Registration Dossier
Registration Dossier
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EC number: 221-244-6 | CAS number: 3040-44-6
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Sep 2018 - Dec 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Sep 2018 - Dec 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 29 Jul 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- certified by Landesamt für Umwelt Rheinland-Pfalz
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: B1056-070518
- Purity: 99.8 area % (GC, RTX-5); 99.9 area-% (GC, DB-Wax UI)
- Purity test date: 30 May 2018
- Expiry date of lot: 07 May 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility. The stability of test substance in deionized water was demonstrated for a period of 7 days at room temperature.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed in a graduated flask depending on the dose group, topped up with deionized water and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.
FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in deionized water - Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 13 - 15 weeks
- Weight at study initiation: males: 371.9 g (mean); females: 207.4 g (mean)
- Housing: individually (exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together.)
- Diet: ad libitum; ground Kliba maintenance diet mouse-rat “GLP” (supplied by Garanovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 21 days
DETAILS OF FOOD AND WATER QUALITY: The drinking water was regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms. On the basis of the analytical findings the drinking water was found to be suitable. The food used in the study was assayed for chemical and for microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
IN-LIFE DATES: From: 18 Sep 2018 To: 18 Oct and 08 Nov 2018 (F0 males); 04 Dec 2018 (F0 females); 27 Nov - 01 Dec 2018 (litters) - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- deionized water
- Details on exposure:
- VEHICLE
- Concentration in vehicle: 0.25, 0.75 and 2.5 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg bw/d - Details on mating procedure:
- - M/F ratio per cage: 1/1 or 1/2 (due to the premature death of one male animal of the control group)
- Length of cohabitation: overnight (from about 16.00 h until 6.30 - 9.00 h of the following morning) for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): single - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analytical verifications of the stability of the test substance in deionized water for a period of 7 days at room temperature had been initiated prior to the start of the study in the same batch. At the beginning (during premating), twice during gestation and once during lactation of the study each 1 sample was taken from the low, mid and high concentration for a concentration control analysis. The samples collected at the beginning of the administration period and during lactation were analyzed via capillary electrophoresis (CE) with indirect UV/VIS detection.
All measured values for the test substance were in the expected range of the target concentrations (90 - 110%) demonstrating the correctness of the preparations. - Duration of treatment / exposure:
- males: 30 days; females: 56 days
The duration of treatment covered a 2-weeks premating and mating period in both sexes, 3 days postmating in males, the entire gestation and approximately 3 weeks of lactation period in females and 4-weeks postmating for sperm negative females up to the day of scheduled sacrifice of the animals. - Frequency of treatment:
- once daily at approximately the same time in the morning (females in labor were not treated)
- Dose / conc.:
- 25 mg/kg bw/day (nominal)
- Remarks:
- low dose group
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Remarks:
- mid dose group
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- high dose group
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: In an acute oral toxicity study in rats, the calculated LD50-value for males and females was 1100 mg/kg bw/d. Severe clinical signs (e.g. dyspnoe, apathy, staggering, trembling, poor general state) and mortality in both sexes were observed at 1000 mg/kg bw/d and above. At the lowest tested dose of 464 mg/kg bw/d, one female died within the first 24 hours.
In a repeated dose test study, the test substance was orally (gavage) applied at concentrations of 0, 150 and 500 mg/kg bw/d for 14 days to four rats per test group and sex. At 500 mg/kg bw/d, one male animal was found dead on study day 10 and one further male was sacrificed in moribund state due to severe clinical findings (gasping, labored respiration, respiration sounds) on study day 10. Furthermore, significantly reduced food consumption, decreased body weights, and hematological as well as gross pathological findings were observed at this dose level. At 150 mg/kg bw/d, no adverse findings were observed.
Based on the above-mentioned effects, the following dose levels were selected: 25, 75 and 250 mg/kg bw/d.
- Fasting period before blood sampling for clinical biochemistry: 16-20 hours - Positive control:
- no
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily
- A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos (protruding eyeball), assessment of the feces excreted during the examination (appearance/consistency), assessment of the urine excreted during the examination, pupil size
BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined on GD 0 - 7, 7 - 4 and 14 - 20.
• Food consumption of the females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
• Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.
WATER CONSUMPTION: Yes
- Time schedule for examinations: once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (female parental animals)
• Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7 and 12-13.
• Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA), Prothrombin time (Hepato Quick’s test) (HQT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)
OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 28 (males) and 55 (females).
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/
arousal level, feces excreted within 2 minutes (appearance/consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response),
coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings
Motor activity measurement:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group
Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: all surviving males at termination
- Parameters examined: Total thyroxine (T4), Thyroid stimulating hormone (TSH) - Oestrous cyclicity (parental animals):
- For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization. For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
- Sperm parameters (parental animals):
- Parameters examined in F0 male parental generation:
testes weight, epididymes weight, prostate weight, weight of seminal vesicles with coagulating glands; histopathological examination of epididymides, prostate, seminal vesicles, testes; spermatogenic staging profiles - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4 pups/sex/litter as nearly as possible); excess pups were sacrificed under isoflurane anesthesia by decapitation, blood was sampled for determination of thyroid hormone concentrations and pups were examined externally, eviscerated and their organs were assessed macroscopically.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, postnatal mortality, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups on PND 13, blood thyroid hormone concentrations, gross necropsy
GROSS EXAMINATION OF DEAD PUPS:
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals (after a treatment period of at least 28 days)
- Maternal animals: All surviving animals (on PND 14)
GROSS NECROPSY
- The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
HISTOPATHOLOGY
The tissues indicated in Table 1 were prepared for microscopic examination.
ORGAN WEIGHTS
- determined in all animals sacrificed on schedule: Epididymides, Ovaries, Prostate (ventral and dorsolateral part together, fixed), Seminal vesicles with coagulating glands (fixed), Testes, Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix
- determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for hematological and clinical chemistry examinations): Adrenal glands (fixed), Brain, Heart, Kidneys, Liver, Spleen, Thymus (fixed) - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
GROSS NECROPSY
- The pups were examined externally and eviscerated, and the organs were assessed macroscopically.
HISTOPATHOLOGY
Thyroid glands/parathyroid glands of one male and one female pup per litter at 13 days of age were fixed in neutral buffered 4% formaldehyde solution for possible further processing.
Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted. - Statistics:
- see table 2
- Reproductive indices:
- Male reproduction data:
- the pairing partners, the number of mating days until vaginal sperm was detected in the female animals
- mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods")
Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- mating, fertility and gestation indices, live birth index, postimplantation loss were calculated for F1 litters (for formulas see "Any other information on materials and methods") - Offspring viability indices:
- - viability index, survival index (for formulas see "Any other information on materials and methods")
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- salivation immediately after dosing (up to 2 hours post dosing) during the whole treatment period in several male and all female animals (not considered to be adverse)
- piloerection in 3/10 females on PND 6 (assessed as treatment-related but not adverse, since this minor finding was occasionally seen in three females during one study phase) - Mortality:
- no mortality observed
- Description (incidence):
- - One male animal of the control showed respiration sounds between premating days 3 - 5 and 7 - 9 and piloerection during premating days 4 - 9 and was sacrificed moribund on premating day 9.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- statistically significantly reduced mean body weight changes in females during lactation (PND 0 – 13: 19.4 g vs. 32.9 g in control) showing a body weight loss in the beginning of the lactation phase (PND 0 – 4: -1.5 g vs. 11.9 g in control) (assessed as treatment-related and adverse) - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- statistically significantly reduced food consumption in females during the entire premating period (up to 10 %), during gestation (GD 0 – 7: 9 %) and during several parts of the lactation period (up to 23 % below control) (assessed as treatment-related and adverse) - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- statistically significantly increased mean water consumption in males during premating days 7 - 10 (about 24 %) (not assessed as treatment-related and adverse, since this minor finding occurred only within a short treatment phase of four days and no further parameter in males was altered)
- reduced water consumption in females during premating (up to 18 % below control, without statistical significance), beginning of gestation (GD 0-1: 21 % below control, without statistical significance) and lactation (up to 23 % below control, with statistical significance) (assessed as treatment-related and adverse, since the reduction in water consumption was present during almost the whole treatment period)
Mid-dose group (75 mg/kg bw/d):
- statistically significantly reduced water consumption in females during PND 6 – 7 (about 17 % below control) (not assessed as treatment-related and adverse, since all other treatment phases were not affected) - Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Low-dose group (25 mg/kg bw/d):
- significantly lower absolute reticulocyte counts in males at the end of the administration period (regarded as incidental and not treatment-related, since this alteration was not dose-dependent) - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- significantly increased glucose levels in females at the end of the administration period (regarded as incidental and not treatment-related, since the mean was within the historical control range (females, glucose 5.58 - 6.98 mmol/L)) - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The one animal, sacrificed in a moribund state, showed hyperkeratosis and squamous cell hyperplasia in the forestomach, and decreased cellularity in the spleen (white pulp), thymus (cortical) and bone marrow of the sternum. All these findings were consistent with the moribund condition of the animal. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- significantly decreased TSH values in males (regarded as incidental and not treatment-related, since the mean was within the historical control range (TSH 4.81 - 9.80 μg/L)) - Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - The male mating index calculated after the mating period for F1 litter was 100 % in the control and test group 2 (75 mg/kg bw/d) and 90 % in test groups 1 (25 mg/kg bw/d) and 3 (250 mg/kg bw/d).
- The female mating index calculated after the mating period for F1 litter was 100 % in the control and test group 2 (75 mg/kg bw/d) and 90 % in test groups 1 (25 mg/kg bw/d) and 3 (250 mg/kg bw/d).
- The male fertility index ranged between 80 % and 100 % without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
- The female fertility index ranged between 88.9 % and 90 % without showing any relation to dosing.
- One control male, one low-dose male, one mid-dose male, and one high-dose male did not generate pregnancy. The apparently infertile male rats did not show relevant gross lesions.
- One control female, two low-dose females, one mid-dose female, and two high-dose females did not become pregnant. The non-pregnant females had no relevant gross lesions or microscopic findings. - Dose descriptor:
- NOAEL
- Remarks:
- systemic tox.
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
- Dose descriptor:
- NOAEL
- Remarks:
- systemic tox.
- Effect level:
- 75 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- water consumption and compound intake
- Dose descriptor:
- NOAEL
- Remarks:
- fertility
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- - The viability index indicating pup survival during lactation (PND 0 - 4) varied between 99.1 %/ 100 % / 99.1 % and 98.8 % in test groups 0 - 3, respectively.
- The pups surviving index indicating pup survival during lactation (PND 4 - 13) was 100 % in all test groups.
Thus, the test substance did not influence pup survival in any of the treated groups (25, 75 and 250 mg/kg bw/d). - Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- no effects observed
- Nipple retention in male pups:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - A few pups showed spontaneous findings at gross necropsy: discolored testis, diaphragmatic hernia and dilated renal pelvis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the treatment.
- Histopathological findings:
- not examined
- Other effects:
- no effects observed
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
- Critical effects observed:
- no
- Reproductive effects observed:
- no
- Conclusions:
- The no observed adverse effect level (NOAEL) for general systemic toxicity was the highest dose of 250 mg/kg bw/d for male and the mid-dose of 75 mg/kg bw/d for female Wistar rats. The NOAEL for reproductive performance and fertility was set to 250 mg/kg bw/d for male and female Wistar rats.
- Executive summary:
The test substance was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 25, 75 and 250 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (deionized water). The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, the entire gestation and approximately 3 weeks of lactation period in females and 4-weeks postmating for sperm negative females up to the day of scheduled sacrifice of the animals.
The oral administration of the test substance resulted in signs of systemic toxicity in females at the highest dose of 250 mg/kg bw/d. In high-dose F0 females, adverse findings consisted of a reduction in water and food consumption and a decrease in body weight change. Male animals showed no adverse signs at any tested dose.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the highest dose of 250 mg/kg bw/d for male and the mid-dose of 75 mg/kg bw/d for female Wistar rats. The NOAEL for reproductive performance and fertility was set to 250 mg/kg bw/d for male and female Wistar rats.
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- developmental toxicity
- Remarks:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Sep 2018 - Dec 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
- Version / remarks:
- 29 Jul 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- certified by Landesamt für Umwelt Rheinland-Pfalz
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: B1056-070518
- Purity: 99.8 area % (GC, RTX-5); 99.9 area-% (GC, DB-Wax UI)
- Purity test date: 30 May 2018
- Expiry date of lot: 07 May 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility. The stability of test substance in deionized water was demonstrated for a period of 7 days at room temperature.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed in a graduated flask depending on the dose group, topped up with deionized water and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.
FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in deionized water - Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- deionized water
- Details on exposure:
- VEHICLE
- Concentration in vehicle: 0.25, 0.75 and 2.5 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg bw/d - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analytical verifications of the stability of the test substance in deionized water for a period of 7 days at room temperature had been initiated prior to the start of the study in the same batch. At the beginning (during premating), twice during gestation and once during lactation of the study each 1 sample was taken from the low, mid and high concentration for a concentration control analysis. The samples collected at the beginning of the administration period and during lactation were analyzed via capillary electrophoresis (CE) with indirect UV/VIS detection.
All measured values for the test substance were in the expected range of the target concentrations (90 - 110 %) demonstrating the correctness of the preparations. - Details on mating procedure:
- - Impregnation procedure: cohoused
- M/F ratio per cage: 1/1 or 1/2 (due to the premature death of one male animal of the control group)
- Length of cohabitation: overnight (from about 16.00 h until 6.30 - 9.00 h of the following morning) for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): single - Duration of treatment / exposure:
- males: 30 days; females: 56 days
The duration of treatment covered a 2-weeks premating and mating period in both sexes, 3 days postmating in males, the entire gestation and approximately 3 weeks of lactation period in females and 4-weeks postmating for sperm negative females up to the day of scheduled sacrifice of the animals. - Frequency of treatment:
- once daily at approximately the same time in the morning (females in labor were not treated)
- Dose / conc.:
- 25 mg/kg bw/day (nominal)
- Remarks:
- low dose group
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Remarks:
- mid dose group
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- high dose group
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: In an acute oral toxicity study in rats, the calculated LD50-value for males and females was 1100 mg/kg bw/d. Severe clinical signs (e.g. dyspnoe, apathy, staggering, trembling, poor general state) and mortality in both sexes were observed at 1000 mg/kg bw/d and above. At the lowest tested dose of 464 mg/kg bw/d, one female died within the first 24 hours. In a repeated dose test study, the test substance was orally (gavage) applied at concentrations of 0, 150 and 500 mg/kg bw/d for 14 days to four rats per test group and sex. At 500 mg/kg bw/d, one male animal was found dead on study day 10 and one further male was sacrificed in moribund state due
to severe clinical findings (gasping, labored respiration, respiration sounds) on study day 10. Furthermore, significantly reduced food consumption, decreased body weights, and hematological as well as gross pathological findings were observed at this dose level. At 150 mg/kg bw/d, no adverse findings were observed.
Based on the above-mentioned effects, the following dose levels were selected: 25, 75 and 250 mg/kg bw/d.
