Phosphinic acid, P-[3-(acetyloxy)-3-cyanopropyl]-P-methyl-, butyl ester

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphinic acid, P-[3-(acetyloxy)-3-cyanopropyl]-P-methyl-, butyl ester

EC Number:

605-460-1

Cas Number:

167004-78-6

Molecular formula:

C11 H20 N O4 P

IUPAC Name:

Phosphinic acid, P-[3-(acetyloxy)-3-cyanopropyl]-P-methyl-, butyl ester

Details on test material:

Molecular weight: 261 g/mol
Molecular formular: C11H2ONO4P

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: supplied by the sponsor, Batch No. CR 21422/01/940801
- Purity, including information on contaminants, isomers, etc.: 88.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: -10(+-5) °C
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: all concentrations from the week 1 and week 3 mixes were analysed for accuracy of concentration. Stability under storage conditions was demonstrated and was within the range of -15% to 10% of nominal dietary concentration.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

CRL:COBS CD (SD) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent, UK
- Age at study initiation: 30 days
- Weight at study initiation: male groups: 98 g to 127 g; female groups: 90 g to 129 g
- Housing: polycarbonate cages with solid sides and mesh floors
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 19 and 23°C
- Humidity (%): between 45 and 65%.
- Photoperiod ( hrs light): 12 hours

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Prior to the start of treatment, a procedure was developed to prepare homogeneous and suitably stable mixtures of the test material in the laboratory rodent diet at the required nominal concentrations.
Test diets were prepared for weeks 1, 2 and 3. On each occasion, for the top dose level, a pre-mix was made using a food processor. This pre-mix was then mixed with an appropriate quantity of laboratory rodent diet and blended in a Turbula T10B mixer to produce diet containing 4000 ppm of test material. Subsequent dietary concentrations were prepared by serial dilution.
Immediately after mixing, the bulk of the prepared quantity of each dietary concentration was stored deep frozen. The remaining portion was fed during the next 2 days, thereafter, frozen diet was fed to the rats every two days.

1st Addendum to Report No. TOX/95/250-4:
The HOE 125 869 percentages of nominal values declined by up to approximately 15% during the freezer storage period of 15 days.
The mean analyses of the test diet samples prepared for dosing were within the range +10% to -15% of nominal.
The mixing process produced homogeneous mixes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At each mix, aliquots of each concentration of the freshly prepared test diets were submitted for analysis of the test material concentration.
All concentrations from the week 1 and week 3 mixes were analysed for accuracy of concentration. In addition, samples of the 80 and 4000 ppm dose levels were taken from the freezer after 8 and 15 days, for analysis to demonstrate stability under these storage conditions. All aliquots were within the range -15% to +10% of nominal dietary concentration that is considered acceptable by AgrEvo UK Limited, with the exception of the 80 ppm day 15 stability sample which was 73.3% of nominal. This discrepancy was considered not to have affected the integrity of the study. Detailed results of these analyses are reported.

Duration of treatment / exposure:

Males were treated for 28 consecutive days and females for 29 days.

Frequency of treatment:

Animals had free access to powdered laboratory rodent diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

nominal in diet

Doses / concentrations 2

Dose / conc.:

80 ppm

Remarks:

nominal in diet; equivalent to 8 mg/kg bw/d

Doses / concentrations 3

Dose / conc.:

400 ppm

Remarks:

nominal in diet; equivalent to 37 mg/kg bw/d

Doses / concentrations 4

Dose / conc.:

2 000 ppm

Remarks:

nominal in diet; equivalent to 182 mg/kg bw/d

Doses / concentrations 5

Dose / conc.:

4 000 ppm

Remarks:

nominal in diet; equivalent to 343 mg/kg bw/d

No. of animals per sex per dose:

5 males / 5 females

Control animals:

yes

Details on study design:

- Rationale for animal assignment (if not random): based on body weight so that each group had similar initial mean body weights and weight distributions.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS:
Animals were observed daily for abnormal behaviour, including neuromuscular co-ordination and physical appearance. Observations were ade once each morning and, on Mondays to Fridays except public and Company holidays, also in the afternoon. More detailed observations were conducted weekly prior to weighing.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed upon receipt, at randomisation, at the start of treatment, at weekly intervals thereafter and at necropsy.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
For each sex, the weekly food consumption of each cage of animals was mearsured at weekly intervals throughout the treatment period. The group mean relative food consumption was calculated. The weekly group mean achieved intake of the test material was also calculated.

