Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 February 2007 to 05 March 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3050
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): MTDID 9580
- Substance type: Clear, colorless liquid
- Physical state: Liquid
- Analytical purity: 99%
- Purity test date: 15 November 2006
- Lot/batch no.: CM 16F176
- Expiration date of the lot/batch: 31 December 2007
- Storage condition of test material: At room temperature in the dark under nitrogen

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: approx 180 g, Females: approx 160 g
- Fasting period before study: No data
- Housing: 5 animals per sex in Macrolon (MIV type) cages.
- Diet (e.g. ad libitum): SM R/M-Z from SSNIFF Spezialdiaten GmbH, Soest, Germany, ad lbitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-22.6
- Humidity (%): 36-74
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 05 February 2007 To: 05 March 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test article was administered unchanged as a daily oral gavage.
DOSES: 0 (control), 50, 450 and 1000 mg/kg/day via oral gavage
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Once daily for at least 28 days, 7 days per week, approximately the same time each day. Animals were dosed up to the day prior to necropsy.
Frequency of treatment:
Once daily for 28 days.
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 50, 450 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
other: Control animals received Milli-Q water daily by oral gavage.
Details on study design:
- Dose selection rationale: Doses were based on the results from a 5-day rangefinding study
- Rationale for animal assignment (if not random): Random
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once on each animal during the study period.
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pupillary reflex was tested in each animal at some point during the study period.
- Dose groups that were examined: All
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately prior to necropsy
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes
- How many animals: All
- Parameters checked: white blood cells, differential leukocyte count, red blood cells, reticulocytes, red blood cell distribution width, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, platelates.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Immediately prior to necropsy.
- Animals fasted: Yes
- How many animals: All
- Parameters checked: Alanine aminotranferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: Eight animals in various dose groups were found dead at the end of the first study week. Necropsy of the animals revealed fluid in the thoracic cavity and dark red discoloration of the lungs. Histopathology relvealed no evidence of major target organ toxicity associated with the deaths. The test substance may have entered the lung in small but significant amounts due to slight variations in intubation velocity of the gavage tube. The clustering of the deaths in the early phase of the study with no subsequent deaths, together with the lack of macroscopic and microscopic findings was consistent with the dosing procedures as the most probable cause of death. Therefore, these deaths were considered unrelated to toxicity exposure via the intended route. No other clinical signs or mortality were observed in the study.
BODY WEIGHT AND WEIGHT GAIN: Body weight gain for the test group animals was similar to controls.
FOOD CONSUMPTION AND COMPOUND INTAKE: Absolute and relative food consumption of females at 1000 mg/kg/day was reduced in weeks 1 and 4.
FOOD EFFICIENCY: NA
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): NA
OPHTHALMOSCOPIC EXAMINATION: No effects were seen upon pupillary reflex examination.
HAEMATOLOGY: No toxicologically relevant results were noted.
CLINICAL CHEMISTRY: No toxicologically relevant results were noted.
URINALYSIS: NA
NEUROBEHAVIOUR: No toxicologically relevant results were noted.
ORGAN WEIGHTS: Increased liver weights and liver to body weight ratios were noted in males at 1000 mg/kg/day. Liver to body weight ratios were also increased in males at 450 mg/kg/day
GROSS PATHOLOGY: Increased incidence and severity of follicular cell hypertrophy in the thyroid of males at 1000 mg/kg/day. This was considered a secondary adaptive change due to liver enlargement and was considered of no toxicological significance.
HISTOPATHOLOGY: NON-NEOPLASTIC: No toxicologically relevant results were noted.
HISTOPATHOLOGY: NEOPLASTIC (if applicable): No toxicologically relevant results were noted.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: See 'Remarks'
Remarks:
At 1000 mg/kg/day, liver weights were increased (1.2-fold greater than control mean) which was supported by an increased incidence/severity of hepatocellular hypertrophy and thyroid follicular hypertrophy. The morphological changes were considered adaptive, however the magnitude of the increase was considered adverse. No toxicologically significant changes in liver weights were noted at 150 and 450 mg/kg/day and the incidence and/or severity of hepatocellular hypertrophy at these dose levels was lower than observed at 1000 mg/kg/day. Based upon the results of this study (in particular the increased liver weight at 1000 mg/kg/day), a No Observed Adverse Effect Level (NOAEL) for the test article of 450 mg/kg/day was established.

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
Based upon the results of this study (in particular the increased liver weight at 1000 mg/kg/day), a No Observed Adverse Effect Level (NOAEL) for the test article of 450 mg/kg/day was established.
Executive summary:

The toxicity potential of the test article was evaluated in male and female Wistar rats administered the test material daily for 28 days via oral gavage following OECD guideline 407 (1995). The test material was administered as received. Based on the results of a 5-day rangefinding study, the animals (5/sex/group) were administered 0 (Milli-U water), 150, 450 and 1000 mg/kg/day via oral gavage. The following parameters were evaluated: clinical signs (daily), functional observation tests (week 4), body weight (weekly), food consumption (weekly), clinical pathology (termination), macroscopy (termination), organ weights and histology on selected tissues. During the first week of the study, deaths occurred in each of the 150 (two males), 450 (one male and one female), and 1000 (one male and three females) mg/kg/day groups. Necropsy revealed fluid in the thoracic cavity and dark red discoloration of the lungs in most cases. Histology revealed no major target organ toxicity in these animals; only a minor acute inflammation in the lining of the thoracic viscera. It was determined that these deaths were consistent with the dosing procedures along with the properties of the test material as the most probable cause of death and were considered unrelated to toxicity via the ingestion route. No clinical signs were noted during the study period and functional observation tests revealed no abnormalities. Females at 1000 mg/kg/day showed reduced food consumption in weeks 1 and 4, but body weights remained similar to controls. At 150 mg/kg/day and higher and number of clinical biochemistry changes were noted (reduced total protein, albumin, cholesterol and bilirubin) that were within or only slightly outside the normal range for rats and were considered to not represent toxicity. At 1000 mg/kg/day, liver weights were increased (1.2-fold greater than control mean) which was supported by an increased incidence/severity of hepatocellular hypertrophy and thyroid follicular hypertrophy. The morphological changes were considered adaptive, however the magnitude of the increase was considered adverse. No toxicologically significant changes in liver weights were noted at 150 and 450 mg/kg/day and the incidence and/or severity of hepatocellular hypertrophy at these dose levels was lower than observed at 1000 mg/kg/day. Based upon the results of this study (in particular the increased liver weight at 1000 mg/kg/day), a No Observed Adverse Effect Level (NOAEL) for the test article of 450 mg/kg/day was established.