- Fasting period before blood sampling for clinical biochemistry: 16-20 hours - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily
- A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, act ivity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos (protruding eyeball), assessment of the feces excreted during the examination (appearance/consistency), assessment of the urine excreted during the examination, pupil size
BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined during the mating period.
• Food consumption of the females with evidence of sperm was determined on GD 0 - 7, 7 - 4 and 14 - 20.
• Food consumption of the females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
• Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.
WATER CONSUMPTION: Yes
- Time schedule for examinations: once a week as representative value over a period of 3 days, with the following exceptions:
• Water consumption was not determined during the mating period.
• Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7 and 12-13.
• Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at PND 14
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 females with litters (in order of delivery) per group
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA), Prothrombin time (Hepato Quick’s test) (HQT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at PND 14
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 females with litters (in order of delivery) per group
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on PND 14
- Organs examined: see table 1
OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 55.
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal
movements/ stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (appearance/consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition
(startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test,
other findings
Motor activity measurement:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five surviving females with litter (in order of delivery) per group - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No - Fetal examinations:
- No
- Statistics:
- see table 2
- Indices:
- Gestation index, Live birth index, Postimplantation loss, Viability index of pups, Survival index of pups (for detailed formulas see "Any other information on materials and methods"
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- salivation immediately after dosing (up to 2 hours post dosing) during the whole treatment period in all female animals (not considered to be adverse)
- piloerection in 3/10 females on PND 6 (assessed as treatment-related but not adverse, since this minor finding was occasionally seen in three females during one study phase) - Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- statistically significantly reduced mean body weight changes in females during lactation (PND 0 – 13: 19.4 g vs. 32.9 g in control) showing a body weight loss in the beginning of the lactation phase (PND 0 – 4: -1.5 g vs. 11.9 g in control) (assessed as treatment-related and adverse) - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- statistically significantly reduced food consumption in females during the entire premating period (up to 10 %), during gestation (GD 0 – 7: 9 %) and during several parts of the lactation period (up to 23 % below control) (assessed as treatment-related and adverse) - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- reduced water consumption in females during premating (up to 18 % below control, without statistical significance), beginning of gestation (GD 0-1: 21 % below control, without statistical significance) and lactation (up to 23 % below control, with statistical significance) (assessed as treatment-related and adverse, since the reduction in water consumption was present during almost the whole treatment period)
Mid-dose group (75 mg/kg bw/d):
- statistically significantly reduced water consumption in females during PND 6 – 7 (about 17 % below control) (not assessed as treatment-related and adverse, since all other treatment phases were not affected) - Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- significantly increased glucose levels in females at the end of the administration period (regarded as incidental and not treatment-related, since the mean was within the historical control range (females, glucose 5.58 - 6.98 mmol/L)) - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- All gross findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Neuropathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- effects observed, treatment-related
- Description (incidence and severity):
- - The post-implantation loss was 3.6 % / 5.8 % / 2.5 % and 19.7 % in test groups 0 - 3, respectively.
- The mean post-implantation loss of test group 3 (250 mg/kg bw/d) showed a high standard deviation of 23.8 % which was caused by the individual values of the females within the group. Three out of eight females showed a rather high post-implantation loss (22 - 67 %), three had values between 7 and 9 % and two showed no post-implantation losses. Although the variation within the group was quite distinct, the mean value of the group was outside the historical control range (0.9 - 16.8 %). Therefore, a relation to treatment cannot be excluded and the finding was assessed as treatment-related and adverse.
- The mean number of F1 pups delivered per dam remained unaffected (11.9 / 11.0 / 12.2 and 9.5 pups/dam in test groups 0 - 3, respectively). - Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- not examined
- Dead fetuses:
- not examined
- Changes in pregnancy duration:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - The mean duration of gestation values varied between 22.0 (control and test group 2 (75 mg/kg bw/d)), 22.1 (test group 1 (25 mg/kg bw/d)) and 22.5** (test group 3 (250 mg/kg bw/d)) [**p<=0.01] days. The mean value of test group 3 was within the range of the historical control data (21.8 - 22.6 days) and was, therefore, not assessed as treatment-related, adverse finding.
- Changes in number of pregnant:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - One control female, two low-dose females, one mid-dose female, and two high-dose females did not become pregnant. The non-pregnant females had no relevant gross lesions or microscopic findings.
- Other effects:
- no effects observed
- Details on maternal toxic effects:
- Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.3 / 11.8 / 12.6 and 11.2 implants/dam in test groups 0 - 3, respectively).
- Dose descriptor:
- NOAEL
- Remarks:
- systemic tox.
- Effect level:
- 75 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- water consumption and compound intake
- Dose descriptor:
- NOAEL
- Remarks:
- fertility
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: no adverse effects up to and including the highest tested dose
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- Fetal body weight changes are not determined in a OECD 422 study. The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study. No runts were seen in all male and female pups of all test groups 0 - 3.
- Reduction in number of live offspring:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - The rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - The viability index indicating pup survival during lactation (PND 0 - 4) varied between 99.1 % / 100 % / 99.1 % and 98.8 % in test groups 0 - 3, respectively.
- The pups surviving index indicating pup survival during lactation (PND 4 - 13) was 100 % in all test groups. - External malformations:
- no effects observed
- Skeletal malformations:
- not examined
- Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - A few pups showed spontaneous findings at gross necropsy: discolored testis, diaphragmatic hernia and dilated renal pelvis (not considered to be associated to the treatment, since these findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences)
- Other effects:
- no effects observed
- Dose descriptor:
- NOAEL
- Remarks:
- developmental tox.
- Effect level:
- 75 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- other: increased post-implantation loss in parental females
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 250 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects as a secondary non-specific consequence of maternal toxicity effects
- Conclusions:
- The no observed adverse effect level (NOAEL) for general systemic toxicity was the highest dose of 250 mg/kg bw/d for male and the mid-dose of 75 mg/kg bw/d for female Wistar rats.
The NOAEL for reproductive performance and fertility was set to 250 mg/kg bw/d for male and female Wistar rats.
The NOAEL for developmental toxicity was 75 mg/kg bw/d. This was based on the increased post-implantation loss in the high-dose females. - Executive summary:
The test substance was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 25, 75 and 250 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (deionized water). The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, the entire gestation and approximately 3 weeks of lactation period in females and 4-weeks postmating for sperm negative females up to the day of scheduled sacrifice of the animals.
The oral administration of the test substance resulted in signs of systemic toxicity in females at the highest dose of 250 mg/kg bw/d. In high-dose F0 females, adverse findings consisted of a reduction in water and food consumption and a decrease in body weight change. Male animals showed no adverse signs at any tested dose.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the highest dose of 250 mg/kg bw/d for male and the mid-dose of 75 mg/kg bw/d for female Wistar rats. The NOAEL for reproductive performance and fertility was set to 250 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 75 mg/kg bw/d. This was based on the increased post-implantation loss in the high-dose females.
