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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-reference
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 3, 2011 to November 22, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Principles of method if other than guideline:
Additional guideline: "OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000" (with no deviations).
GLP compliance:
yes
Specific details on test material used for the study:
Specific Gravity / Density: 1.14 g/cm3. pH (1% in water, indicative range): 5.5 – 5.5 (determined at NOTOX). Stability at higher temperatures: yes, maximum temperature 40°C. Stability in vehicle (Polyethylene glycol): at least 6 hours at room temperature over the concentration range 20 to 200 mg/mL.
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality).
Remarks:
Charles River Deutschland, Sulzfeld, Germany.
Details on species / strain selection:
Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Animals:
Total number of animals: 20 males, 20 females (females were nulliparous and non-pregnant). Age at start of treatment: approximately 6 weeks.
Identification: earmark and tattoo. Randomization: by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean. Acclimatization period: at least 5 days before the start of treatment under laboratory conditions. Health inspection: prior to commencement of treatment to ensure that the animals were in a good state of health.
- Allocation:
5 males and 5 females per group. 4 groups: 0, 100, 300, and 1000 mg/kg bw/day. Dose volume: 5mL/kg bw/day
- Conditions:
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 18.5 – 22.3ºC), a relative humidity of 40-70% (actual range: 41 - 86%) and 12 hour light/12 hour dark cycle. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity (with a maximum of 4 hours). Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
- Accommodation:
Group housing of 5 animals per sex in Makrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet:
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived. During motor activity measurements, animals had no access to food.
Water:
Free access to tap water except during motor activity measurements. Certificates of analysis (performed quarterly) were examined and archived.
Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.
Route of administration:
oral: gavage
Details on route of administration:
Method: oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Vehicle:
polyethylene glycol
Details on oral exposure:
Dose volume 5 mL/kg body weight. Actual dose volumes were adjusted weekly according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analysed in the formulations of Group 2 (100 mg/kg bw/day), Group 3 (300 mg/kg bw/day) and Group 4 (1000 mg/kg bw/day) were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Duration of treatment / exposure:
28 days (All animals were dosed up to the day prior to necropsy).
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Controls
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
in agreement with target concentrations
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
in agreement with target concentrations
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
in agreement with target concentrations
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Based on the results of a 5-day range finding study (NOTOX Project 497635), the dose levels for this 28-day oral gavage study were selected to be 0, 100, 300 and 1000 mg/kg.
- Study outline: the test substance, formulated in polyethylene glycol 400, was administered daily for at least 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.
- Evaluated parameters: chemical analyses of formulations were conducted once after the study to assess accuracy, homogeneity and stability over 6 hours. The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.
Positive control:
no
Observations and examinations performed and frequency:
- Mortality / Viability: at least twice daily.
- Clinical signs: at least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. Clinical observations were conducted immediately after dosing, except for those performed in a standard arena which were performed prior to dosing. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades are reported, as well as the percentage of animals affected in summary tables.
- Functional Observations: during week 4 of treatment, the following tests were performed on all animals (abbreviations mentioned in the respective tables indicated between brackets): - hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent), and - motor activity test (recording period: 1 hour under normal laboratory light conditions for individual animals, using a computerized monitoring system (Kinder Scientific LLC, Poway, USA)). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head. Functional observation tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
Body weights and food consumption: weekly.
Water consumption: subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Sacrifice and pathology:
- Animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
- The organ weights and terminal body weight were recorded from the animals on the scheduled day of necropsy.
- All organ and tissue samples, as defined under Histopathology (following), were processed, embedded in paraffin wax (Klinipath, Duiven, The Netherlands) cut to slides at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
- The following slides were examined by a pathologist: - all tissues collected at the scheduled sacrifice from all group 1 and 4 animals, - liver of all animals of groups 2 and 3 (males and females), based on (possible) treatment-related changes in this organ in group 4, - all gross lesions.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Histopathology was subjected to a peer review.
Other examinations:
Blood samples were collected under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.. Animals were deprived of food overnight (for a maximum of 20 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin (0.5 mL) for clinical biochemistry parameters. An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids (tubes: Greiner BioOne GmbH, Kremsmünster, Austria).
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one ttest) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. - The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. - The exact Fisher-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test (Ref. 5) to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Hunched posture was observed for all females at 1000 mg/kg bw/dayduring the second half of the study, starting from Day 19.
- A slight to moderate degree of salivation was shown by females at 100 mg/kg bw/day and all animals at 300 and 1000 mg/kg bw/day. Salivation is often noted in rats of this age and strain following oral gavage and the salivation observed in this study was considered to be a physiological response rather than a sign of systemic toxicity.
- One control female showed scales and scabs in the neck region. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.
- No clinical signs were observed for control males and males at 100 mg/kg bw/day.
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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- A prolonged prothrombin time (PT) was observed for males at 1000 mg/kg bw/day when compared to control males. The following statistically significant differences between controls and animals at 1000 mg/kg bw/day were considered not to represent a change of toxicological significance, because the differences were slight in nature and the values of the animals at 1000 mg/kg bw/day remained well within the range considered normal for rats of this age and strain: increased relative monocyte counts in males and lower haemoglobin levels and lower haematocrit in females.
- Lower relative neutrophil counts in females at 100 mg/kg bw/day and lower red blood cell counts in females at 300 mg/kg bw/day were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher total bilirubin levels in males and females at 1000 mg/kg bw/day,
- Higher cholesterol levels in males at 300 mg/kg bw/day and males and females at 1000 mg/kg bw/day,
- Higher bile acids levels in females at 1000 mg/kg bw/day and (not statistically significant) in males at 300 and 1000 mg/kg bw/day.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Functional observations:
- No toxicologically significant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
- Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.
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):
Macroscopic examination:
Necropsy did not reveal any toxicologically relevant alterations. The incidence of the macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included: reduced size of the right seminal vesicle (one control male and one male at 1000 mg/kg bw/day), foci on the thymus (one control female, one male at 100 mg/kg bw/day and one female at 300 mg/kg bw/day), diaphragmatic hernia of the right medial lobe of the liver (one male at 300 mg/kg bw/day), a yellowish, hard nodule in the body of the left epididymis (one male at 300 mg/kg bw/day), pelvic dilation of the right kidney (two males at 1000 mg/kg bw/day), presence of fluid in the uterus (one or two females of all groups), scab formation on the skin of the back of the neck (one control female), dark red discolouration of the left mandibular lymph node (one female at 100 mg/kg bw/day), tan foci on the right side of the clitoral gland (one female at 300 mg/kg bw/day and one female at 1000 mg/kg bw/day) and an enlarged left mandibular lymph node (one female at 1000 mg/kg bw/day).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A minor treatment-related microscopic finding, vacuolation of Kupffer cells, was present in the livers of rats at 300 and 1000 mg/kg bw/day. All males and females at 1000 mg/kg bw/day had minimal vacuolation of Kupffer cells. Four of five males and two of five females at 300 mg/kg bw/day also had minimal vacuolation of Kupffer cells. There were no incidences at 100 mg/kg bw/day. Vacuolation was minimal in the sense that the observation was barely noticeable.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat. (dissolved fraction)
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
clinical biochemistry
clinical signs
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
other: changes in bile acids
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Results summary:

