Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed GLP and OECD guidline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG breeding colony
- Age at study initiation: approx . 6 weeks
- Weight at study initiation (mean):
Male: Group 1: 132.5, Group 2: 131.8, Group 3: 132.0, Group 4: 126.2 g
Female: Group 1: 123.4, Group 2: 123.4, Group 3: 122.0, Group 4: 118.2 g
- Fasting: when kept in diuresis cages
- Housing: fully air-conditioned, in Makrolon (TM) cages, in groups of 5 animals
- Diet (e.g. ad libitum): Altromin 1324 rat diet (TM), ad libitum, exept for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum, exept for the period in which the animals were kept in diuresis cages
- Acclimation period: approx. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability was evaluated
Duration of treatment / exposure:
28 applications within 29 days
Frequency of treatment:
once a day, 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0
Basis:
actual ingested
Remarks:
Doses / Concentrations:
40
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a preliminaryy study groups of three male and three female rats received Nitroanissiure TTR at a dose level of 1000 mg/kg body weight per day over a period of 14 days. No symptomes occurred. Developooent of body weight was not impaired. The animals killed at the end of the observation period showed no macroscopically visible changes. Based on these results Nitroanisslure TTR was tested in the present study at the dose levels of 0, 40, 200 and 1000 mg/kg body weight per day.

- Rationale for animal assignment (if not random):
Randomisation
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily, and at weekends and on public holidays once daily

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study and then twice weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: 2 times per week. The values refer to the intervalls between one measurement and the next. They are converted to the food consumption per 100 g bodyweight over a 24 hour period.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: once weekly over a period of 16 hours and was calculated as water consumption / animal / 16 h

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The an imals were examined weekly for opacity of the refracting media of the eyes


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study, hematological examinations were performed on all animals
- Anaesthetic used for blood collection: Yes
- Animals fasted: No
- How many animals: all
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: animals were killed by cutting of the vena cava cranialis in deep narcosis and exsanguinated
- Animals fasted: No
- How many animals: all
- Parameters checked in table were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: a few days before termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations: The animals were examined weekly for neurological disturbances
- Battery of functions tested: sensory activity / grip strength / motor activity / other: no

OTHER: The an imals were examined weekly for damage to the oral mucosa and impairment of dental growth.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The folowing parameters were compared statistically with the control group values at the leyel of significance p = 0.05:

- Body weights at the designated measurement t imes
- Hematological data
- Clinical chemistry parameters (without GGT and bilirubin)
- Urine analysis (pH-value and specific weight)
- absolute organ weights and organ to body weight ratios

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Increased salivat ion was observed sporadically in the highest dose group
Mortality:
mortality observed, treatment-related
Description (incidence):
Increased salivat ion was observed sporadically in the highest dose group
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):
effects observed, treatment-related
Description (incidence and severity):
increased from day twelve in highest dose group
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
increased aspartate aminotransferase (ASAT/ GOT) values in females and increased alanine aminotransferase (ALAT/GPT) values in both sexes of the 1000 mg/ kg body weight group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
pH-value of the urine was decreased in both sexes of the high dose group and in males of the intermediate dose group
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behaviour and general health con dition of the other animals remained normal
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weights were increased in males of the 200 mg/kg body weight group and in both sexes of the 1000 mg/kg body weight group, showing a dose-dependency in males
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
slight liver damage in the animals of the high dose group, consisting of hepatocellular hypertrophy, increased mitotic indices and increased incidences of stellate cell proliferation
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the findings in this oral 28d repeated dose toxicity study the "no observed adverse effect level" is considered to be 200 mg/kg body weight for male and female Wistar rats.
Executive summary:

Groups of 5 male and 5 female Wlstar rats received Nitroanissäure TTR by oral gavage at dose levels of 0, 40, 200 or 1000 mg/kg body weight per day for 28 days and were necropsied at day 29. Behaviour and state of health were observed dally in all groups. Body weights and food consumption were recorded twice weekly, and water consulmption once weekly. Hematologlcal examinations and clinical chemistry were carried out at the termination of the study. Urine analysis was also performed at the end of the study. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs weighed and the organ to body weight ratios calculated. The major organs and tissues were processed for hi stopathological examination and checked for microscopically visible changes. Body weights, hematologlcal and clinical chemistry data, urine data (pH value, specific weight), absolute and relative organ weights were analysed with the aid of a statistical program to show differences compared with the controls.

Increased salivation was observed sporadically in the highest dose group starting from day five up to the end of the study. Behaviour and general health condition of the other animals remained normal throughout the study . Body weight gain and food consumption remained unaffected by the treatment during the whole study. Water consumption was increased from day twelve up to end of study in the highest dose group. pH-value of the urine was decreased in both sexes of the high dose group and in males of the intermediate dose group, probably due to the acidity of the test compound. Hematological examinations showed no compound related effect. Clinical chemistry investigations revealed increased aspartate aminotransferase (ASAT/ GOT) values in females and increased alanine aminotransferase (ALAT/GPT) values in both sexes of the 1000 mg/ kg body weight group. Relative liver weights were increased in males of the 200 mg/kg body weight group and in both sexes of the 1000 mg/kg body weight group, showing a dose-dependency in males. No compound-related macroscopically visible changes were found at necropsy. The histopathological examinations revealed evidence for slight liver damge in the animals of the high dose group, consisting of hepatocellular hypertrophy, increased mitotic indices and increased incidences of stellate cell proliferation.

In conclusion repeated application of 1000 mg Nitroanissäure TTR / kg body weight caused slight liver damage, reflected in elevation of ASAT- and ALAT-values and in histopathological changes . These findings were accompanied with increased liver to body weight ratios. Additionally increased salivation and Increased water consumption was observed. After repeated treatment with 200 mg Nitroanissäure TTR / kg body welght, increased liver to body weight ratios were still observed in males. However, these effects were not accompanied by any change in clinical chemistry, which might be indicative for liver damage. Furthermore, histopathological examinations revealed no changes of liver structure. No compound-related effects occurred in females. Nitroanissäure TTR caused no detectable effects at the dose of 40 mg/kg body weight in male and female Wistar rats when administered 28 times during 29 days. With regard to the present study the "no observed effect level" is 40 mg/kg body weight per day in males and 200 mg/kg body weight per day in females. The the "no observed adverse effect level" is considered to be 200 mg/kg body weight for both sexes.