Table 3: Summary Pregnancy Status Report - Reproduction
|
Test Group 0/F 0 mg/kgbw/d |
Test Group 1/F 25 mg/kgbw/d |
Test Group 2/F 75 mg/kgbw/d |
Test Group 3/F 250 mg/kgbw/d |
|
No. of females at start |
N |
10 |
10 |
10 |
10 |
No. of females mated |
N |
10 |
10 |
10 |
10 |
Without evidence of mating |
N |
0 |
1 |
0 |
1 |
- Pregnant |
N |
0 |
0 |
0 |
0 |
- Not pregnant |
N |
0 |
1 |
0 |
1 |
Females with defined Day 0 pc |
N |
10 |
9 |
10 |
9 |
Pregnant |
N |
9 |
8 |
9 |
8 |
- sacrificed scheduled |
N |
9 |
8 |
9 |
8 |
Not pregnant |
N |
1 |
2 |
1 |
2 |
- sacrificed scheduled |
N |
1 |
2 |
1 |
2 |
Pregnant, not delivering |
N |
0 |
0 |
0 |
0 |
Delivering |
N |
9 |
8 |
9 |
8 |
-- With liveborn pups |
N |
9 |
8 |
9 |
8 |
|
% |
100.0 |
100.0 |
100.0 |
100.0 |
-- With all pups stillborn |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Table 4: Summary Delivery Report
|
Test Group 0/F 0 mg/kgbw/d |
|
Test Group 1/F 25 mg/kgbw/d |
Test Group 2/F 75 mg/kgbw/d |
Test Group 3/F 250 mg/kgbw/d |
|
|
No. of females at start |
N |
10 |
|
10 |
10 |
10 |
|
No. of females mated |
N |
10 |
f- |
10 |
10 |
10 |
|
|
% |
100.0 |
|
100.0 |
100.0 |
100.0 |
|
Pregnant |
N |
9 |
f- |
8 |
9 |
8 |
|
|
% |
90.0 |
|
80.0 |
90.0 |
80.0 |
|
Without delivery |
N |
1 |
|
2 |
1 |
2 |
|
- Pregnant |
N |
0 |
|
0 |
0 |
0 |
|
- Not pregnant |
N |
1 |
|
2 |
1 |
2 |
|
-- Delivering |
N |
9 |
f- |
8 |
9 |
8 |
|
|
% |
100.0 |
|
100.0 |
100.0 |
100.0 |
|
-- With liveborn pups |
N |
9 |
f- |
8 |
9 |
8 |
|
Gestation Index |
% |
100.0 |
|
100.0 |
100.0 |
100.0 |
|
Gestation days |
Mean |
22.0 |
n |
22.1 |
22.0 |
22.5 |
** |
|
S.d. |
0.0 |
|
0.4 |
0.0 |
0.5 |
|
|
N |
9 |
|
8 |
9 |
8 |
|
-- With stillborn pups |
N |
1 |
f+ |
0 |
1 |
1 |
|
|
% |
11.1 |
|
0.0 |
11.1 |
12.5 |
|
-- With all pups stillborn |
N |
0 |
f+ |
0 |
0 |
0 |
|
|
% |
0.0 |
|
0.0 |
0.0 |
0.0 |
|
Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
f=FISHER-EXACT; n=DUNNETT
Table 5: Summary Litter Report
|
Test Group 0/F 0 mg/kgbw/d |
|
Test Group 1/F 25 mg/kg bw/d |
Test Group 2/F 75 mg/kgbw/d |
Test Group 3/F 250 mg/kgbw/d |
|
Total Number of Pregnant Females |
N |
9 |
|
8 |
9 |
8 |
Total number of litters |
N |
9 |
|
8 |
9 |
8 |
With liveborn pups |
N |
9 |
f- |
8 |
9 |
8 |
|
% |
100.0 |
|
100.0 |
100.0 |
100.0 |
With stillborn pups |
N |
1 |
f+ |
0 |
1 |
1 |
|
% |
11.1 |
|
0.0 |
11.1 |
12.5 |
With all pups stillborn |
N |
0 |
f+ |
0 |
0 |
0 |
|
% |
0.0 |
|
0.0 |
0.0 |
0.0 |
Implantation Sites |
N |
111 |
|
94 |
113 |
90 |
|
Mean |
12.3 |
x- |
11.8 |
12.6 |
11.2 |
|
S.d. |
1.9 |
|
1.7 |
1.6 |
3.9 |
|
N |
9 |
|
8 |
9 |
8 |
Pups delivered |
N |
107 |
|
88 |
110 |
76 |
|
Mean |
11.9 |
x- |
11.0 |
12.2 |
9.5 |
|
S.d. |
2.0 |
|
1.2 |
1.6 |
4.0 |
|
N |
9 |
|
8 |
9 |
8 |
Postimplantation Loss |
Mean% |
3.6 |
x+ |
5.8 |
2.5 |
19.7 |
|
S.d. |
5.7 |
|
6.7 |
5.4 |
23.8 |
|
N |
9 |
|
8 |
9 |
8 |
Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
f=FISHER-EXACT; x=WILCOX
Table 6: Summary Litter Report - Pup Status
Test Group 0/F 0 mg/kgbw/d |
|
Test Group 1/F 25 mg/kgbw/d |
Test Group 2/F 75 mg/kgbw/d |
Test Group 3/F 250 mg/kgbw/d |
||
Pups liveborn |
N |
106 |
|
88 |
109 |
73 |
|
% |
99.1 |
|
100.0 |
99.1 |
96.1 |
|
Mean |
11.8 |
x- |
11.0 |
12.1 |
9.1 |
S.d. |
1.8 |
|
1.2 |
1.6 |
3.8 |
|
|
N |
9 |
|
8 |
9 |
8 |
Pups stillborn |
N |
1 |
|
0 |
1 |
3 |
|
% |
0.9 |
|
0.0 |
0.9 |
3.9 |
|
Mean |
0.1 |
x+ |
0.0 |
0.1 |
0.4 |
S.d. |
0.3 |
|
0.0 |
0.3 |
1.1 |
|
|
N |
9 |
|
8 |
9 |
8 |
Perinatal Loss |
Mean% |
0.7 |
x+ |
0.0 |
0.9 |
2.9 |
|
S.d. |
2.1 |
|
0.0 |
2.8 |
8.2 |
|
N |
9 |
|
8 |
9 |
8 |
Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
f=FISHER-EXACT; x=WILCOX
Table 7: Summary Litter Report - Dead Pups
Test Group 0/F 0 mg/kgbw/d |
Test Group 1/F 25 mg/kgbw/d |
Test Group 2/F 75 mg/kgbw/d |
Test Group 3/F 250 mg/kgbw/d |
||
Litters with liveborn pups |
N |
9 |
8 |
9 |
8 |
Pups delivered |
N |
107 |
88 |
110 |
76 |
found dead [pup] / Dead |
N |
1 |
0 |
1 |
0 |
|
% |
0.9 |
0.0 |
0.9 |
0.0 |
stillborn / Dead |
N |
1 |
0 |
1 |
3 |
|
% |
0.9 |
0.0 |
0.9 |
3.9 |
Alive / Alive |
N |
106 |
88 |
109 |
73 |
|
% |
99.1 |
100.0 |
99.1 |
96.1 |
cannibalized [pup] / Dead |
N |
0 |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
1.3 |
sacrificed scheduled [pup] / Dead |
N |
72 |
64 |
72 |
56 |
|
% |
67.3 |
72.7 |
65.5 |
73.7 |
culled / Dead |
N |
33 |
24 |
36 |
16 |
|
% |
30.8 |
27.3 |
32.7 |
21.1 |
Litters not surviving Day 13 |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test
(one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
Table 8: Summary Litter Report - Pups Died
|
Test Group 0/F 0 mg/kgbw/d |
|
Test Group 1/F 25 mg/kgbw/d |
|
Test Group 2/F 75 mg/kgbw/d |
|
Test Group 3/F 250 mg/kgbw/d |
|
Litters with liveborn pups |
N |
9 |
|
8 |
|
9 |
|
8 |
Pups delivered |
N |
107 |
|
88 |
|
110 |
|
76 |
Days 0 To 0 |
N |
0 |
|
0 |
|
0 |
|
0 |
|
% |
0 |
|
0 |
|
0 |
|
0 |
Days 1 To 4 |
N |
1 |
|
0 |
|
1 |
|
1 |
|
% |
0.9 |
|
0 |
|
0.9 |
|
1.3 |
Days 5 To 7 |
N |
0 |
|
0 |
|
0 |
|
0 |
|
% |
0 |
|
0 |
|
0 |
|
0 |
Days 8 To 13 |
N |
0 |
|
0 |
|
0 |
|
0 |
|
% |
0 |
|
0 |
|
0 |
|
0 |
Pups surviving days 0 To 4 |
N |
105 |
|
88 |
|
108 |
|
72 |
Viability Index |
Mean% |
99.1 |
x- |
100.0 |
|
99.1 |
|
98.8 |
|
S.d. |
2.6 |
|
0.0 |
|
2.6 |
|
3.5 |
|
N |
9 |
|
8 |
|
9 |
|
8 |
Pups surviving days 4 To 13 |
N |
72 |
|
64 |
|
72 |
|
56 |
Survival Index |
Mean% |
100.0 |
NA |
100.0 |
|
100.0 |
|
100.0 |
|
S.d. |
0.0 |
|
0.0 |
|
0.0 |
|
0.0 |
|
N |
9 |
|
8 |
|
9 |
|
