Ammonium 2-amino-4-(hydroxymethylphosphinyl)butyrate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun 2006 - Feb 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

May 1981

Deviations:

yes

Remarks:

no histopathological examination, temperatures ranged from 18-23°C

GLP compliance:

yes (incl. QA statement)

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

Specific details on test material used for the study:

50% aqueous solution

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)IGS BR

Details on species / strain selection:

The rat was chosen as the test species because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD® (SD)IGS BR strain was used because of the historical control data available in this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Total number: 45 males and 45 females, 23 each were allocated to the study
- Age at study initiation: control and low dose: 61-74 days; high dose: 75-88 days
- Weight at study initiation: males (control and low dose): 337-376 g; females (control and low dose): 197-252 g; males (high dose): 385-471 g; females (high dose): 211-276 g
- Fasting period before study: overnight before blood sampling and during exposure
- Housing: groups of five
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 25 days (control and low dose); 39 days (high dose)

DETAILS OF FOOD AND WATER QUALITY: Routinely checked for chemical and microbiological contaminants

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): monitored
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 June 2006 To: 14 Aug 2006

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 0.26 - ca. 2.71 µm

Remarks on MMAD:

Group 2: MMAD (µm) was between 0.26 and 2.71. Inhalable fraction % <7 µm: 92; GSD 2.15
Group 3: MMAD (µm) was between 0.26 and 2.57. Inhalable fraction % <7 µm: 91; GSD 2.29

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The inhalation system comprised a snout-only inhalation exposure chamber, restraining tubes a concentric jet atomiser, a glass elutriator and a syringe pump. The snout-only inhalation chamber was a modular apparatus of aluminium alloy construction comprising a base unit, a variable number of animal exposure sections and a top section incorporating a central aerosol inlet surrounded by a tangential air inlet.
- Method of holding animals in test chamber: Restraining tube
- Source and rate of air: not specified
- Method of conditioning air: Air supply and extract were monitored using in-line flowmeters.
- System of generating particulates/aerosols: A polypropylene syringe of Test Article was positioned onto a syringe driver, which delivered Test Article to the inlet of a concentric jet atomiser via a polythene needle.
- Temperature, humidity, pressure in air chamber: Temperature was recorded manually, every 30 minutes during exposure; a small negative pressure
- Air flow rate: 1 l/min
- Air change rate: not specified
- Method of particle size determination: Marple Personal Cascade Impactor
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetry
- Samples taken from breathing zone: yes

OTHER:
All animals (including spares) were subjected to restraint procedures and exposed to air only (‘Sham dosing’) for 5 or 6 days, in order to accustom animals to the restraining procedure

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations
The achieved chamber concentrations of Glufosinate and total particulate were calculated from the mass of Glufosinate and total material deposited on the open face filter sample and the volume of chamber atmosphere sampled

Duration of treatment / exposure:

4 w

Frequency of treatment:

6 h/d, 5 d/w

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.05 mg/L air

Remarks:

0.056 mg/l (actual dose exposed) equivalent to 0.109 mg/l of the technical liquid

Doses / concentrations 2

Dose / conc.:

0.1 mg/L air

Remarks:

0.105 mg/l (actual dose exposed) equivalent to 0.205 mg/l of the technical liquid

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on an inhalation study performed previsouly with the solid form.
- Rationale for administration route: The inhalation route of administration was chosen to simulate the conditions of potential human exposure.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
- Other:

Positive control:

not included

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On the days of exposure (pre-exposure, during exposure, up to 2 h post-exposure, as late as possible in the working day)
- Time schedule for general health: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: One week pre-exposure, on exposure starting date, weekly throughout treatment, and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/rat/week: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure and during week 4 of treatment
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 4 of treatment (before dosing)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocytes (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt). Additional blood samples were taken to examine prothrombin time (PT) and activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 4 of treatment (before dosing)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea
Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot). Electrophoretic protein fractions; (Alb), alpha1 globulin (a1), alpha2 globulin (a2), beta globulin (Beta), gamma globulin (Gamma) were analysed with agarose gel, albumin

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes / No / Not specified
- Time schedule for analysis:
- Dose groups that were examined:
- Number of animals:
- Parameters checked in table [number] were examined.
LUNG BURDEN: Yes / No / Not specified
- Time schedule for analysis:
- Dose groups that were examined:
- Number of animals:
- Parameters checked in table [number] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
- a full macroscopic examination of the tissues was performed
- The following organs were weighed: adrenals, heart, kidneys, liver, lungs including bronchi, spleen, testes

HISTOPATHOLOGY: No

Other examinations:

Tissue glutamine synthetase: These samples were the subject of a separate study (not reported herein) to investigate possible effects on glutamine synthetase activity.

