Registration Dossier

Administrative data

Endpoint:
immunotoxicity: short-term oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct - Dec 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.7800
Version / remarks:
Aug 1998
Deviations:
no
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-(hydroxymethylphosphinyl)butyrate

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Rj:WI (IOPS HAN)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 7 weeks
- Weight at study initiation: 220 g to 263 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 (light: 7 am to 7 pm)

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
- Reason for choice of route: The oral route (admixture with diet) was selected as it is an accepted route of exposure by regulatory authorities and since it is a possible route of human exposure.

DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with (Type of food): Certified rodent powdered and irradiated diet A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France)
- Storage temperature of food: -18 degrees celsius
:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity of the test substance in diet was verified before the study for the lowest and highest concentrations to demonstrate adequate formulation procedures. The mean value obtained from the homogeneity check was taken as measured concentration. Dietary level of the test substance was also verified for the intermediate concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
continuous
Doses / concentrationsopen allclose all
Dose / conc.:
500 ppm (nominal)
Remarks:
(equating approximately to 36.4 mg/kg bw/day of the test substance)
Dose / conc.:
2 000 ppm (nominal)
Remarks:
(equating approximately to 147 mg/kg bw/day of the test substance)
Dose / conc.:
6 000 ppm (nominal)
Remarks:
(equating approximately to 428 mg/kg bw/day of the test substance)
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were set after evaluation of the results of previous toxicity studies with this substance. The dietary levels selected for this 28-day immunotoxicity study were 0, 500, 2000 and 6000 ppm.
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Rationale for animal assignment (if not random): random
- Reason for selection of species and gender: There is no evidence of immunotoxicity with the test substance in rats or mice. Therefore, the selection of species and gender for the immunotoxicity study is based on which one is more sensitive to the compound using other toxicologic endpoints. In the existing toxicology database, No Observed Adverse Effect Levels (NOAEL’s) for male Wistar rats in repeated oral exposure studies are generally lower than those for both sexes of mice or female rats. Thus, male Wistar rat is selected as the species and gender for immunotoxicity testing.

Examinations

Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: mortality and clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during acclimatization and at least weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yess
- Time schedule: Once weekly

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

OPHTHALMOSCOPIC EXAMINATION: No

IMMUNOLOGY: Yes
- Time schedule for collection of blood: On Day 30
- Anaesthetic used for blood collection: Yes (by inhalation of Isoflurane)
- Animals fasted: No
- How many animals: all animals
- Parameters checked: the level of SRBC-specific immunoglobulin M in response to antigen administration.

CLINICAL CHEMISTRY: No

URINALYSIS: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, examination of all major organs, tissues and body cavities. Selected organs were weighed (spleen and thymus)
- Time of termination: On day 30
HISTOPATHOLOGY: No
Cell viabilities:
SPLEEN: No
THYMUS: No
BONE MARROW: No
Humoral immunity examinations:
ANTIBODY PLAQUE FORMING CELLS (PFC) ASSAY: No
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
- Method: Sheep Red Blood Cell (SRBC) Sensitization
- Dose groups: all dose groups
- No. of animals: all animals
Specific cell-mediated immunity:
ONE-WAY MIXED LYMPHOCYTE CULTURE (MLC) ASSAY: No
DELAYED-TYPE HYPERSENSITIVITY (DTH) REACTION: No
CYTOTOXIC T-LYMPHOCYTE (CTL) ASSAY: No
Non-specific cell-mediated immunity:
NATURAL KILLER (NK) CELL ACTIVITY: No
MACROPHAGE NUMBER AND FUNCTION: No
Positive control:
Cyclophosphamide at 3.5 mg/kg bw/day. Cyclophosphamide, a known immunosuppressive agent (batch number 068K1131: a white powder, 98.0% purity) was used throughout the study. Information on the chemical characterization of cyclophosphamide was documented by the supplier Sigma-Aldrich, USA. Cyclophosphamide was stored in an air-tight, light-resistant container at +5°C (±3°C).
Statistics:
F-test, modified t-test, t-test, Bartlett test, ANOVA, Kruskal-Wallis, Dunnett, Dunn, Mann-Whitney

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- 6000 ppm: Piloerection and ocular discharge between Days 17 and 29 and wasted appearance between study Days 17 and 22 in a single male rat.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 6000 ppm: Mean body weight was reduced by 6 to 7% throughout the study (p≤0.01 on study Days 8 and 15). This effect was attributable to a reduced mean body weight gain between study Days 1 and 8 (28 g compared to a gain of 49 g in the controls; p≤0.01). Thereafter, mean body weight gain per day was similar to the controls.
- 2000 ppm: mean body weight was reduced by 5 to 6% throughout the study (not statistically significant). This effect was attributable to a slightly reduced mean body weight gain between study Days 1 and 8 (37 g compared to a gain of 49 g in the controls; p≤0.01). Thereafter, mean body weight gain per day was similar to the controls.
- 500 ppm: Mean body weight was reduced by 3 to 4% throughout the study (not statistically significant). This effect was attributable to a slightly reduced mean body weight gain between study Days 1 and 8 (39 g compared to a gain of 49 g in the controls; p≤0.01). Thereafter, mean body weight gain per day was similar to the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 6000 ppm: Mean food consumption/day was reduced by 19 and 10% on study Days 8 and 15, respectively (p≤0.01), and was similar to the control group afterwards.
- 2000 ppm: Mean food consumption/day was slightly reduced by 12% (p≤0.01) during the first week of treatment in comparison to the control group and was unaffected afterwards.
- 500 ppm: Mean food consumption/day was slightly reduced by 9% (p≤0.01) during the first week of treatment in comparison to the control group and was unaffected afterwards.
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Description (incidence and severity):
There was no treatment-related change in the level of SRBC-specific IgM present in the blood 4 days after intravenous injection of SRBC up to 6000 ppm.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no changes in mean organ weights when compared to the controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- 6000 ppm: Pelvic dilatation in the kidney was observed in 4/10 animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Specific immunotoxic examinations

Cell viabilities:
not examined
Humoral immunity examinations:
no effects observed
Description (incidence and severity):
There was no treatment-related change noted in anti-SRBC IgM concentrations up to 6000 ppm.
Specific cell-mediated immunity:
not examined
Non-specific cell-mediated immunity:
not examined
Other functional activity assays:
not examined

Effect levels

Dose descriptor:
NOEL
Effect level:
6 000 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
immunology

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Compared to controls, no impairment of the immunological IgM response following immunization with SRBC was observed in male Wistar rats treated with glufosinate-ammonium in the diet at dose levels up to 6000 ppm for at least 28 days (corresponding to 428 mg/kg body weight/day). Therefore, glufosinate-ammonium was considered not to have any immunotoxic potential.