8 |
Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
x=WILCOX; NA=No Test Applicable
Table 9: Individual Delivery Report
Group |
Female |
Pregnant Status |
Gestation Days |
Dead N |
Alive N |
Total N |
Implantation Sites N |
Post Implantation Loss % |
Live Birth Index % |
Test Group 0/ F 0 mg/kg bw/d |
101 |
Pregnant |
22 |
0 |
12 |
12 |
12 |
0.00 |
100.00 |
102 |
Pregnant |
22 |
0 |
13 |
13 |
13 |
0.00 |
100.00 |
|
103 |
Pregnant |
22 |
0 |
11 |
11 |
13 |
15.38 |
100.00 |
|
104 |
Pregnant |
22 |
0 |
10 |
10 |
11 |
9.09 |
100.00 |
|
105 |
Pregnant |
22 |
0 |
11 |
11 |
11 |
0.00 |
100.00 |
|
106 |
Pregnant |
22 |
0 |
12 |
12 |
13 |
7.69 |
100.00 |
|
107 |
Pregnant |
22 |
1 |
15 |
16 |
16 |
0.00 |
93.75 |
|
108 |
Pregnant |
22 |
0 |
9 |
9 |
9 |
0.00 |
100.00 |
|
109 |
Pregnant |
22 |
0 |
13 |
13 |
13 |
0.00 |
100.00 |
|
110 |
GD 0; No Implants; No Pups |
- |
- |
- |
- |
0 |
- |
- |
|
Test Group 1/ F 25 mg/kg bw/d |
111 |
Pregnant |
22 |
0 |
11 |
11 |
13 |
15.38 |
100.00 |
112 |
Pregnant |
22 |
0 |
12 |
12 |
12 |
0.00 |
100.00 |
|
113 |
Pregnant |
22 |
0 |
10 |
10 |
10 |
0.00 |
100.00 |
|
114 |
Pregnant |
22 |
0 |
12 |
12 |
12 |
0.00 |
100.00 |
|
115 |
No GD 0; No Implants |
- |
- |
- |
- |
0 |
- |
- |
|
116 |
Pregnant |
23 |
0 |
9 |
9 |
9 |
0.00 |
100.00 |
|
117 |
Pregnant |
22 |
0 |
12 |
12 |
14 |
14.29 |
100.00 |
|
118 |
Pregnant |
22 |
0 |
12 |
12 |
13 |
7.69 |
100.00 |
|
119 |
Pregnant |
22 |
0 |
10 |
10 |
11 |
9.09 |
100.00 |
|
120 |
GD 0; No Implants; No Pups |
- |
- |
- |
- |
0 |
- |
- |
|
Test Group 2/ F 75 mg/kg bw/d |
121 |
Pregnant |
22 |
0 |
9 |
9 |
9 |
0.00 |
100.00 |
122 |
Pregnant |
22 |
0 |
13 |
13 |
13 |
0.00 |
100.00 |
|
123 |
Pregnant |
22 |
0 |
13 |
13 |
14 |
7.14 |
100.00 |
|
124 |
Pregnant |
22 |
0 |
12 |
12 |
12 |
0.00 |
100.00 |
|
125 |
Pregnant |
22 |
0 |
14 |
14 |
14 |
0.00 |
100.00 |
|
126 |
Pregnant |
22 |
0 |
14 |
14 |
14 |
0.00 |
100.00 |
|
127 |
Pregnant |
22 |
0 |
11 |
11 |
13 |
15.38 |
100.00 |
|
128 |
GD 0; No Implants; No Pups |
- |
- |
- |
- |
0 |
- |
- |
|
129 |
Pregnant |
22 |
0 |
12 |
12 |
12 |
0.00 |
100.00 |
|
130 |
Pregnant |
22 |
1 |
11 |
12 |
12 |
0.00 |
91.67 |
|
Test Group 3/ F 250 mg/kg bw/d |
131 |
Pregnant |
22 |
0 |
12 |
12 |
12 |
0.00 |
100.00 |
132 |
Pregnant |
22 |
0 |
9 |
9 |
16 |
43.75 |
100.00 |
|
133 |
Pregnant |
23 |
3 |
10 |
13 |
13 |
0.00 |
76.92 |
|
134 |
Pregnant |
22 |
0 |
7 |
7 |
9 |
22.22 |
100.00 |
|
135 |
Pregnant |
23 |
0 |
10 |
10 |
11 |
9.09 |
100.00 |
|
136 |
Pregnant |
22 |
0 |
11 |
11 |
12 |
8.33 |
100.00 |
|
137 |
Pregnant |
23 |
0 |
1 |
1 |
3 |
66.67 |
100.00 |
|
138 |
GD 0; No Implants; No Pups |
- |
- |
- |
- |
0 |
- |
- |
|
139 |
Pregnant |
23 |
0 |
13 |
13 |
14 |
7.14 |
100.00 |
|
140 |
No GD 0; No Implants |
- |
- |
- |
- |
0 |
- |
- |
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 29 Jul 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- certified by Landesamt für Umwelt Rheinland-Pfalz
- Limit test:
- no
Test material
- Reference substance name:
- 2-piperidinoethanol
- EC Number:
- 221-244-6
- EC Name:
- 2-piperidinoethanol
- Cas Number:
- 3040-44-6
- Molecular formula:
- C7H15NO
- IUPAC Name:
- 2-piperidinoethanol
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: B1056-070518
- Purity: 99.8 area % (GC, RTX-5); 99.9 area-% (GC, DB-Wax UI)
- Purity test date: 30 May 2018
- Expiry date of lot: 07 May 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility. The stability of test substance in deionized water was demonstrated for a period of 7 days at room temperature.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed in a graduated flask depending on the dose group, topped up with deionized water and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.
FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in deionized water
Test animals
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 13 - 15 weeks
- Weight at study initiation: males: 371.9 g (mean); females: 207.4 g (mean)
- Housing: individually (exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together.)
- Diet: ad libitum; ground Kliba maintenance diet mouse-rat “GLP” (supplied by Garanovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 21 days
DETAILS OF FOOD AND WATER QUALITY: The drinking water was regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms. On the basis of the analytical findings the drinking water was found to be suitable. The food used in the study was assayed for chemical and for microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
IN-LIFE DATES: From: 18 Sep 2018 To: 18 Oct and 08 Nov 2018 (F0 males); 04 Dec 2018 (F0 females); 27 Nov - 01 Dec 2018 (litters)
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- deionized water
- Details on oral exposure:
-
VEHICLE
- Concentration in vehicle: 0.25, 0.75 and 2.5 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg bw/d - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analytical verifications of the stability of the test substance in deionized water for a period of 7 days at room temperature had been initiated prior to the start of the study in the same batch. At the beginning (during premating), twice during gestation and once during lactation of the study each 1 sample was taken from the low, mid and high concentration for a concentration control analysis. The samples collected at the beginning of the administration period and during lactation were analyzed via capillary electrophoresis (CE) with indirect UV/VIS detection.