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes


Blood for haematology and clinical chemistry analyses was obtained on study day 28 of treatment from the retro-orbital sinus of each animal, under light ether anaesthesia. The brain was collected at necropsy from all animals for acetylcholinesterase estimation on study days 29 and 30.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
NECROPSY: Animals were killed by exsanguination via the abdominal aorta under deep ether anaesthesia. All animals were examined thoroughly and macroscopic abnormalities recorded. The brain was extracted immediately, weighed and placed in a pre-measured volume of ice-cold Sorenson phosphate assay buffer. Care was taken to minimise contamination of the brain surface by test material that may have been present on the fur of the animal. After collection, the brain was homogenised and analysed for acetylcholinesterase activity.

Organ weights: The following organs from animals surviving to scheduled termination were weighed at necropsy : Liver, Testes with epididymides, Heart, Kidneys, Brain, Adrenals, Spleen

Tissue sampling: The following organs and tissues from all animals were fixed in 10% neutral buffered formalin :
Liver, Spleen, Adrenals, Testes, Heart, Kidneys, Any other tissue showing macroscopic abnormalities

HISTOPATHOLOGY: Yes
Tissue processing: Following fixation, nominal 5 (I sections of all organs and tissues from decedents and each animal of the control and high dose groups were prepared and stained with haematoxylin and eosin.
Microscopic examination: All tissue sections were examined by a pathologist using a light microscope.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

One male of the 4000 ppm dose group died during blood sampling. No other deaths occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 4000 ppm, there was an overall reduction in body weight gain of 54% in males and 31% in females, compared with the control animals. Terminal body weights at the high dose level were 24% and 11% below the control values for males and females respectively.
Body weight gain at the high dose was mainly affected in the first week of treatment, thereafter the differential remained more or less constant throughout the rest of the study.

Body weight gain was unaffected at the remaining treatment levels.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

At 4000 ppm, food consumption was reduced by 33% and 17% in males and females, respectively, compared to the control.
Relative food consumption was markedly reduced over the first week of treatment in both sexes at 4000 ppm, thereafter values were comparable across the groups .

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effect of treatment was observed. On day 28, lower white cell parameters were observed in male animals at all treated levels. This apparent effect was due to unusually high values obtained for the control males. Not with standing, all values for treated animals were within the background control range. Other statistically significant observations were not considered to be dose related or were within normal limits when compared to historical controls.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effect of treatment was observed. Other statistically significant observations were not considered to be dose related or were within normal limits when compared to historical controls.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were seen in any treatment group. Some statistically significant differences in relative organ weights are considered to be related to the markedly lower terminal body weights.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No compound induced changes were observed. All findings reported are considered to be within the spontaneous background range for this age and strain of rat.

Effect levels

Dose descriptor:

NOEL

Effect level:

182 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Group mean test material-intake (mg/kg bw/day)

 

Dose level (ppm)	80	400	2000	4000
Males	7	36	181	313
Females	8	37	182	372
Combined sexes	8	37	182	343

Applicant's summary and conclusion

Conclusions:

The no observed effect level (NOEL) for the combined sexes 2000 ppm, equivalent to a daily intake of 182 mg/kg bw/day.

Executive summary:

Groups of 5 male and 5 female Sprague Dawley rats were fed diet containing 0,
80, 400, 2000 and 4000 ppm (equivalent to 0, 8, 37, 182 and 343 mg/kg/day
respectively) for 28 days.
Animals were observed daily for clinical signs, whilst body weight and food consumption were measured weekly. Biochemistry and haematology were carried out on study day 28. 

At 4000 ppm, overall body weight gain was reduced by 54% in males and 31% in females, compared with the control group. Food consumption was also reduced at the high dose level by 33% and 17% in males and females, respectively. There were no other treatment-related effects at this dose level.

No treatment related effects were observed at 80, 400 or 2000 ppm. 

The no observed effect level (NOEL) for both sexes was 2000 ppm (equivalent to 182 mg/kg bw/d).