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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January - February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Guideline:
other: - OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Guanidine phosphate (1:1)
- Physical state: White crystal solid
- CAS no.: 5423-22-3
- Storage condition of test material: In refrigerator (2-8°C) in the dark in well-sealed container

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: appr. 6 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: group housing in Macrolon cages
- Diet: free excess to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements and overnight prior to necropsy, animals had no access to food.
- Water: Free access to tap water except during motor activity measurements.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): appr. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16JAN2013 To: 25FEB 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing, and were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion after the in-life phase of the study, according to a validated method (TNO project 20292/05). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions, or 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
28 days.
Frequency of treatment:
Once daily, 7 d/w.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on the results of a 8-day range finding study (WIL Research Project 501084).
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily in the cage. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION:
- Weekly.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- Organ weights of surviving animals were recorded (according to test guide line)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were fasted overnight with a maximum of 20 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined:
- Tissues/organs checked: According to test guidelines (+ deviations)

ORGAN WEIGHTS:
Organs checked according to test guidelines (+deviations)

HISTOPATHOLOGY: Yes (see table) / No / No data
According to test guidelines (+ deviations)
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The animals treated at 800 mg/kg bw showed lethargy, hunched posture, swelling of the abdomen, piloerection, pale faeces, lean appearance and salivation. Hunched posture, piloerection, slight salivation and lean appearance was noted for the male found dead on day 13 and no clinical signs were seen in the other male found dead on day 3. One female at 200 mg/kg showed piloerection and lean appearance (day 10), which was transient and therefore not considered toxicologically relevant. No further clinical signs were noted for any other animal. Two control animals, one male and one female, had scabs on the neck or shoulder. This is occasionally seen and since these animals were not exposed to the test substance, this finding was not considered toxicologically relevant.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males treated with 800 mg/kg guanidine phosphate (1:1) were found dead during the study on day 3 and day 13. No further mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain was noted in males at 800 mg/kg, predominantly in the first two weeks. No other deviation from expected body weight gain was found.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after allowance for body weight was slightly lower in males and females at 800 mg/kg compared to control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Lower and higher white blood cells counts were found in males and females resp. In females, also lower eosinophil levels and lower platelet counts were detected.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 800 mg/kg bw/d, increased creatinine level (males), increased inorganic phosphate level (males and females), decreased chloride and glucose level (females) and increased alanine aminotransferase, calcium, potassium and bile acids levels (females) were detected.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Higher absolute and relative kidney and liver weights were noted in males and females, except for the absolute liver weight in males which was comparable to controls. The absolute and relative adrenal weights were higher in males only.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were treatment-related macroscopic findings present at the highest dose males and females only which comprised enlarged kidneys and thickened small intestine (duodenum, jejunum, ileum). In females at 800 mg/kg a firm mass was noted in the stomach.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related morphologic alterations consisted of a variety of changes in the kidneys, consisting of presence of tubular basophilia, concomitant with tubules showing desquamation of tubular epithelial cells, as well as foci of degeneration and necrosis of tubular epithelial cells. All these changes were predominantly present within the outer medulla and/or medullary rays (within the cortex). As reaction to the degenerative changes, peritubular fibrosis and lymphoid cell infiltrates was noted among animals in the 800 mg/kg treated group.
Outspoken mineralization was noted at the corticomedullary junction in a single female of the 800 mg/kg treated group.
Further, tubular dilation was observed in most animals of this group. This change is mainly thought responsible for the enlargement of the kidneys of animals in this group, grossly.

Although generally mild, duodenal hypertrophy was noted among animals in the 800 mg/kg treated group. Grossly, the duodenal hypertrophy resulted in thickening of the small intestine in two animals. Mild hypertrophy of the adrenal cortex was present in the males of the 800 mg/kg treated group.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mortality and macroscopic and histopathologic effects on kidneys, liver, duodenum and adrenals at 800 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of a sub-acute study (oral; 28 days exposure) performed according to OECD/EC guidelines and GLP principles, a No Observed Adverse Effect Level (NOAEL) for guanidine phosphate (1:1) of 200 mg/kg was established.

Executive summary:

A sub-acute study was performed according to OECD/EC guidelines and GLP principles. Male and female Wistar rats were exposed via oral gavage to 0, 50, 200 or 800 mg/kg bw/ day. Two males treated at 800 mg/kg were found dead on Days 3 and 13. No further mortality occurred. In the highest dose group, animals showed lethargy (with lower motor activity in females), hunched posture, swelling of the abdomen, piloerection, pale faeces, lean appearance and salivation. Reduced body weight gain was noted in males at 800 mg/kg, paralleling slightly lower food consumption in rats of the highest dose group. No other deviations in expected body weights and body weight gain were noted. Higher relative kidney and liver weights were noted in males and females. There were treatment-related macroscopic findings present at 800 mg/kg only (enlarged kidneys and thickened small intestine (duodenum, jejunum, ileum)). This coincided with changes in clinical biochemistry parameters at 800 mg/kg bw/ d such as higher creatinine level in males, higher inorganic phosphate level (both sexes), and in females lower chloride, glucose, alanine aminotransferase, calcium, potassium and bile acids levels.

Based on these data, a No Observed Adverse Effect Level (NOAEL) for guanidine phosphate (1:1) of 200 mg/kg was established. Since the effects were not found to be significantly toxic (not significantly different from the effects seen at the acute testing), the test substance is not classified according to Regulation (EC) No 1272/2008.