Ammonium 2-amino-4-(hydroxymethylphosphinyl)butyrate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jul - Oct 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

August 1982

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

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

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 23 - 34 g, mean weigt 28.8 g (males), 21 - 29 g, mean weight 25.4 g (females)
- Assigned to test groups randomly: yes
- Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet (e.g. ad libitum): rat / mice diet Altromin 1324 (Altromin GmbH, Lage / Lippe), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 10 %
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionized water
- Concentration of test material in vehicle: 1.0 %, 2.0 % and 3.5 %
- Amount of vehicle (if gavage or dermal): 10 mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test compound dilutions were prepared fresh. Appropriate amounts were weight in a flask, mixed with deionized water and topped up to the calibration mark. A solution was formed.

Duration of treatment / exposure:

24, 48, or 72 h

Frequency of treatment:

single treatment

Post exposure period:

24, 48, or 72 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw (total dose)

Doses / concentrations 4

Dose / conc.:

350 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Endoxan
- Route of administration: gavage
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow, erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose levels for micronucleus testing were selected on the basis of a preliminary study to determine the acute toxicity and the maximal applicable dose. Oral administration of 400 mg test substance per kg body weight has caused partial lethality. Therefore, 350 mg/kg bw was selected for the main study as top dose level.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were treated once with the test compound and according to the test procedure were killed 24, 48 or 72 hours after administration of the test compound.

DETAILS OF SLIDE PREPARATION: For each animal, about 3 mL foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.
Staining procedure:
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided increase).
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two-sided).

Statistics:

The statistical evaluations were performed using "Diamant" computer program Version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95 % level of significance. Actual data were also compared with historical controls.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals survived after application of 0, 100 and 200 mg test substance per kg boy weight. Application of 350 mg resulted in death of 2 females out of 30 animals treated. These animals were replaced by two females treated with 350 mg/kg bw and survived the treatment. The following signs of toxicity were observed with some of the animals at the top dose level: increased spontaneous activity, aggressivity, tactile hyperaesthesia, motor excitation, uncoordinated gait, narrowed palpebral fissures, clonic convulsions.
The two animals which did not survive the treatment of 350 mg test substance had panting, uncoordinated gait and abdominal position.
The dissection of the animals revealed no abnormal macroscopic findings with the except of yellow (foamy) liquid in the intestinal tract of 4/30 animals of the 350 mg/kg bw dose level.
The bone marrow smears were examined for the occurance of micronuclei in red blood cells.
The incidence of micronucleated polychromatic erythrocytes in the dose groups of the test substance was within the normal range of the negative control groups. Due to a low spontaneous control value in the negative control group (72 h killing time) a small statistically significant increase in the number of micronucleated polychromatic erythrocytes seemed to occur in female animals at 350 mg/kg bw. These numbers too were in the normal range of negative control values and were considered as of no toxicological significance. The number of normochromatic erythrocytes with micronuclei did not differ significantly from the values of the simultaneous control animals for each of the three killing times investigated. With most treatment groups the ratio of polychromatic erythrocytes to normocytes remained unaffected by the test compound. Only in females at the 72 hours killing time a statistically significant reduction in the ratio of polychromatic erythrocytes to normocytes was observed at 200 and 350 mg/kg bw.
Cyclophosphamid (Endoxan) induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity of the test system.

