Registration Dossier

Toxicological information

Neurotoxicity

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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

Constituent 1
Chemical structure
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 / concentrationsopen allclose all
Dose / conc.:
20 ppm
Dose / conc.:
200 ppm
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.