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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted according to internationally recognised test methods. For read across justification see Section 13.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material : CP12
- Physical state: Liquid
- Lot/batch No.: 0012
- Expiration date of the lot/batch: 2015-08-01
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy SpA
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Males: 289-345 g; Females: 189-238 g
- Fasting period before study: No
- Housing: Polysulphone solid bottom cages, Group caged except for in pairs (1 male/1 female) for mating and females individually caged during gestation
- Diet (e.g. ad libitum): Mucedola 4RF21 pelleted diet available ad libitum
- Water (e.g. ad libitum): Municipal supply tap water available ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-10-18 To: 2012-12-09

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared daily by dissolution/suspension in 0.5% aqueous carboxymethylcellulose.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Proposed formulation procedure checked for concentration and homogeneity in the range from 10 to 100 mg/mL to confirm that the method was suitable. All levels were within 80-120% of nominal for concentration and the CV for homogeneity was < 20%. Stability after 24 hours at room temperature was verified in the range from 10 to 100 mg/mL and determined to be within acceptable limits (80-120% of nominal of concentration and CV for homogeneity < 20%).
Samples of the formulations, prepared on Weeks 1 and 6, were analysed to check for homogeneity and concentration. Results were within acceptable limits (90-110% of nominal for concentration and the CV for homogeneity was < 10%).

Duration of treatment / exposure:
Males: from 14 days before pairing for a total of approximately 5 weeks
Females: from 14 days before pairing to day 3 post partum (total of approximately 6 weeks)
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/day
Basis:
other: in vehicle
No. of animals per sex per dose:
10 males & 10 females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on data from 2 week dose-ranging screen
- Rationale for animal assignment (if not random): Random, stratified body weight


Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for signs of ill health and moribund condition
- Cage side observations were included.: Yes - Daily, 2 or 3 times following dose administration

DETAILED CLINICAL OBSERVATIONS: Yes (functional observation battery)
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Weekly; Females: Weekly from allocation to study, gestation days 0, 7, 14 and 20, post partum Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No - Not applicable as animals dosed by gavage

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: No - Not applicable - Not a dietary study

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No - Not applicable as animals dosed by gavage:

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males - after 33 days of treatment; Females - after day 4 of lactation. Terminal blood samples taken from the abdominal vena cava
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes - overnight
- How many animals: 5 males/5 females/group
- Parameters examined: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Males - after 33 days of treatment; Females - after day 4 of lactation. Terminal blood samples taken from the abdominal vena cava
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes - overnight
- How many animals: 5 males/5 females/group
- Parameters examined: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

URINALYSIS: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes - Macroscopic examination of organs and tissues including organ weights: adrenal glands, brain, epididymides, heart, kidney, liver, ovaries (with oviduct), spleen, testes, thymus.
- Preservation: adrenal glands, bone marrow, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum (including Peyer’s patches, kidneys, liver, Lungs (including mainstem bronchi), lymph nodes (cervical and mesenteric), nasal cavity, oesophagus, ovaries with oviducts, pituitary gland, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating gland, spinal column, spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus and cervix, vagina. All tissues fixed and preserved in 10% neutral buffered formalin with the exception of testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol.

HISTOPATHOLOGY: Yes (5 males/5 females examined from high dose and control groups) - adrenal glands, bone marrow, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum (including Peyer’s patches, kidneys, liver, Lungs (including mainstem bronchi), lymph nodes (cervical and mesenteric), ovaries with oviducts, pituitary gland, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating gland, spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus and cervix, vagina.

Other examinations:
No
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
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
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Reduced urea and bile acids, increased globulin in males
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY – No mortality occurred in the study. Clinical observations for neurotoxicity assessment (removal of animals from the home cage and open arena) did not reveal changes attributable to the tested substance. Hairloss and/or salivation were observed in some males treated at 1000 mg/kg/day and in single females treated at 300 or 1000 mg/kg/day) .

BODY WEIGHT AND WEIGHT GAIN - Body weight and body weight gain were unaffected by treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE - Food consumption was unaffected by treatment.

HAEMATOLOGY - One male treated at 300 mg/kg/day and 2 treated at 1000 mg/kg /day exhibited slightly reduced lymphocytes, monocytes, eosinophils and basophils. One female treated at 300 mg/kg/day and 2 treated at 1000 mg/kg/day exhibited slightly decreased erythrocytes, haemoglobin and haematocrit associated with reticulocytosis. A statistically significant difference in erythrocytes noted between control and treated males were not dose-related, therefore considered unrelated to treatment.

CLINICAL CHEMISTRY - Males treated at 300 or 1000 mg/kg/day showed a dose-related decrease of urea and bile acids and increased globulin. Urea was also decreased in males dosed at 100 mg/kg/day and in two females treated at 1000 mg/kg/day. The other statistically significant differences between control and treated animals (protein and sodium in males, glucose and albumin/globulin ratio in females) were of minimal severity or observed in the low and/or medium groups, therefore they were considered incidental.

GROSS PATHOLOGY - No treatment-related changes were noted. Those changes that were observed had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age.

ORGAN WEIGHTS – No differences were noted in organ weights.

HISTOPATHOLOGY: NON-NEOPLASTIC – No treatment-related findings were noted. Changes that were observed had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age. Examples of such findings included liver inflammatory cell foci and thymus atrophy. A single case of moderate mucosa hyperplasia associated with inflammation was seen in the forestomach of a single female treated at 1000 mg/kg/day. This was considered to be an incidental finding unrelated to treatment.

A detailed qualitative evaluation of testes performed on 5 randomly selected control and high dose males took into account the tubular stages of the spermatogenic cycle, in order to identify potential treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. Regular layering in the germinal epithelium was noted.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall lack of effects on: clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A combined repeat-dose toxicity reproduction/developmental toxicity screen has been undertaken according to OECD TG 422 methods. Dose levels of 0, 100, 300 and 1000 mg/kg/day were investigated. No treatment-findings were noted during the in-vivo phase. Changes in clinical pathological investigations were considered of no toxicological significance. No treatment-related changes were observed at post mortem examinations. On the basis of these findings, the NOAEL (No Observed Adverse Effect Level) for general toxicity is considered to be 1000 mg/kg/day for both males and females.
Executive summary:

A combined repeat-dose toxicity reproduction/developmental toxicity screen has been undertaken according to OECD TG 422 methods. Dose levels of 0, 100, 300 and 1000 mg/kg/day were investigated. No treatment-findings were noted during the in-vivo phase. Changes in clinical pathological investigations were considered of no toxicological significance. No treatment-related changes were observed at post mortem examinations. On the basis of these findings, the NOAEL (No Observed Adverse Effect Level) for general toxicity is considered to be 1000 mg/kg/day for both males and females.