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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 August 1980 - September 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was not conducted under GLP, but was performed according to methods equivalent or similar to OECD guideline 408.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981
Reference Type:
publication
Title:
A subchronic toxicity study of Phosflex 51B in Sprague-Dawley rats
Author:
Freudenthal RI, Brandwene D, Clous W
Year:
2001
Bibliographic source:
International Journal of Toxicology, 20, 269-274

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no
Remarks:
QA statement included
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Phosflex 51B
- Physical state: Liquid
- Analytical purity: No data
- Composition of test material, percentage of components: Confidential information
- Lot/batch No.: Confidential information
- Expiration date of the lot/batch:
- Stability under test conditions: Highly stable in a cool, dry and well ventilated area

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: approx. 6 weeks
- Weight at study initiation:
Male: 213-217 (mean of dosing groups)
Female: 154-159 (mean of dosing groups)
- Housing: Standard laboratory conditions
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 40-60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of test substance premixed with portion of basal diet and blended. Premix was thoroughly mixed with remainder of basal diet to obtain appropriate dietary concentration. Corn oil was incorporated into test and control diets as dust suppressant (1% w/w).

DIET PREPARATION
- Rate of preparation of diet (frequency): every 3-4 weeks
- Mixing appropriate amounts with (Type of food): Purina Certified Rodent Chow #5002
- Storage temperature of food: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration and homogeneity were analyzed by the EHC Analytical Chemistry Laboratory every 3 to 4 weeks
Duration of treatment / exposure:
90 days
Frequency of treatment:
Continuously
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100, 400, 1600 ppm (or mg/kg diet)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Male: 5.0-10.3, 19.8-41.0, 81.9-164.5 mg/kg bw
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Female: 6.0-10.4, 23.1-41.4, 96.4-168.0 mg/kg bw
Basis:
nominal in diet
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on the data from a 28-day Dietary Range-Finding Study with Phosflex 51B in Rats (T-10430)
Positive control:
Not relevant

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: General appearance, behavior, signs of toxicity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, once a week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to study initiation, after 45 days and at termination of study
- Anaesthetic used for blood collection: Yes (2,2-dichloro-1, 1-difluoroethyl methyl ether)
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters checked: hematocrit, hemoglobin, eythrocyte count, leukocyte count (total and differential), platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to study initiation, at the mid-point of the study and at termination of study
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters checked: aspartate aminotransferase, alanine aminotransaminase, sorbitol dehydrogenase, alkaline phosphatase, cholesterol, total bilirubin, direct bilirubin, total protein, albumin, globulin, blood urea nitrogen, glucose, lactate dehydrogenase, calcium, potassium, sodium, phosphorus

URINALYSIS: Yes
- Time schedule for collection of urine: at mid-point and at termination of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, microscopic examination of sediment

NEUROTOXICITY: Yes
Cholinesterase activity was measured in 20 rats (10 males and 10 females) from each dose group and the control group after 45 days (blood chemistry) and at termination (blood chemistry and in brain). The following parameters were used to determine neurotoxicity: red blood cell cholinesterase, plasma cholinesterase and brain cholinesterase.
Sacrifice and pathology:
GROSS PATHOLOGY/NECROPSY
All tissues, organs and lesions from the animals of the 0 and 1600 ppm dose groups were examined. Only the heart, liver, kidneys and lesions were examined from animals of the 100 and 400 ppm dose groups.

ORGAN WEIGHTS
Brain, heart, liver, kidneys, adrenals and ovaries or testes (without epididymides)

HISTOPATHOLOGY
Control and high dose group: posterior femoral musculature, sternum, lungs, trachea, heart (including ascending aorta), spleen, thymus, mesenteric lymph nodes, mandibular salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, pancreas, right and left lobes of the liver, kidneys, urinary bladder, uterus, cervix, vagina, prostate, thyroids, parathyroids, brain, ovaries, testes and epididymides, pituitary, adrenals, eyes and Harderian glands
Other examinations:
Not relevant
Statistics:
- One-way ANOVA and Dunnett's test for continuous variables
- Appropriate statistical test for frequency data

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
effects were not treatment related
Mortality:
no mortality observed
Description (incidence):
effects were not treatment related
Body weight and weight changes:
no effects observed
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:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
effects were considered negligible
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Description (incidence and severity):
effects were considered negligible
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
effects not considered toxicological
Gross pathological findings:
no effects observed
Description (incidence and severity):
effects were not treatment related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
effects were not treatment related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Deaths of males were 0/20, 0/20, 0/20 and 0/20 for the control, low, medium and high dosing group, respectively. For females this was, 1/20, 1/20, 1/20 and 0/20, respectively. No treatment related clinical signs were observed.

BODY WEIGHT
No effects on body weight and body weight gain were observed in the treatment group, as compared to the control group.

FOOD CONSUMPTION
No effects on food consumption were observed in the treatment group, as compared to the control group.

HAEMATOLOGY
A significant decrease in the terminal white blood cell count was noted in males of the mid-dose group as compared to the control group. However, no dose-response relation was present and the value was determined to be even lower in the pretest control group.

A significant increase was noted in interim platelet count in females of the mid-dose group, as compared to the control group. However, this effect was not present at the end of the study (terminal measurement) and therefore considered not to be of toxicological significance.