All measured values for the test substance were in the expected range of the target concentrations (90 - 110%) demonstrating the correctness of the preparations. - Duration of treatment / exposure:
- males: 30 days; females: 56 days
The duration of treatment covered a 2-weeks premating and mating period in both sexes, 3 days postmating in males, the entire gestation and approximately 3 weeks of lactation period in females and 4-weeks postmating for sperm negative females up to the day of scheduled sacrifice of the animals. - Frequency of treatment:
- once daily at approximately the same time in the morning (females in labor were not treated)
Doses / concentrationsopen allclose all
- Dose / conc.:
- 25 mg/kg bw/day (nominal)
- Remarks:
- low dose group
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Remarks:
- mid dose group
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- high dose group
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: In an acute oral toxicity study in rats, the calculated LD50-value for males and females was 1100 mg/kg bw/d. Severe clinical signs (e.g. dyspnoe, apathy, staggering, trembling, poor general state) and mortality in both sexes were observed at 1000 mg/kg bw/d and above. At the lowest tested dose of 464 mg/kg bw/d, one female died within the first 24 hours.
In a repeated dose test study, the test substance was orally (gavage) applied at concentrations of 0, 150 and 500 mg/kg bw/d for 14 days to four rats per test group and sex. At 500 mg/kg bw/d, one male animal was found dead on study day 10 and one further male was sacrificed in moribund state due to severe clinical findings (gasping, labored respiration, respiration sounds) on study day 10. Furthermore, significantly reduced food consumption, decreased body weights, and hematological as well as gross pathological findings were observed at this dose level. At 150 mg/kg bw/d, no adverse findings were observed.
Based on the above-mentioned effects, the following dose levels were selected: 25, 75 and 250 mg/kg bw/d.
- Fasting period before blood sampling for clinical biochemistry: 16-20 hours - Positive control:
- no
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily
- A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos (protruding eyeball), assessment of the feces excreted during the examination (appearance/consistency), assessment of the urine excreted during the examination, pupil size
BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined on GD 0 - 7, 7 - 4 and 14 - 20.
• Food consumption of the females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
• Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.
WATER CONSUMPTION: Yes
- Time schedule for examinations: once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (female parental animals)
• Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7 and 12-13.
• Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA), Prothrombin time (Hepato Quick’s test) (HQT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)
NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")
OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 28 (males) and 55 (females).
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings
Motor activity measurement:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group
Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: all surviving males at termination
- Parameters examined: Total thyroxine (T4), Thyroid stimulating hormone (TSH) - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes (see table 1) - Other examinations:
- Organ weights:
determined in all animals sacrificed on schedule: Epididymides, Ovaries, Prostate (ventral and dorsolateral part together, fixed), Seminal vesicles with coagulating glands (fixed), Testes, Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix
determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for hematological and clinical chemistry examinations): Adrenal glands (fixed), Brain, Heart, Kidneys, Liver, Spleen, Thymus (fixed) - Statistics:
- see table 2
Results and discussion
Results of examinations
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- salivation immediately after dosing (up to 2 hours post dosing) during the whole treatment period in several male and all female animals (not considered to be adverse)
- piloerection in 3/10 females on PND 6 (assessed as treatment-related but not adverse, since this minor finding was occasionally seen in three females during one study phase) - Mortality:
- no mortality observed
- Description (incidence):
- - One male animal of the control showed respiration sounds between premating days 3 - 5 and 7 - 9 and piloerection during premating days 4 - 9 and was sacrificed moribund on premating day 9.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- statistically significantly reduced mean body weight changes in females during lactation (PND 0 – 13: 19.4 g vs. 32.9 g in control) showing a body weight loss in the beginning of the lactation phase (PND 0 – 4: -1.5 g vs. 11.9 g in control) (assessed as treatment-related and adverse) - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- statistically significantly reduced food consumption in females during the entire premating period (up to 10 %), during gestation (GD 0 – 7: 9 %) and during several parts of the lactation period (up to 23 % below control) (assessed as treatment-related and adverse) - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- statistically significantly increased mean water consumption in males during premating days 7 - 10 (about 24 %) (not assessed as treatment-related and adverse, since this minor finding occurred only within a short treatment phase of four days and no further parameter in males was altered)
