Ammonium 2-amino-4-(hydroxymethylphosphinyl)butyrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2000 - Feb 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium 2-amino-4-(hydroxymethylphosphinyl)butyrate

EC Number:

278-636-5

EC Name:

Ammonium 2-amino-4-(hydroxymethylphosphinyl)butyrate

Cas Number:

77182-82-2

Molecular formula:

C5H12NO4P.H3N

IUPAC Name:

ammonium 2-amino-4-[hydroxy(methyl)phosphoryl]butanoate

Method

Target gene:

his and trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Aroclor 1254-induced rat liver
- method of preparation of S9 mix: prepared from male Sprague Dawley rats induced with Aroclor 1254. Stored frozen at -80°C, and thawed just prior to incorporation into the top agar
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 ml 10% S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes

Test concentrations with justification for top dose:

An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of the test substance at 1.6, 8, 40, 200, 1000 and 5000 µg/plate. Toxicity was observed by a marked reduction in revertant numbers seen on all plates treated at 200 µg/plate.

Test concentrations were:
Experiment 1: 0.08, 0.4, 2, 10, 50, 250 µg/plate
Experiment 2: S.typh. strains: 6.25, 12.5, 25, 50, 100, 200 µg/plate; E. coli: 3.125, 6.25, 12.5, 25, 50, 100 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile high purity water

- Justification for choice of solvent/vehicle: not given

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 1 h
- Exposure duration/duration of treatment: 3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Colony counting

Evaluation criteria:

Evaluation criteria
The test article was considered to be mutagenic if:
1. the assay was valid
2. Dunnett's test gave a significant response (p < 0.01) and the data set(s) showed a significant dose correlation
3. the positive responses were reproducible.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 250 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 250 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 250 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 250 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

True negative controls validity:

not valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 250 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Evidence of toxicity observed on all plates treated at 1000 and 5000 µ/plate . A marked reduction in revertant numbers at the next highest test dose (200 ng/plate) was considered as further clear evidence of toxicity at this dose level

STUDY RESULTS
- Concurrent vehicle negative and positive control data : valid

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value 0.05

Ames test:
- Signs of toxicity : yes
- Individual plate counts : see table
- Mean number of revertant colonies per plate and standard deviation : see table

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see attachment
- Negative (solvent/vehicle) historical control data: see attachment

Any other information on results incl. tables

Table 1: Summary of mean revertant colonies (-S9) - Experiment 1

Substance	Dose Level µg/plate	TA98	TA100	TA1535	TA1537	WP2uvrA
		Mean±SD	Mean±SD	Mean±SD	Mean±SD	Mean±SD
WATER	100 µl	29±8 (M)	136±6 (M)	20±3 (M)	16±4 (M)	12±3 (M)
Test substance	0.08	37±6	129±5	17±5	21 ±6	9±1
		(M)	(M)	(M)	(M)	(M)
	0.4	36± 7	141 ± 15	23±2	17 ±6	12±3
		(M)	(M)	(M)	(M)	(M)
	2	37±5	130±9	18 ± 3	23±4	10±4
		(M)	(M)	(M)	(M)	(M)
	10	25±6	158 ± 15	18±4	16±1	12±6
		(M)	(M)	(M)	(M)	(M)
	50	22± 10	136 ± 11	23±4	16±5	7±2
		(M)	(M)	(M)	(M)	(M)
	250	14±5	86± 8	6±2	13 ± 3	-
		(M)	(M)	(M)	(M)	(1)
Positivecontrols	Compound   DoseLevel	2NF	NaN3	NaN3	AAC	NQO
		5µg	2 µg	2µg	50µg	10 µg
	Mean +/- SD	1477 +/- 146	826 +/- 45	710 +/- 16	329 +/- 25	1049 +/- 20

SD Standard deviation

2NF 2-Nitrofluorene

NaN3 Sodium azide

AAC 9-Aminoacridine

NQO 4-Nitroquinoline 1-oxide

T : Toxic, no revertant colonies

M : Plate counted manually

Table 2: Summary of mean revertant colonies (+S9) - Experiment 1

 

Substance	Dose Level µg/plate	TA98	TA100	TA1535	TA1537	WP2uvrA
		Mean±SD	Mean±SD	Mean±SD	Mean±SD	Mean±SD
WATER	100 µI	38 ± 11	162± 12	20±3	19±5	16±5
Test substance	0.08	39± 10	153 ± 10	19±3	16±2	17± 5
					(M)
	0.4	32±11	167±4	25 ± 1	17±2	14±4
	2	37± 12	163 ±6	21 ±5	24±5	13 ±6
	10	41 ±5	167± 8	19±2	9±5	11±5
					(M)
	50	31 ± 1	158 ± 7	19±3	11±4	6±2
			(M}
	250	14±6	75±7	5±1	11 ±5	4±1
		(M)			(M)
Positivecontrols	Compound   DoseLevel       Mean +/- SD	B[a]P	AAN	AAN	AAN	AAN
		10 µg	5 µg	5 µg	5 µg	10 µg
		562 +/- 27	2504 +/- 65	230 +/- 14	540 +/- 15	601 +/- 70

SD Standard deviation

B[a]P Benzo[a]pyrene

AAN 2-Aminoanthracene

M : plate counted manually

Table 3: Summary of mean revertant colonies (-S9) - Experiment 2

Substance	Dose level µg/plate	TA98	TA100	TA1S3S	TA1S37	WP2uvrA
		Mean±SD	Mean±SD	Mean±SD	Mean±SD	Mean±SD
WATER	100µI	32±5	101 ± 17	27±5	21 ±6	18 ±2
Test substance	3.125   6.25   12.5   25   50   100   200	-	-	-	-	24±3
		36±8	114 ± 18	28±5	23±5	14±3
		29±5 (M)	132 ± 14	26±5	15 ± 3	19±2
		31 ± 12	134± 18	46±5	20±5	14±2
		16 ± 1	104±22	37± 11 (M)	18±4	17±3
		20±2	97±9	33 ± 11	17±3	11 ±2
		11 ± 3	58± 8	22±8	15 ±6	-
Positive controls	Compound   Doselevel       Mean±SD	2NF	NaN3	NaN3	AAC	NQO
		5µg	2µg	2µg	50µg	2µg
		1382±77	765±43	671 ± 8	185 ± 83	1148 ± 79

 

SD Standard deviation

 

NQO 4-Nitroquinoline 1-oxide 2NF 2-Nitrofluorene

NaN3 Sodium azide AAC 9-Aminoacridine

 

M: Plate counted manually

Table 3: Summary of mean revertant colonies (+S9) - Experiment 2

Substance	Dose Level µg/plate	TA98	TA100	TA1535	TA1537	WP2uvrA
		Mean±SD	Mean±SD	Mean±SD	Mean±SD	Mean±SD
WATER	100 µl	42±6	116 ± 17	24± 7	19±6	26±4
Test substance	3.125	-	-	-	-	14±4
	6.25	43± 11	112±9	21±4	15 ±7	17±6
	12.5	34±7	102 ± 10	27± 10	13 ±2	19 ±6
	25	22±4	101 ± 15	19±9	24±6	21±4
	50	17±6	94±3	14 ± 3	27±2	7±1
	100	14±4	94±12	16±7	23±5	11 ±4
						(M)
	200	14±3	48±4	6±2 (M)	9±4 (M)	-
Positivecontrols	Compound   Doselevel       Mean±SD	B[a]P	AAN	AAN	AAN	AAN
		10 µg	5µg	5µg	5 µg	10 µg
		208±62	201 ± 36	84±25	100 ±27	161 ± 15

 

SD Standard deviation

B[a]P Benzo[a]pyrene

AAN 2-Aminoanthracene

M : plate counted manually