CLINICAL CHEMISTRY
A dose-related decrease was observed for the terminal LDH value of the treated males, being significant for the high-dose males.
A significant increase in terminal sodium levels was observed in the mid and high-dose males, as compared to the control group. However, this effect observed was considered to be minimal and not of toxicological significance as the levels in the pretest control group were comparable. Terminal total protein for the high-dose males and albumin for all treated males were significantly increased when compared to the control group. These effects were also considered to be minimal and not of toxicological relevance (no dose-response relation present). Terminal globulin was decreased for the mid-dose males, but no dose response relation was seen. The interim phosphorus value was significantly decreased for all treated males, and this decrease was still present in the high-dose males after termination. Due to the recovery and the fact that no dose response relation was seen, the effect was considered to be of minimal toxicological relevance. Total bilirubin was only significantly decreased at the interim measurement and not after termination. The terminal SGOT value was significantly decreased for the high-dose males, but no dose-related response was observed and therefore the effect was not considered toxicologically relevant. All of the above alterations in clinical chemistry parameters of males were considerd negligible.

A dose-related decrease in LDH values was also observed in females, being significant at the interim measurement in all treated females and at the terminal measurement in the mid and high dose females.
As compared to the control group, a significant decrease in interim sodium levels for all treated females was noted, but this effect was disappeared at termination and the levels were comparable to those in the pretest control group. A significant increase in calcium levels was noted at termination for the mid and high dose females, but this was minimal and no dose-related response was observed. At termination, the phosphorus levels of the mid and high dose females were significantly increased as compared to the control group, showing a dose-response relation. This is however expected as a result of metabolism of the test substance, which is a phosphate. A significant decrease in terminal globulin values was observed in mid and high dose females, but the values were comparable to those of the pretest control group and therefore not considered of toxicological relevance.

URINALYSIS
An increase in protein present in urine was noted at the interim measurement timepoint in mid and high dose males and all female groups. This was not observed at termination and therefore the effect was found to be not of toxicological relevance.

NEUROTOXICITY
Red blood cell cholinesterase activity was significantly decreased for the mid-dose males at termination. The effect was not considered of toxicological relevance as no dose-related response could be detected and the standard deviation of the mean was large. For females the cholinesterase activity was only significantly decreased in the mid-dose group at the interim measurement. Both effects were considered negligible.

A significant decrease in plasma cholinesterase activity was observed for mid and high dose males at the interim measurement, and for high-dose females at both measuring timepoint. The effect seen in males was considered negligible, but for females a dose-related decrease was identified.

Brain cholinesterase was found to be decreased for low dose males only, and no dose-related response was observed. Therefore this effect was considered negligible.

ORGAN WEIGHTS
Absolute liver weight was significantly increased for the male and female high dose animals. An indication for a dose-related response was found, especially in males. The absolute adrenal weight was significantly increased for females of the high dose group only.

Relative liver weight was significantly increased for males and females of the high dose group and males of the mid-dose group. Relative kidney weight was significantly increased for males of the high dose group only, with an indication for a dose-dependent response. The same accounted for relative adrenal weight in the high dose females. These effects were not considered toxicologically relevant in absence of changes in blood chemistry.

GROSS PATHOLOGY
Macroscopy findings were limited to discolouration/foci in liver and lung in all groups. No treatment related findings were recorded.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment related findings were recorded.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 600 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects
Dose descriptor:
NOAEL
Effect level:
107.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects
Dose descriptor:
NOAEL
Effect level:
124.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects
Dose descriptor:
NOEL
Effect level:
400 mg/kg diet
Sex:
male/female
Basis for effect level:
other: Increase in liver and adrenal weight at 1600 ppm (mg/kg)
Dose descriptor:
NOEL
Effect level:
26.6 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Increase in liver and adrenal weight at 1600 ppm (mg/kg)
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Increase in liver and adrenal weight at 1600 ppm (mg/kg)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Test diet analysis revealed that the actual concentration of the test substance in the diet was 92-97% of the theoretical concentration.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the 90-day administration of Phosflex 51B by diet in rats showed a biologically significant increase in liver and adrenal weights in the high-dose group, but this was not regarded as an adverse effect. The statistically significant differences in hematology and clinical chemistry values and in red blood cell, plasma and brain cholinesterase activities between control and treated animals were minimal, inconsistent and considered not to be of biological significance. Therefore, a NOAEL of 107.5 and 124.8 mg/kg bw/day (equivalent to 1600 ppm) was established for males and females, respectively.
Executive summary:

Phosflex 51B was administered to rats in concentrations of 0, 100, 400 and 1600 ppm by diet (= mg/kg food). Mortality, clinical signs, body weight and food consumption were recorded. In addition, hematology, clinical chemistry, neurotoxicity and histopathology were studied.

No treatment related mortality and clinical signs were noted in the animals. The statistically significant differences in hematology and clinical chemistry values and in red blood cell, plasma and brain cholinesterase activities between control and treated animals were minimal, inconsistent and considered not to be of biological significance. A biologically significant increase in liver and adrenal weights (only females) was noted in the high-dose groups, but this was not regarded as a toxic and therefore not an adverse effect.

Under the conditions of this study, the 90-day administration of Phosflex 51B by diet in rats showed to induce no adverse effects. Therefore, a NOAEL of 107.5 and 124.8 mg/kg bw/day (equivalent to 1600 ppm) was established for males and females, respectively.