Any other information on results incl. tables

Table 2: Summary of findings in bone marrow erythrocytes

Sex	Dose [mg/kg bw]	Sampling after dosing	No. of animals	Erythrocytes					Erythrocytes with micronuclei
				Poly	Normo	P/N			Poly (Mean)					Normo (Mean)
				Mean	Mean	Mean	SD		NO	%	SD		Mut.I.	NO	%	SD		Mut.I.
Male	Control 100 200 350 P.Cont.	24 h 24 h 24 h 24 h 24 h	5 5 5 5 5	1000 1000 1000 1000 1000	1000 1000 1000 1000 1000	1.03 1.09 1.01 0.91 0.92	0.11 0.10 0.10 0.22 0.09	I - I - I - I - I	3 4 2 2 65	0.34 0.36 0.24 0.20 6.46	0.05 0.23 0.17 0.07 1.34	I - I - I - I *A	1.0 1.1 0.7 0.6 19.0	1 1 1 1 2	0.14 0.06 0.08 0.14 0.22	0.13 0.05 0.11 0.11 0.11	I - I - I - I - I	1.0 0.4 0.6 1.0 1.6
Female	Control 100 200 350 P.Cont.	24 h 24 h 24 h 24 h 24 h	5 5 5 5 5	1000 1000 1000 1000 1000	1000 1000 1000 1000 1000	1.31 1.37 1.29 1.29 0.88	0.16 0.21 0.06 0.26 0.15	A - (A) - (I) - (I) * (I)	1 2 1 2 31	0.08 0.22 0.08 0.16 3.14	0.13 0.11 0.08 0.15 0.56	I - I - I - I *A	1.0 2.8 1.0 2.0 39.3	1 2 2 1 2	0.12 0.16 0.16 0.12 0.18	0.08 0.09 0.09 0.16 0.04	I - I - I - I - I	1.0 1.3 1.3 1.0 1.5
Male	Control 100 200 350	48 h 48 h 48 h 48 h	5 5 5 5	1000 1000 1000 1000	1000 1000 1000 1000	0.84 1.08 1.08 0.96	0.24 0.15 0.10 0.10	I - I - I - I	3 3 3 3	0.32 0.34 0.30 0.32	0.13 0.13 0.12 0.23	I - I - I - I	1.0 1.1 0.9 1.0	2 1 1 2	0.20 0.08 0.14 0.22	0.16 0.08 0.21 0.16	I - I - I - I	1.0 0.4 0.7 1.1
Female	Control 100 200 350	48 h 48 h 48 h 48 h	5 5 5 5	1000 1000 1000 1000	1000 1000 1000 1000	1.17 1.03 1.12 1.26	0.17 0.21 0.22 0.15	I - I - I - I	2 2 2 2	0.20 0.16 0.22 0.22	0.16 0.15 0.13 0.13	I - I - I - I	1.0 0.8 1.1 1.1	3 1 1 3	0.26 0.14 0.10 0.30	0.15 0.11 0.07 0.20	I - I - I - A	1.0 0.5 0.4 1.2
Male	Control 100 200 350	72 h 72 h 72 h 72 h	5 5 5 5	1000 1000 1000 1000	1000 1000 1000 1000	1.01 1.17 0.89 0.83	0.18 0.18 0.14 0.24	I - I - I - I	2 2 3 4	0.24 0.24 0.32 0.38	0.09 0.13 0.16 0.13	I - I - I - I	1.0 1.0 1.3 1.6	2 0 1 2	0.22 0.04 0.06 0.24	0.13 0.05 0.09 0.13	I - I - I - I	1.0 0.2 0.3 1.1
Female	Control 100 200 350	72 h 72 h 72 h 72 h	5 5 5 5	1000 1000 1000 1000	1000 1000 1000 1000	1.44 1.22 0.58 0.72	0.19 0.24 0.15 0.15	A - (A) * (A) * (A)	1 1 2 3	0.14 0.12 0.24 0.34	0.05 0.13 0.11 0.15	I - I - I * I	1.0 0.9 1.7 2.4	2 1 2 1	0.22 0.14 0.18 0.14	0.08 0.09 0.08 0.17	I - I - I - I	1.0 0.6 0.8 0.6

Mut.I. = Mutagenic Index = ery with micronuclei in dose group/ery with micronuclei in control

P.Cont. = Positive Control = Endoxan (50 mg/kg bw)

Results in brackets since control lies outside normal range

NT = control not treated

- = no difference from control (p > 0.5)

I = within the normal range

A = outside the normal range

* = significantly different from control (p < 0.5)