At 1000 mg/kg bw/day, minimal vacuolation of Kupffer cells (barely noticeable) was present in the livers of all males and females and was accompanied with a prolonged prothrombin time in males, higher total bilirubin levels in males and females and higher bile acids levels in males and females. Although most levels were only slightly above the range considered normal for rats of this age and strain, the change in bile acids levels in females was remarkable. In addition, hunched posture was observed for females at 1000 mg/kg bw/day during the second half of the study. Taking all findings into account, the combination of effects in animals at 1000 mg/kg bw/day was considered to be adverse in nature.

At 300 mg/kg bw/day, minimal vacuolation of Kupffer cells (barely noticeable) was observed among several animals and was accompanied with slightly higher cholesterol levels and higher bile acids levels. These findings were considered not to be adverse in nature based on the slight nature of these findings and the absence of any other toxicologically relevant findings.

Animals at 100 mg/kg bw/day did not show toxicologically significant changes in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

From the results presented in this report, a No Observed Adverse Effect Level (NOAEL) for the test substance of 300 mg/kg bw/day was established.

Conclusions:
Wistar rats were treated with the test substance for 28 consecutive days by daily oral gavage at dose levels up to 1000 mg/kg. Under the study conditions, a NOAEL of 300 mg/kg was established.
Executive summary:

A study was conducted to determine the chronic toxicity of the test substance according to OECD Guideline 407, EU Method B.7 and EPA OPPTS 870.3050. Groups of 5 male and 5 female Wistar rats were treated with the test substance (dissolved in polyethylene glycol) for 28 consecutive days by oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Chemical analyses of formulations were conducted once after the study and confirmed accuracy, homogeneity and stability over 6 h. The following parameters were evaluated: clinical signs daily, functional observation tests in Week 4, bodyweight and food consumption weekly, clinical pathology and macroscopy at termination, organ weights and histopathology on a selection of tissues. At 1000 mg/kg bw/day, minimal vacuolation of Kupffer cells (barely noticeable) was present in the livers of all males and females and was accompanied with a prolonged prothrombin time in males, higher total bilirubin levels in males and females and higher bile acids levels in males and females. Although most levels were only slightly above the range considered normal for rats of this age and strain, the change in bile acids levels in females was remarkable. In addition, hunched posture was observed for females at 1000 mg/kg bw/day during the second half of the study. Taking all findings into account, the combination of effects in animals at 1000 mg/kg bw/day was considered to be adverse. At 300 mg/kg bw/day, minimal vacuolation of Kupffer cells (barely noticeable) was observed among several animals and was accompanied with slightly higher cholesterol levels and higher bile acids levels. These findings were not considered not to be adverse due to their slight nature and the absence of any other toxicologically relevant effects. Animals at 100 mg/kg bw/day did not show toxicologically significant changes in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). Under the study conditions, the NOAEL was determined to be 300 mg/kg bw/day (Stitzinger, 2011).

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion