	140	117
Solvent control		100	55	100	114
		100	61	100	158
Positive control (EMS 0.5 µL/mL w/o S9 mix, DMN 0.3 µL/mL with S9 mix)		62	651	19	863
Test material	0.20	103	66	-	-
(µL/mL)	0.25	96	107	-	-
	0.30	55	201	-	-
	0.35	81	203	-	-
	0.40	50	297	-	-
	1.00	-	-	80	261
	1.10	-	-	45	348
	1.20	-	-	48	380

-: not tested

EMS: ethylmethylsulfonate

DMN: dimethylnitrosamine

Applicant's summary and conclusion

Conclusions:

In this study, there was a concentration related positive response of induced mutant colonies over background.

Executive summary:

In a mammalian cell gene mutation assay performed similarly to the OECD TG 490, L5178Y TK+/- mouse lymphoma cells cultured in vitro were exposed to the test item diluted in water in the presence and absence of Rat S9 mammalian metabolic activation.  

The following concentrations were used:

 

Experiment I

Without S9 mix: 31.3, 62.5, 125, 250, and 500 µL/L

With S9 mix: 250, 500, and 1000 µL/L

 

Experiment II

Without S9 mix: 200, 250, 300, 350, and 400 µL/L

With S9 mix: 1000, 1100, and 1200 µL/L

 

The highest chosen dose reduced the growth rate by 50-90% based on the results of the preliminary cytotoxicity test (for experiment I) and on the results of experiment I (for experiment II).

 

 

An increase in mutation frequency was seen at concentrations of 0.25 µL/mL or greater, reaching significant levels at 0.30 µL/mL. In the presence of an activation system, the test item is less toxic and a significant increase in mutation frequency was seen at concentrations greater or equal to 1.10 µL/mL.

 

The positive controls did induce the appropriate response.  

 

In this study, there was a concentration related positive response of induced mutant colonies over background.

 

This study is classified as acceptable.