Ammonium 2-amino-4-(hydroxymethylphosphinyl)butyrate

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun - Aug 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

July 21, 1997

GLP compliance:

yes

Limit test:

no

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:

rat

Strain:

Wistar

Remarks:

SPF quality

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: 162 - 232 grams (males), 126 - 169 grams (females)
- Housing: individually in Makrolon type-3 cages with wire mesh tops and standardized softwood bedding
- Diet (e.g. ad libitum): pelleted standard Provimi Kliba 3433 rat maintenance diet, ad libitum
- Water (e.g. ad libitum): community tap water from Itingen, ad libitum
- Acclimation period: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every two weeks
- Mixing appropriate amounts with (Type of food): standard Provimi Kliba 3433 rat maintenance diet
- Storage temperature of food: at room temperature in disposable paper bags

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, homogeneity and content of the test article in the feed were determined in samples taken from each diet preparation. The analysis were performed by RCC Ltd., Environmental Chemistry & Pharmanalytics Division, according to a HPLC-method provided by the Sponsor.

Duration of treatment / exposure:

37 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 ppm

Doses / concentrations 2

Dose / conc.:

200 ppm

Doses / concentrations 3

Dose / conc.:

2 000 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentration of the test substance was based on the results of an acute neurotoxicity study in the rat. Concentrations of the test substance were selected to cause clear inhibition of the target enzymeglutamine synthetase at least at the highest concentration, in the brain, liver and kidneys. The 100-fold range between the low and high concentrations was expected to show a clear dose-effect-relationship for both compounds.

Examinations

Observations and clinical examinations performed and frequency:

VIABILITY / MORTALITY: Yes
- Time schedule: 4 x daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: once during pretest; once daily during treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during pretest and treatment period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: Yes

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters checked in table No.1were examined.
- Description of procedures: Body temperature, landing foot splay, grip strength
- Same technicians used throughout testing: No data
- Technicians were blind to treatment status of animals: No data
- Time schedule for examinations: study days 3 - 5, 10 - 12, 24 - 26, 31 - 33, 38 - 40 and 45 - 47

LOCOMOTOR ACTIVITY: Yes
- Type of equipment used: commercially available Activity Monitor AM 1052 system (Benwick Electronic Equipment Design Manufacture, England)
- Length of session, number and length of subsessions: 60 minute period at intervals of 15 minutes

LEARNING AND MEMORY TESTING: Yes
(2) Equipment used
- Type of equipment (including manufacturer, if available): opaque plexiglas-box with two obstacles
(3) Testing and training procedures
- Number of trials per day: 6

Sacrifice and (histo)pathology:

- Time point of sacrifice: between study day 51 and study day 53
- Number of animals sacrificed: all animals
- Parameters measured: brain (cerembrum, cerebellum, pons), spinal cord (in total), sciatic nerve, tibial nerve, heart, kidney, liver, gross lesions
- Procedures for perfusion: After opening of the abdominal and thoracic cavities, 1 mL of heparin solution was injected into the left heart chamber. Thereafter the right heart chamber was opened and the animal was perfused with Ringer solution (5 - 10 min) followed by infusion with 4 % formaldehyde solution (15 - 20 min).
- Number of animals perfused: all animals
- Type of staining: hematoxylin and eosin
- Methodology of preparation of sections: all organ and tissue samples were processed, embedded and cut at a thickness of 2 - 4 micrometers and stained with hematoxylin and eosin.
- Thickness: 2 - 4 µm

Statistics:

The following statistical methods were used to analyze the locomotor activity, grip strength, body weight, organ weights and ratios, as well as clinical laboratory data:
At RCC Ltd., the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex (body weight, organ weights and ratios). The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
Locomotor activity (after 15, 30, 45 and 60 minutes, and total 1-hour activity), grip strength (hindlimb and forelimb), body temperature and landing footsplay were analyzed statistically at RTI. Due to the multiple testing problem inherent in this analysis, exposed versus control group p-values were adjusted using bootstrap resampling methods (Westfall and Young, 1993) implemented in the MULTTEST procedure of SAS 6.12 5SAS Institute, 1989, 1996) as described in the statistical analysis report. In this study, the bootstrap multiple testing procedure was applied to the eight comparisons of exposed groups to controls, separately for the set of outcomes relating to locomotor activity, grip strength, body temperature, and landing footsplay at each study week, by sex.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During pretest, diarrhea was noted in one male of group 2 and soft feces were evident in one female of this dose group.
These findings were considered to be unrelated to treatment with the test article due to their isolated incidence.
Urinating / defecation was observed in several animals treated with the test substance and in some control animals during pretest and treatment period. This was considered to be not test article related.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the mean body weights and body weight gains of all test substance-treated animals. All values were within the range commonly recorded for this strain and age.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the mean daily food consumption of test substance-treated animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant dose-related inhibition of glutamine synthetase (GS) activity was observed in the liver of both sexes and the kidney of males only at all concentrations tested. Inhibition of GS was also observed in the brain tissue of males at 200 and 2000 ppm and of females at 2000 ppm.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Locomotor activity: At week 2, male locomotor activity was statistically significantly reduced in the 20 ppm group vs. cntrols at each of the 15 and 30 minutes time points. Also at week 2, female locomotor activity was significantly reduced in the 20, 200 and 2000 ppm groups vs. controls at 15 minutes only. There was no evidence of a dose-effect relationship in the amplitude / severity of the changes observed and a similar change was not at week 4. In addition all the individual values were within the normal range observed in pretest or in other studies conducted with animals of this strain and age. Therefore, the findings observed in week 2 were considered to be spurious and of no toxicological or biological significance.
- Rearing: No test article-related effects on rearing were observed in all animals treated with the test substance.
- Grip strength: There were no statistically significant differences in animals treated with the test substance as compared to control groups.
- Body temperature: There were no statistically significant differences in animals treated with the test substance as compared to control groups.
- Landing foot splay: The landing foot splay distance of the test substance-treated animals was unaffected from the test article treatment.
- Water maze test: In the water maze test, consisting of learning, memory and relearning phases, no effects on any treated animals were observed compared with the negative controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At necropsies, the only treatment-related observation was a statistically significant minimal increase in kidney / body weight ratio for males treated with 2000 ppm test substance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were evident at scheduled necropsy in any animals treated with the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related microscopic findings were noted in any of the organs or tissues examined (brain, spinal cord, sciatic nerve, tibial nerve, heart, kidney and liver) of animals treated with the test substance.
The few microscopic findings recorded were recognized as the usual spontaneous background lesions which is regularly noted in rats of this strain and age, considered incidental and of no biological or toxicological significance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Inhibition of glutamine synthetase

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

200 ppm

System:

nervous system

Organ:

brain

Treatment related:

yes

Any other information on results incl. tables

Table 3: Mean test article intake in mg/kg/day (nominal value)

Group	Males	Females
1 (0 ppm)	-	-
2 (20 ppm test substance)	1.5	1.8
3 (200 ppm test substance)	14.9	17.1
4 (2000 ppm test substance)	143.3	161.5

Table 4: Glutamine Synthetase Activity expressed in Percent of Control Group (Control, 100 %)

	Liver		Kidney		Brain
Dose [ppm]	Males	Females	Males	Females	Males	Females
20	67**	77*	75**	98	96	102
200	44**	55**	64**	93	93*	100
2000	35**	37**	65**	94	75**	73**

*/** Dunnett-test based ion pooled variance sig. at 5 % or 1 % level

Table 5: Food Consumption (g/animal/day) Summary

			Group 1 Negative control	Group 2 20 ppm	Group 3 200 ppm	Group 4 2000 ppm
Males	Pretest
	Days 8 – 15 Weeks 2/3	Mean SD N	20.9 1.6 10	20.5 2.1 10	20.0 1.9 10	20.9 3.0 10
	Days 15 – 22 Weeks 3/4	Mean SD N	23.4 1.7 10	23.8 2.0 10	22.7 1.7 10	23.8 2.7 10
	Mean of means over Pretest		22.1	22.2	21.4	22.4
	Treatment
	Days 1 – 8 Weeks 1/2	Mean SD N	22.7 2.2 10	23.9 2.1 10	21.6 2.7 10	21.1 2.7 10
	Days 8 – 15 Weeks 2/3	Mean SD N	23.6 2.1 10	25.5 2.2 10	23.7 3.0 10	25.0 2.4 10
	Days 15 – 22 Weeks 3/4	Mean SD N	23.9 2.4 10	24.8 2.2 10	23.0 2.6 10	24.2 3.0 10
	Days 22 – 29 Weeks 4/5	Mean SD N	24.2 2.2 10	23.8 3.1 10	22.1 2.2 10	23.8 2.1 10
	Days 29 – 36 Weeks 5/6	Mean SD N	22.6 1.9 10	24.1 1.9 10	22.2 2.2 10	23.5 2.2 10
	Mean of means over Treatment		23.4	24.4	22.5	23.5
Females	Pretest
	Days 8 – 15 Weeks 2/3	Mean SD N	14.7 1.1 10	13.7 1.4 10	13.7 1.3 10	13.5 1.5 10
	Days 15 – 22 Weeks 3/4	Mean SD N	17.1 1.4 10	16.1 1.3 10	16.5 1.2 10	17.2 1.4 10
	Mean of means over Pretest		15.9	14.9	15.1	15.3
	Treatment
	Days 1 – 8 Weeks 1/2	Mean SD N	16.8 1.5 10	17.0 2.0 10	16.3 1.8 10	14.7 1.5 10
	Days 8 – 15 Weeks 2/3	Mean SD N	17.1 1.7 10	17.7 1.2 10	17.4 1.5 10	17.1 1.1 10
	Days 15 – 22 Weeks 3/4	Mean SD N	18.5 1.4 10	17.8 1.8 10	17.6 1.8 10	17.0 1.4 10
	Days 22 – 29 Weeks 4/5	Mean SD N	18.5 1.4 10	16.5 1.8 10	16.5 2.0 10	16.2 2.3 10
	Days 29 – 36 Weeks 5/6	Mean SD N	17.0 2.5 10	17.5 1.5 10	17.2 1.4 10	17.4 1.3 10
	Mean of means over Treatment		17.6	17.3	17.0	16.5