- reduced water consumption in females during premating (up to 18 % below control, without statistical significance), beginning of gestation (GD 0-1: 21 % below control, without statistical significance) and lactation (up to 23 % below control, with statistical significance) (assessed as treatment-related and adverse, since the reduction in water consumption was present during almost the whole treatment period)
Mid-dose group (75 mg/kg bw/d):
- statistically significantly reduced water consumption in females during PND 6 – 7 (about 17 % below control) (not assessed as treatment-related and adverse, since all other treatment phases were not affected) - Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Low-dose group (25 mg/kg bw/d):
- significantly lower absolute reticulocyte counts in males at the end of the administration period (regarded as incidental and not treatment-related, since this alteration was not dose-dependent) - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- significantly increased glucose levels in females at the end of the administration period (regarded as incidental and not treatment-related, since the mean was within the historical control range (females, glucose 5.58 - 6.98 mmol/L)) - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Low-dose group (25 mg/kg bw/d):
- weakly significantly increased absolute and relative spleen weights of males (regarded as incidental, since these changes did not show dose dependency) - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- All gross findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Neuropathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The one animal, sacrificed in a moribund state, showed hyperkeratosis and squamous cell hyperplasia in the forestomach, and decreased cellularity in the spleen (white pulp), thymus (cortical) and bone marrow of the sternum. All these findings were consistent with the moribund condition of the animal. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- High-dose group (250 mg/kg bw/d):
- significantly decreased TSH values in males (regarded as incidental and not treatment-related, since the mean was within the historical control range (TSH 4.81 - 9.80 μg/L))
Effect levels
open allclose all
- Dose descriptor:
- NOAEL
- Effect level:
- 75 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- water consumption and compound intake
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
Target system / organ toxicity
- Critical effects observed:
- no
Any other information on results incl. tables
Table 3: Summary water consumption per animal and day
Males |
Test Group 0/M 0 mg/kgbw/d |
Test Group 1/M 25 mg/kgbw/d |
Test Group 2/M 75 mg/kgbw/d |
Test Group 3/M 250 mg/kgbw/d |
||
Pre-mating |
d 0 -> 3 |
Mean [g] |
17.0 n |
19.1 |
19.5 |
18.3 |
S.d. |
5.9 |
3.6 |
4.5 |
5.5 |
||
N |
10 |
10 |
10 |
10 |
||
Deviation Vs Control [%] |
|
12.7 |
14.8 |
7.7 |
||
d 7 -> 10 |
Mean [g] |
16.8 n |
20.1 |
19.3 |
20.8 * |
|
S.d. |
2.8 |
3.4 |
3.6 |
3.1 |
||
N |
9 |
10 |
10 |
10 |
||
Deviation Vs Control [%] |
|
19.4 |
14.9 |
23.9 |
||
Females |
Test Group 0/F 0 mg/kgbw/d |
Test Group 1/F 25 mg/kgbw/d |
Test Group 2/F 75 mg/kgbw/d |
Test Group 3/F 250 mg/kgbw/d |
||
Pre-mating |
d 0 -> 3 |
Mean [g] |
15.8 n |
16.8 |
16.8 |
13.0 |
S.d. |
2.2 |
2.8 |
3.1 |
2.7 |
||
N |
10 |
10 |
10 |
10 |
||
Deviation Vs Control [%] |
|
6.0 |
6.6 |
-17.9 |
||
d 7 -> 10 |
Mean [g] |
16.6 n |
17.6 |
18.1 |
14.2 |
|
S.d. |
3.1 |
2.7 |
3.6 |
3.3 |
||
N |
10 |
10 |
10 |
10 |
||
Deviation Vs Control [%] |
|
6.2 |
9.1 |
-14.6 |
||
Gestation |
d 0 -> 1 |
Mean [g] |
17.4 n |
17.6 |
17.5 |
13.8 |
S.d. |
2.8 |
3.3 |
4.8 |
3.2 |
||
N |
9 |
8 |
9 |
8 |
||
Deviation Vs Control [%] |
|
1.0 |
0.5 |
-20.6 |
||
d 6 -> 7 |
Mean [g] |
23.6 n |
26.8 |
27.0 |
25.3 |
|
S.d. |
2.0 |
4.7 |
5.5 |
5.8 |
||
N |
9 |
8 |
9 |
8 |
||
Deviation Vs Control [%] |
|
13.3 |
14.3 |
7.2 |
||
d 13 -> 14 |
Mean [g] |
30.3 n |
31.6 |
33.9 |
36.6 |
|
S.d. |
7.3 |
4.9 |
6.5 |
9.8 |
||
N |
9 |
8 |
9 |
8 |
||
Deviation Vs Control [%] |
|
4.3 |
11.9 |
20.8 |
||
d 19 -> 20 |
Mean [g] |
32.4 n |
33.4 |
31.0 |
36.5 |
|
S.d. |
2.7 |
5.6 |
4.3 |
7.4 |
||
N |
9 |
8 |
9 |
8 |
||
Deviation Vs Control [%] |
|
3.1 |
-4.3 |
12.7 |
||
Lactation |
d 1 -> 2 |
Mean [g] |
34.4 n |
31.5 |
33.6 |
30.0 |
S.d. |
3.9 |
4.7 |
3.7 |
5.3 |
||
N |
9 |
8 |
9 |
8 |
||
Deviation Vs Control [%] |
|
-8.4 |
-2.1 |
-12.8 |
||
d 3 -> 4 |
Mean [g] |
46.2 n |
41.2 |
42.6 |
35.7 ** |
|
S.d. |
5.0 |
7.3 |
7.6 |
6.5 |
||
N |
9 |
8 |
9 |
8 |
||
Deviation Vs Control [%] |
|
10.9 |
-7.8 |
-22.8 |
||
d 6 -> 7 |
Mean [g] |
51.4 n |
46.9 |
42.9 * |
42.2 * |
|
S.d. |
5.6 |
5.8 |
5.1 |
7.8 |
||
|
N |
9 |
8 |
9 |
8 |
|
Deviation Vs Control [%] |
|
-8.6 |
-16.5 |
-17.8 |
||
d 9 -> 10 |
Mean [g] |
58.8 n |
54.8 |
55.0 |
48.6 * |
|
S.d. |
8.0 |
5.4 |
4.7 |
9.0 |
||
N |
9 |
8 |
9 |
8 |
||
Deviation Vs Control [%] |
|
-6.8 |
-6.5 |
-17.4 |
||
d 12 -> 13 |
Mean [g] |
63.0 n |
62.2 |
57.7 |
52.2 |
|
S.d. |
7.6 |
7.6 |
9.1 |
12.1 |
||
N |
9 |
8 |
9 |
8 |
||
Deviation Vs Control [%] |
|
-1.2 |
-8.4 |
-17.1 |
||
Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
d = day; n=DUNNETT
Table 4: Summary food consumption per animal and day
Males |
Test Group 0/M 0 mg/kgbw/d |
Test Group 1/M 25 mg/kgbw/d |
Test Group 2/M 75 mg/kgbw/d |
Test Group 3/M 250 mg/kgbw/d |
||
Pre-mating |
d 0 -> 7 |
Mean [g] |
19.6 n |
22.1 |
22.1 |
20.7 |
S.d. |
6.3 |
1.5 |
2.6 |
1.6 |
||
N |
10 |
10 |
10 |
10 |
||
Deviation Vs Control [%] |
|
12.4 |
12.7 |
5.5 |
||
d 7 -> 13 |
Mean [g] |
21.7 n |
23.0 |
22.9 |
23.1 |
|
S.d. |
1.5 |
1.9 |
2.8 |
1.2 |
||
N |
9 |
10 |
10 |
10 |
||
Deviation Vs Control [%] |
|
6.3 |
5.9 |
6.8 |
||
d 0 -> 13 |
Mean [g] |
21.6 n |
22.5 |
22.5 |
21.8 |
|
S.d. |
1.5 |
1.6 |
2.6 |
1.3 |
||
N |
9 |
10 |
10 |
10 |
||
Deviation Vs Control [%] |
|
4.1 |
4.1 |
1.0 |
||
Females |
Test Group 0/F 0 mg/kgbw/d |
