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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
480-680-7
EC Name:
-
Cas Number:
120128-90-7
Molecular formula:
Hill formula: C8H16N2O3 CAS formula: C8H16N2O3
IUPAC Name:
480-680-7
Test material form:
other: Clear colourless to yellowish liquid

Test animals

Species:
rat
Strain:
other: Wistar strain, Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean. (males: 156-176 grams; females: 93-123 grams)
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 – 23.4
- Humidity (%): 41 - 95
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12
Temporary deviations from the maximum level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 17 October 2011 - 19 January 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for density and purity of the test substance.

VEHICLE:
Water

DOSE VOLUME:
5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight
Details on mating procedure:
no mating
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted during the treatment phase, according to a validated method (NOTOX project 454117). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations; in weeks 1, 6 and 13). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation prepared for use in Week 6. This was considered to have been introduced during sample pretreatment, since the response was not observed in the duplicate test sample. In all other formulations of Group 1, no test substance was detected.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
At least 90 days.
Frequency of treatment:
Once daily, 7 d/w.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000
Basis:
actual ingested
No. of animals per sex per dose:
10
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 14-day oral range finding study with PC-2011-350 by daily gavage in the rat (NOTOX project 497623).

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing. Once prior to start of treatment and at weekly intervals during the treatment phase pre-dose clinical observations were performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs was recorded.
Arena observations were conducted prior to dosing instead of after dosing. No peak effect of occurrence of clinical signs was noted during the dose range finding study. Therefore, the timing of conduct of the arena observations was considered not critical.

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

FOOD EFFICIENCY: yes
- 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
Food consumption was measured off-line on Day 9 instead of Day 8. Based on conducted measurements, an adequate assessment of the food intake could be made. A correction was made afterwards in the calculated food intake data to account for the difference in measurement period of food intake during the first two weeks.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: yes
- Time schedule for examinations: at pretest: all animals (including spare animals), at week 13: Groups 1 and 4.

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

CLINICAL CHEMISTRY: yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: During week 12-13 of treatment
- Dose groups that were examined: all animals of Groups 1 and 4
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.
Postmortem examinations (parental animals):
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 were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS: yes
Organs checked according to test guidelines

HISTOPATHOLOGY: yes
According to test guidelines
For one animal of Group 4 one mandibular lymph node was available for histopathology. Sufficient data was available for histopathological evaluation.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Details on results (P0)

MORTALITY AND CLINICAL SIGNS
No mortality occurred during the study period.
No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted included scabs, a broken tail apex, elongated teeth (indicated in the tables as “broken upper incisors”) and salivation. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

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

FOOD EFFICIENCY
Food consumption before or after allowance for body weight was similar between treated and control animals.

OPHTHALMOSCOPIC EXAMINATION
No toxicologically significant ophthalmology findings were noted. Incidental ophthalmology findings at pretest or in week 13 consisted of pinpoint or multifocal corneal opacities, central lens opacity, persistent hyaloid vessel remnants, posterior cataract due to hyaloid remnants, and focal corneal oedema. The nature and incidence of these findings was within the range considered to be normal for rats of this age and strain, and therefore considered to be of no toxicological relevance.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes in haematological parameters were considered to be of no toxicological significance as they occurred in the absence of a (clear) dose-related trend and remained within the range considered normal for rats of this age and strain. These changes consisted of lower white blood cell (WBC) red blood cell counts in males at 1000 mg/kg, higher mean corpuscular haemoglobin (MCH) in males and females at 1000 mg/kg, higher mean corpuscular volume (MCV) in males at 100 and 300 mg/kg, lower reticulocyte counts in females at 300 mg/kg, lower haematocrit level in females at 100 mg/kg, higher mean corpuscular haemoglobin concentration (MCHC) in females at 100, 300 and 1000 mg/kg, and higher platelet counts in females at 300 and 1000 mg/kg.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as they occurred in the absence of a (clear) dose-related trend and remained within the range considered normal for rats of this age and strain. These changes consisted of lower sodium and chloride levels in males at 100 mg/kg, higher total bilirubin and urea levels in females at 1000 mg/kg, higher creatinine levels in females at 100, 300 and 1000 mg/kg, and lower potassium levels in females at 100 mg/kg.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals of the control group and at 1000 mg/kg. Motor activity was similar between the 1000 mg/kg and control group. Both groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
No toxicologically significant changes were noted in organ weights and organ to body weight ratios. Statistically significant changes in organ weights were considered not to be a sign of toxicity as they occurred in the absence of a (clear) dose-related trend and remained within the range considered normal for rats of this age and strain. These changes consisted of lower absolute and relative spleen and prostate weight in males at 100 mg/kg, lower absolute and relative seminal vesicle weight in males at 100 and 300 mg/kg, higher absolute brain weight in females at 300 mg/kg, and lower absolute and relative spleen weight in female at 300 mg/kg.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations. The incidence of macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included red foci on the lungs, an accessory liver on the caudate lobe, pelvic dilation of the kidneys, flaccid seminal vesicles, a tan focus on the preputial or clitoral glands, tan discolouration of the clitoral glands, enlarged spleen with irregular surface, a bent tail bone, a yellowish hard or soft nodule on the epididymal adipose tissue, fluid in the uterus, red discolouration of the mandibular lymph nodes and alopecia.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance. All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no effects on reproductive organs up to the limit dose

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was established.
Executive summary:

In a subchronic toxicity study (according to OECD Guideline 408), the testsubstance was administered to 10 Wistar rats/sex/dose by gavage at dose levels of 0, 100, 300 and 1000 mg/kg for 90 consecutive days.

There were no compound related effects in mortality, clinical signs, functional observations, ophthalmoscopy, body weight and weight gain, food consumption and food efficiency, hematology, clinical chemistry, organ weights, gross and histologic pathology or neurobehaviour.

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was established.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirements for a OECD 408 study in rats.