Table 6: Body Weights (gram) Summary

			Group 1 Negative control	Group 2 20 ppm	Group 3 200 ppm	Group 4 2000 ppm
Males	Pretest
	Day 8 Week 2	Mean SD N	193 15.9 10	195 20.8 10	189 12.5 10	193 26.5 10
	Day 15 Week 3	Mean SD N	228 17.2 10	238 19.3 10	228 12.7 10	235 28.3 10
	Treatment
	Day 1 Week 1	Mean SD N	258 20.0 10	268 19.4 10	254 14.5 10	265 29.0 10
	Day 8 Week 2	Mean SD N	282 22.8 10	296 22.4 10	277 19.8 10	286 30.9 10
	Day 15 Week 3	Mean SD N	300 24.8 10	319 23.4 10	296 23.7 10	307 32.8 10
	Day 22 Week 4	Mean SD N	317 26.5 10	342 25.4 10	312 25.3 10	327 35.1 10
	Day 29 Week 5	Mean SD N	331 28.4 10	361 24.4 10	326 26.8 10	343 36.4 10
	Day 36 Week 6	Mean SD N	342 29.7 10	373 25.6 10	340 29.0 10	356 37.2 10
Females	Pretest
	Day 8 Week 2	Mean SD N	142 11.9 10	147 11.4 10	145 8.9 10	151 10.7 10
	Day 15 Week 3	Mean SD N	158 11.2 10	164 10.8 10	162 9.6 10	169 13.0 10
	Treatment
	Day 1 Week 1	Mean SD N	174 11.4 10	176 11.6 10	177 10.0 10	184 12.8 10
	Day 8 Week 2	Mean SD N	183 11.8 10	190 12.7 10	187 10.8 10	189 14.3 10
	Day 15 Week 3	Mean SD N	190 12.0 10	197 11.7 10	196 12.0 10	199 14.5 10
	Day 22 Week 4	Mean SD N	199 13.5 10	205 11.9 10	206 11.8 10	209 16.5 10
	Day 29 Week 5	Mean SD N	205 13.1 10	210 12.9 10	212 12.3 10	214 14.1 10
	Day 36 Week 6	Mean SD N	209 12.9 10	218 10.8 10	218 12.6 10	219 14.0 10

Table 7: Weekly Outside Cage Observations, Summary Data Males

			Group 1			Group 2			Group 3			Group 4
Number of animals			10	10	10	10	10	10	10	10	10	10	10	10
Findings / Study week		Score	P	2	5	P	2	5	P	2	5	P	2	5
APP	Piloerection Salivation Hunched posture	1 – 3 1 – 3 1
MOT	Ataxia Tremor / twitching Prostration Circling Spasm	1 – 3 1 – 3 1 1 1 – 3
BEH	Hyperactivity Somnolence Increased reactivity Inc. exploration Red. Grooming Vocalization Urinating / defecation	1 – 3 1 – 3 1 – 3 1 – 3 1 – 3 1 – 3 1	5-	4-	5-	2-	2-	3-	1-		3-	4-		2-
RES	Dyspnea Tachypnea Bradypnea	1 1 1
REF	Blink Pinna Iridic light reflex Push-off (hind legs) Pain response Startle / hearing	1 1 1 1 1 1
MIS	Lacrimation Limbs cyanotic Mydriasis Miosis Exophthalmos Red. Muscle tone Ptosis Emaciation	1 – 3 1 1 1 1 1 – 3 1 – 3 1 – 3

APP = Appearance

MOT = Motor

BEH = Behavioral

RES = Respiration

MIS = Miscellaneous

REF = Reflexes

P = Pretest examination (during acclimatization)

n = Number of affected animals

n- = Mean severity between 1.00 and 1.67

n+ = Mean severity between 1.68 and 2.33

n! = Mean severity between 2.34 and 3.00

Table 8: Weekly Outside Cage Observations, Summary Data Females

			Group 1			Group 2			Group 3			Group 4
Number of animals			10	10	10	10	10	10	10	10	10	10	10	10
Findings / Study week		Score	P	2	5	P	2	5	P	2	5	P	2	5
APP	Piloerection Salivation Hunched posture	1 – 3 1 – 3 1
MOT	Ataxia Tremor / twitching Prostration Circling Spasm	1 – 3 1 – 3 1 1 1 – 3
BEH	Hyperactivity Somnolence Increased reactivity Inc. exploration Red. Grooming Vocalization Urinating / defecation	1 – 3 1 – 3 1 – 3 1 – 3 1 – 3 1 – 3 1		2-	4-						2-			1-
RES	Dyspnea Tachypnea Bradypnea	1 1 1
REF	Blink Pinna Iridic light reflex Push-off (hind legs) Pain response Startle / hearing	1 1 1 1 1 1
MIS	Lacrimation Limbs cyanotic Mydriasis Miosis Exophthalmos Red. Muscle tone Ptosis Emaciation	1 – 3 1 1 1 1 1 – 3 1 – 3 1 – 3

APP = Appearance

MOT = Motor

BEH = Behavioral

RES = Respiration

MIS = Miscellaneous

REF = Reflexes

P = Pretest examination (during acclimatization)

n = Number of affected animals

n- = Mean severity between 1.00 and 1.67

n+ = Mean severity between 1.68 and 2.33

n! = Mean severity between 2.34 and 3.00

Table 9: Water Maze Test

		Pretest			After 4 weeks	After 5 weeks
Group	Sex	Learning test [%]	Memory test [%]	Relearning test [%]	Learning test [%]	Memory test [%]	Relearning test [%]
1 2 3 4	Males	100 100 100 100	100 100 100 100	100 100 100 100	100 100 100 100	100 100 100 100	100 100 100 100
1 2 3 4	Females	100 100 100 100	100 100 100 100	100 100 100 100	100 100 100 100	100 100 100 100	100 100 100 98.33

Table 10: Organ Weights (gram) Summary at Necropsy

			Group 1 Negative control	Group 2 20 ppm	Group 3 200 ppm	Group 4 2000 ppm
Males	Body weight	Mean SD N	343.060 9.682 5	369.000 28.175 5	350.540 48.045 5	342.280 27.546 5
	Brain	Mean SD N	1.81 0.08 5	1.89 0.08 5	1.91 0.08 5	1.91 0.06 5
	Heart	Mean SD N	1.083 0.108 5	0994 0.099 5	1.015 0.108 5	1.030 0.163 5
	Liver	Mean SD N	11.54 0.64 5	11.44 1.61 5	11.47 1.13 5	10.91 1.40 5
	Kidneys	Mean SD N	2.05 0.21 5	2.09 0.08 5	2.13 0.16 5	2.37 0.24 5
Females	Body weight	Mean SD N	216.900 13.430 5	226.780 17.115 5	230.860 17.542 5	210.700 18.699 5
	Brain	Mean SD N	1.79 0.05 5	1.84 0.04 5	1.81 0.04 5	1.78 0.05 5
	Heart	Mean SD N	0.728 0.029 5	0.859 0.089 5	0.799 0.038 5	0.726 0.061 5
	Liver	Mean SD N	7.16 0.73 5	6.99 0.69 5	7.31 0.55 5	6.80 0.46 5
	Kidneys	Mean SD N	1.36 0.05 5	1.48 0.09 5	1.53 0.10 5	1.45 0.15 5

Applicant's summary and conclusion

Conclusions:

No NOEL for male and female rats was determined in this study due to inhibition of GS activity in the liver of both sexes and the kidney of males only, at all concentrations tested. In addition inhibition of GS activity was observed in the brain tissue of males treated with GA at the two highest concentrations (200 and 2000 ppm) and females treated with 2000 ppm GA. Increased kidney weight was also noted for males of the highest dosage group. With regard to mode of action analysis, it is concluded that the inhibition of GS in mammals is a reversible effect, however
the 25% and 27% inhibition observed in males and females in the brain is considered adverse. Thus, the NOAEL for male and female rats was 200 ppm (i.e 14.9 and 17.1 mg/kg bw/d for males and females, respectively). No clinical signs were noted in this study.