Test Group 1/F 25 mg/kgbw/d |
Test Group 2/F 75 mg/kgbw/d |
Test Group 3/F 250 mg/kgbw/d |
||||
Pre-mating |
d 0 -> 7 |
Mean [g] |
15.5 n |
15.1 |
15.1 |
14.1 ** |
||
S.d. |
1.2 |
0.9 |
0.9 |
0.9 |
||||
N |
10 |
10 |
10 |
10 |
||||
Deviation Vs Control [%] |
|
-2.8 |
-2.5 |
-9.4 |
||||
d 7 -> 13 |
Mean [g] |
16.3 n |
16.1 |
16.0 |
14.6 * |
|||
S.d. |
1.6 |
1.9 |
1.2 |
1.0 |
||||
N |
10 |
10 |
10 |
10 |
||||
Deviation Vs Control [%] |
|
-0.9 |
-1.6 |
-10.3 |
||||
d 0 -> 13 |
Mean [g] |
15.9 n |
15.6 |
15.5 |
14.3 * |
|||
S.d. |
1.3 |
1.3 |
1.0 |
0.9 |
||||
N |
10 |
10 |
10 |
10 |
||||
Deviation Vs Control [%] |
|
-1.9 |
-2.1 |
-9.8 |
||||
Gestation |
d 0 -> 7 |
Mean [g] |
20.3 n |
19.2 |
19.3 |
18.4 * |
||
S.d. |
1.4 |
1.0 |
1.8 |
1.3 |
||||
N |
9 |
8 |
9 |
8 |
||||
Deviation Vs Control [%] |
|
-5.4 |
-5.2 |
-9.1 |
||||
d 7 -> 14 |
Mean [g] |
21.9 n |
21.1 |
21.6 |
20.7 |
|||
S.d. |
1.4 |
1.2 |
1.7 |
2.0 |
||||
N |
9 |
8 |
9 |
8 |
||||
Deviation Vs Control [%] |
|
-3.6 |
-1.5 |
-5.7 |
||||
d 14 -> 20 |
Mean [g] |
22.9 n |
23.1 |
22.9 |
22.2 |
|||
S.d. |
1.6 |
1.9 |
1.9 |
2.1 |
||||
N |
9 |
8 |
9 |
8 |
||||
Deviation Vs Control [%] |
|
1.1 |
0.3 |
-2.7 |
||||
d 0 -> 20 |
Mean [g] |
21.6 n |
21.0 |
21.2 |
20.4 |
|||
S.d. |
1.4 |
1.2 |
1.8 |
1.7 |
||||
N |
9 |
8 |
9 |
8 |
||||
Deviation Vs Control [%] |
|
-2.7 |
-2.1 |
-5.9 |
||||
Lactation |
d 1 -> 4 |
Mean [g] |
35.6 n |
32.9 |
33.5 |
27.5 ** |
||
S.d. |
2.5 |
2.7 |
2.8 |
6.8 |
||||
N |
9 |
8 |
9 |
8 |
||||
Deviation Vs Control [%] |
|
-7.7 |
-6.0 |
-22.7 |
||||
d 4 -> 7 |
Mean [g] |
43.8 n |
40.7 |
40.2 |
35.6 ** |
|||
S.d. |
2.1 |
2.5 |
2.3 |
8.7 |
||||
N |
9 |
8 |
9 |
8 |
||||
Deviation Vs Control [%] |
|
-7.1 |
-8.2 |
-18.7 |
||||
d 7 -> 10 |
Mean [g] |
52.4 n |
51.7 |
50.0 |
45.4 |
|||
S.d. |
3.2 |
3.1 |
3.9 |
12.4 |
||||
N |
9 |
8 |
9 |
8 |
||||
Deviation Vs Control [%] |
|
-1.4 |
-4.6 |
-13.3 |
||||
d 10 -> 13 |
Mean [g] |
58.3 n |
57.8 |
55.6 |
49.6 |
|||
S.d. |
4.2 |
3.8 |
5.0 |
13.1 |
||||
N |
9 |
8 |
9 |
8 |
||||
Deviation Vs Control [%] |
|
-0.9 |
-4.7 |
-14.9 |
||||
d 1 -> 13 |
Mean [g] |
47.5 n |
45.8 |
44.8 |
39.6 * |
|||
S.d. |
2.5 |
2.6 |
3.2 |
9.8 |
||||
N |
9 |
8 |
9 |
8 |
||||
Deviation Vs Control [%] |
|
-3.7 |
-5.7 |
-16.8 |
||||
Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
d = day; n=DUNNETT
Table 5: Summary body weight changes [g]
Males |
Test Group 0/M 0 mg/kgbw/d |
Test Group 1/M 25 mg/kgbw/d |
Test Group 2/M 75 mg/kgbw/d |
Test Group 3/M 250 mg/kgbw/d |
||
Pre-mating |
d 0 -> 7 |
Mean |
-1.3 n |
5.4 |
6.3 |
2.4 |
S.d. |
30.3 |
4.3 |
4.8 |
5.3 |
||
N |
10 |
10 |
10 |
10 |
||
d 7 -> 13 |
Mean |
8.0 n |
8.9 |
10.2 |
11.0 |
|
S.d. |
3.7 |
3.2 |
6.1 |
4.7 |
||
N |
9 |
10 |
10 |
10 |
||
d 0 -> 13 |
Mean |
16.3 n |
14.4 |
16.5 |
13.3 |
|
S.d. |
5.9 |
6.7 |
7.5 |
7.6 |
||
N |
9 |
10 |
10 |
10 |
||
Mating |
d 7 -> 14 |
Mean |
8.6 n |
7.4 |
12.2 |
7.3 |
S.d. |
2.7 |
3.6 |
5.8 |
4.9 |
||
N |
9 |
10 |
10 |
10 |
||
Females |
Test Group 0/F 0 mg/kgbw/d |
Test Group 1/F 25 mg/kgbw/d |
Test Group 2/F 75 mg/kgbw/d |
Test Group 3/F 250 mg/kgbw/d |
||||||
Pre-mating |
d 0 -> 7 |
Mean |
0.3 n |
0.9 |
-1.7 |
-1.0 |
||||
S.d. |
4.8 |
5.2 |
4.2 |
6.6 |
||||||
N |
10 |
10 |
10 |
10 |
||||||
d 7 -> 13 |
Mean |
7.1 n |
8.4 |
11.9 |
7.7 |
|||||
S.d. |
7.9 |
3.7 |
4.8 |
5.1 |
||||||
N |
10 |
10 |
10 |
10 |
||||||
d 0 -> 13 |
Mean |
7.4 n |
9.3 |
10.2 |
6.7 |
|||||
S.d. |
5.9 |
5.3 |
4.0 |
7.8 |
||||||
N |
10 |
10 |
10 |
10 |
||||||
Gestation |
d 0 -> 7 |
Mean |
26.1 n |
25.1 |
22.1 |
23.0 |
||||
S.d. |
6.6 |
4.4 |
2.8 |
4.0 |
||||||
N |
9 |
8 |
9 |
8 |
||||||
d 7 -> 14 |
Mean |
25.7 n |
21.6 |
26.5 |
24.6 |
|||||
S.d. |
3.3 |
3.7 |
5.4 |
5.8 |
||||||
N |
9 |
8 |
9 |
8 |
||||||
d 14 -> 20 |
Mean |
59.4 n |
58.2 |
61.2 |
51.0 |
|||||
S.d. |
7.4 |
10.4 |
7.0 |
17.9 |
||||||
N |
9 |
8 |
9 |
8 |
||||||
d 0 -> 20 |
Mean |
111.3 n |
105.0 |
109.8 |
98.5 |
|||||
S.d. |
13.7 |
11.4 |
9.8 |
23.3 |
||||||
N |
9 |
8 |
9 |
8 |
||||||
Lactation |
d 0 -> 4 |
Mean |
11.9 n |
8.8 |
7.7 |
-1.5 * |
||||
S.d. |
11.9 |
9.0 |
8.2 |
10.3 |
||||||
N |
9 |
8 |
9 |
8 |
||||||
d 4 -> 7 |
Mean |
5.9 n |
7.1 |
4.9 |
4.8 |
|||||
S.d. |
7.5 |
9.1 |
7.2 |
7.4 |
||||||
N |
9 |
8 |
9 |
8 |
||||||
d 7 -> 10 |
Mean |
10.0 n |
12.1 |
12.7 |
12.4 |
|||||
S.d. |
4.7 |
2.9 |
5.4 |
3.2 |
||||||
N |
9 |
8 |
9 |
8 |
||||||
d 10 -> 13 |
Mean |
5.1 n |
8.6 |
5.1 |
3.7 |
|||||
S.d. |
5.2 |
4.4 |
6.4 |
4.8 |
||||||
N |
9 |
8 |
9 |
8 |
||||||
d 0 -> 13 |
Mean |
32.9 n |
36.6 |
30.4 |
19.4 * |
|||||
S.d. |
6.1 |
9.5 |
12.1 |
13.1 |
||||||
N |
9 |
8 |
9 |
8 |
||||||
Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
d = day; n=DUNNETT
Applicant's summary and conclusion
- Conclusions:
- The no observed adverse effect level (NOAEL) for general systemic toxicity was the highest dose of 250 mg/kg bw/d for male and the mid-dose of 75 mg/kg bw/d for female Wistar rats.
- Executive summary:
The test substance was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 25, 75 and 250 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (deionized water). The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, the entire gestation and approximately 3 weeks of lactation period in females and 4-weeks postmating for sperm negative females up to the day of scheduled sacrifice of the animals.
The oral administration of the test substance resulted in signs of systemic toxicity in females at the highest dose of 250 mg/kg bw/d. In high-dose F0 females, adverse findings consisted of a reduction in water and food consumption and a decrease in body weight change. Male animals showed no adverse signs at any tested dose.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the highest dose of 250 mg/kg bw/d for male and the mid-dose of 75 mg/kg bw/d for female Wistar rats.
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