Statistics:

- Bartlett's test: body weight, blood chemistry and hematology, organ weights
- Fisher's exact test, Mantel test, Dunnett's test, Bartlett's test, William's test, square-root transformation, Shirley's test: body weight, organ weight, and clinical pathology data

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 0.205 mg/l: Treatment-related clinical signs following exposure to the test substance were noted in males and females. The number and incidence of these signs was greatest post exposure on Day 2 and pre exposure on Day 3. Irritable – slight to moderate (5/5 males, 1/5 females); nervous – slight to moderate (3/5 males, 2/5 females) and underactive – slight to moderate (2/5 males, 3/5 females) behaviour was observed intermittently pre, during and postdose in the majority of animals. Repetitive sideways movements of the head – slight to marked (1/5 males, 2/5 females) were observed following 2 exposures to the test substance. Other signs recorded for animals included salivation, piloerection, vocalising, eyes partially closed, fast/or shallow breathing and standing on hind limbs. Ungroomed coats in all males. All males were noted to have several bite marks at the first check on Day 3. Following exposure on Day 3, males were housed individually.
- 0.109 mg/l: In females: eyes partially closed (3/5), fast breathing (1/5), underactive – slight (1/5), vocalising (1/5), salivation (1/5), aggressive (1/5), hunched posture (1/5) and pallor – slight (1/5). Vocalising, salivation and aggression were all recorded in the same animal. These signs most commonly resolved by the final check on Day 3. A single female displayed repetitive sideways movements of the head on Day 3 and 4 of exposures. Ungroomed coats in 2/5 females

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- 0.205 mg/l: One female was killed on humane grounds on Day 3 of exposure due to clinical signs of, piloerection, unsteady gait, partially closed
eyelids, hunched posture and pallor.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean bodyweight gain for male exposed rats was similar to or less than Control for 0.109 mg/l and 0.205 mg/l, respectively. Mean bodyweight gain for females exposed rats was statistically significantly more than Control. In view of the inconsistent responses for the 2 sexes and the lack of dose relationship it is considered that the differences in weight gain are fortuitous.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 0.205 mg/l: Mean haematocrit, haemoglobin, mean cell haemoglobin, mean cell haemoglobin concentration and mean cell volume were all slightly higher for males and females. Although the degree of elevation was slight (typically 1.03 to 1.09Xcontrol), statistical significance was achieved for most parameters at this dose level and in both sexes. Large unstained cell counts were statistically significantly higher in males and females. However, these changes were very small and are not considered to constitute an adverse effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean urea levels were lower in all treated groups of males and females (by up 0.79x control) with a dose related trend apparent. Statistical significance was attained for females given 0.205 mg/L. The toxicological significance of this finding is unknown.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- 0.205 mg/l: Increased aggression was evident in males following exposure on Day 2. Irritable – slight to moderate (5/5 males, 1/5 females); nervous – slight to moderate (3/5 males, 2/5 females) and underactive – slight to moderate (2/5 males, 3/5 females) behaviour was observed intermittently pre, during and postdose in the majority of animals. Repetitive sideways movements of the head – slight to marked (1/5 males, 2/5 females) were observed following 2 exposures to the test substance. Vocalising and standing on hind limbs.
- 0.109 mg/l: In females: underactive – slight (1/5), vocalising (1/5), aggressive (1/5), hunched posture (1/5) and pallor – slight (1/5). Vocalising and aggression were all recorded in the same animal. These signs most commonly resolved by the final check on Day 3. A single female displayed repetitive sideways movements of the head on Day 3 and 4 of exposures.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 0.205 mg/l: In females, dose related increase in absolute and relative lung weights and statistically significant increase in absolute lung weight in males, statistically significant increase in absolute and relative kidney weights in males
- 0.109 mg/l: In females, dose related increase in absolute and relative lung weights, statistically significant increase in absolute lung weight in males, statistically significant increase in absolute and relative kidney weights in males

However, in the absence of a correlation between the sexes, and the absence of microscopic examination to corroborate this finding, the toxicological significance of increased kidney weights is unknown. A dose related increase in lung weights of both male and females rats was noted at termination and is considered to be related to exposure to the test material. In the absence of microscopic examination the aetiology of this increase in weight is unknown.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

0.109 mg/L air

Based on:

test mat. (total fraction)

Remarks:

technical liquid

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

0.205 mg/L air

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes