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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:
June-July 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Relatively well reported study according to guideline/standard; however, analysis of formulations was not completely satisfying.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test compound: PDTN
Appearance: white crystalline solid
Chemical name: Acetonitrile,2,2',2'',2'''-(1,3-propanediyldinitrilo)tetrakis-
Batch no: JNN98038
Composition: PDTN 99.2±1.0% m/m; water 0.5±0.1% m/m
Storage: at room temperature in the dark
Expiry date: 1 June 2000

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: mean weight males ca. 220-230 g; mean weight females ca. 179-184 g
- Fasting period before study: not required
- Housing: in groups of 5 per sex in stainless steel suspended cages with wire mesh floors
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: at least 5 days (no exact data reported)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 21 (no exact data reported)
- Humidity (%): ca. 50 (no exact data reported)
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 26 June To: 24 July 1998

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: formulations (w/w) were prepared daily within 4 h prior to treatment. Adjustment was made for the specific gravity of the vehicle.


VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations
- Concentration in vehicle: 10, 40, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): no info
- Purity: no info
- Specific gravity: 1.127
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared on 24 September 1998 (2 months post-treatment) were analysed to check stability, homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations). The samples were prepared in an identical manner as during the treatment period of the 28-day study.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily oral gavage
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a 5-day RF study (0, 50, 200 and 1000 mg/kg bw; based on increased liver weight)
- Rationale for animal assignment (if not random): computer randomisation based on BW
- Rationale for selecting satellite groups: not used
- Post-exposure recovery period in satellite groups: not used
- Section schedule rationale (if not random): no info
Positive control:
Not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: arena observations (prior to start and on days 8, 15, 22 and 28)

BODY WEIGHT: Yes
- Time schedule for examinations:on days 1, 8, 15, 22 and 28

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: ???????

WATER CONSUMPTION: Yes (subjective appraisal only)

OPHTHALMOSCOPIC EXAMINATION: No (not required in 28-day study)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes (ca. 20 h)
- How many animals: all
- Parameters: RBC, HB, HCT, MCV, MCH, MCHC, platelets, RDW, WBC, differential leukocyte count, prothrombin time, partial
thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes (ca. 20 h)
- How many animals:all
- Parameters: ALAT, ASAT, total bilirubin, cholesterol, creatinine, glucose, urea, total protein, albumin, ALP, Na, K, Cl, Ca, inorganic
phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes (FOB)
- Time schedule for examinations: week 4
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (hearing), pupillary reflex, static righting reflex, grip strength, activity test

OTHER: none
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes; Adrenal glands, Brain, Caecum, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum, Kidneys,
Liver, Lung, Lymph nodes - mandibular, mesenteric, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary
gland, Prostate gland, Rectum, Sciatic nerve, Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow,
Stomach, Testes, Thymus, Thyroid including parathyroid, Trachea, Urinary bladder, Uterus, All gross lesions

ORGAN WEIGHTS: adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus
Other examinations:
No
Statistics:
The following statistical methods were used to analyse the data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnet t-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 when the data could not be assumed to follow a normal distribution.
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.
References:
- C.W. Dunnett, A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
- R.G. Miller, Simultaneous Statistical Inference, Springer Verlag, New York (1981)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY: One female dosed at 1000 rng/kg/day was found dead on day 23. The cause of death was
considered to be due to urinary tract infection and not considered to be treatment-related.
Piloerection and hunched posture were noted among males and females treated at 1000 mg/kg/day. Red discolouration of the urine was noted in all females of this dose group on a few days only. Severe brown staining of the fur was noted in males and females
treated at 1000 mglkglday. This finding was also noted to a lesser extent in all males treated at 200 mglkg/day and incidentally in the control and 50 mglkg dose groups. Based on the incidence and severity in the 1000 mglkg dose group, a relationship with
treatment, most likely a secondary, non-specific effect, cannot be excluded. Excessive salivation was observed in one control and
the majority of treated animals. This sign is often noted in rats of this age and strain following oral gavage and not considered test
compound-related.

BODY WEIGHT AND WEIGHT GAIN: Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

FOOD CONSUMPTION: There were no differences in food consumption before or after allowance for body weight between treated and control animals which were considered to have resulted from treatment with PDTN.

FOOD EFFICIENCY: no changes

WATER CONSUMPTION: no info

OPHTHALMOSCOPIC EXAMINATION: not examined

HAEMATOLOGY: no treatment-related changes

CLINICAL CHEMISTRY: A statistically significant increase was noted in alanine aminotransferase activity of males and females
treated at 1000 mglkglday. The values of three males and one female were outside the normal range of historical background data
for rats of this age and strain.

URINALYSIS: not examined.

NEUROBEHAVIOUR: Any findings noted in the functional observation tests and the variation in motor activity did not indicate a
relation with treatment.

ORGAN WEIGHTS: Absolute and relative liver weights were slightly increased in males and females treated at 1000 mg/kg bw.
Statistical significance was only reached in relative liver weight in males.

GROSS PATHOLOGY: Macroscopic observations at necropsy did not reveal clear treatment-related findings. Enlarged kidneys with a pelvic dilation, enlarged urinary bladder with a blackbrown contents and a black-brown and thickened vagina were noted in the
female that died during the study. Watery fluid in the uterus, found in one female treated at 50 mg/kg/day, is related to a stage in the oestrous cycle and is a normal finding. Alopecia or scab formation, noted among treated animals were considered to be within the
range of biological variation for rats of this age and strain and not to represent a change of toxicologica1 significance.

HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related findings observed in the 1000 mg/kg/day dose group included minimal to slight centrilobular hepatocellular hypertrophy in the liver of 4/5 males and 2/5 females. The female that died during the study period revealed marked hydronephrosis, moderate tubular dilation and pyelonephritis in the kidney, moderate acute inflammation of the
urinary bladder and slight inflammation of the vagina. There were no microscopic findings recorded in the 50 or 200 mglkglday
treated rats which could be attributed to treatment with the test substance.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): not examined

HISTORICAL CONTROL DATA (if applicable): not needed

OTHER FINDINGS: no

Effect levels

Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
From the results of the present 28-day study, the NOAEL was 200 mg/kg bw/day.
Executive summary:

Subacute 28 -day oral toxicity with PDTN by daily gavage in the rat.

Based on the results of a 5-day range finding study, the dose levels for the 28 -day toxicity study were selected to be also 0, 50, 200 and 1000 mg/kg/day. The study was based on the following guidelines: EEC Directive 96/54/EEC, B.7, and OECD 407.

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. The following parameters were evaluated: clinical signs (daily); functional observation tests (week 4); body weight and food consumption (weekly); clinical pathology and macroscopy (at termination); organ weights and histopathology on a selection of tissues. Accuracy, stability and homogeneity of test substance formulations in polyethylene glycol were demonstrated by analyses. Slightly high values for the accuracy of group 2 formulations were detected due to technical problems.

50 rng/kg/day: No treatment-related findings noted.

200 mg/kg/day: No treatment-related findings noted

1000 mg/kg/day:

1) Clinical signs included hunched posture, piloerection and severe brown staining of the fur (males and females) and red discolouration of the urine (females).

2) Increased alanine aminotransferase activity was noted in males and females.

3) Statistically significantly increased relative liver weights were noted in males. Relative liver weights were also slightly higher in females

4) Centrilobular hepatocyte hypertrophy was noted microscopically in 4/5 males and 2/5 females.

From the results presented in this report a No Observed Adverse Effect Level (NOEL) of 200 mg/kg/day was concluded.