Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-11-27 to 1991-06-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations.

Data source

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

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
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed into a glass flask on an analytical balance and the vehicle (w/w) added. Homogeneity of test substance in vehicle: By the use of an electric shaker. From chemical analysis the test substance appeared to be a homogeneous suspension in water at all concentrations used in this study. Frequency of test substance formulation: Daily immediately prior to dosing.

DIET PREPARATION
no feeding study

VEHICLE
- Justification for use and choice of vehicle (if other than water): distilled water
- Concentration in vehicle: 25 - 180 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Appendix 2 of the original study report, summary:
The concentrations of the substance in water were determined for the subacute 28-day oral toxicity study in the rat, using a Spectrophotometric method. The substance formed a homogeneous suspension in water at all concentrations tested. The accuracy of preparation testing revealed that the concentrations analyzed were in agreement with the concentrations prepared.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
900 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose range finder: levels of 50, 150 and 750 mg/kg/day) to provide a basis for selection of dose levels for a study of longer duration. No differences of biological significance were observed in clinical appearance, body weight, food consumption, macroscopic appearance or liver weights between the treated groups. However, mortality was noted in the oral LD50 study (RCC NOTOX 017752) among animals treated at 1000 mg/kg body weight. Based on these observations, a high treatment level of 900 mg/kg/day was selected for a study o f 28 days duration.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not appicable
- Post-exposure recovery period in satellite groups: not appicable
- Section schedule rationale (if not random): not reported
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality / Viability: Twice daily. Animals showing pain, distress or discomfort and which were considered not transient in nature or was likely to become more severe were sacrificed for human reasons.
Food consumption: Weekly
- Cage side observations checked in table were included. Yes.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily. Severity of observations were graded.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and on the day preceding termination, prior to overnight fasting.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
not applicable

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No calculations conducted, however the raw data needed for such a computation (i.e. food consumption and body weight gain) are presented in the original study report.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes were examined following instillation of tropicamide solution (5 mg(ml) before commencement of treatment and during the last week of treatment.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected immediately prior to post mortem examination, between 8.00 and 10.00 a.m..
- Anaesthetic used for blood collection: Yes (no identity)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No. 1] were examined.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Statistics:
The following statistical methods were used to analyse the body weight, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was
applied for the comparison of the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p<0.05 was accepted as the (I lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) i n the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the
basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistis values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
900 mg/kg/d: Clinical signs of ill health were noted in males and females. One male was found dead on day 8 and 1 male was killed in extremis on day 9 of treatment.

BODY WEIGHT AND WEIGHT GAIN
900 mg/kg/d: Markedly low body weights and body weight gain was noted in males over the 4 week study period. Females were noted with slightly low body weight gain after 3 and 4 weeks of treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
900 mg/kg/d: Decreased food consumption was noted in males over the study period. Relative food consumption of males was noted as low after 1 week of treatment and high after weeks 2, 3 and 4 of treatment.

OPHTHALMOSCOPIC EXAMINATION
900 mg/kg/d: Increased numbers of white blood cells were noted in males only.

HAEMATOLOGY
900 mg/kg/d: Increased numbers of white blood cells were noted in males only.

CLINICAL CHEMISTRY
900 mg/kg/d: Decreased serum creatinine in males and decreased total protein in males/females. Increased total bilirubin, alanine aminotransferase and alkaline phosphatase in males/females and increased aspartate aminotransferase in males

GROSS PATHOLOGY
* 200 mg/kg/d: Macroscopically observed black areas were noted in the lungs of 1 female.
900 mg/kg/d: Macroscopically observed black areas were noted in the lungs of 1 female. Other findings included ascites, distended stomach with red areas, distended urinary bladder, small pale liver with irregular surface and small seminal vesicles.

HISTOPATHOLOGY: NON-NEOPLASTIC
* 200 mg/kg/d: Microscopically observed cellular reaction was noted in the lungs of the above female.
900 mg/kg/d: Microscopically observed lesions consisted of: hepatocellular changes and b i l e duct hyperplasia noted in the liver ; increased incidence and degree of vacuolation noted in the adrenal cortex; depletion of the splenic white pulp; cellular cellular reaction and accumulation of test
substance noted i n the lungs; haemorrhage i n the stomach (215 males); colloid depletion in the seminal vesicles (115 males) and interstitial edema in the pancreas (1/5 males).


* Note following explanation on the observed effects in dose group 200 mg/kg/d, as provided by the original study report:
Page 22: Regurgitation of test substance was noted intermittently in animals receiving 200 or 900 mg/kg/day and on a few occasions in males receiving 50 mg/kg/day. Since this phenomenon is very rare in rats and possibly may be associated with the above mentioned irritant effect of the test substance, it was considered not to be of toxicological significance.
Page 26: Macroscopically observed black areas were noted i n the lungs of 1 female receiving 900 mg/kg/day and 1 female receiving 200 mg/kg/day. In both animals cellular reactions were noted histopathologically. Since accumulation of test material was apparent at microscopic examination, in the female receiving 900 mg/kg/day, these changes in the lungs may represent the presence of foreign body granulomas.
It is therefore concluded that the test animals accidentally took up some test substance into their lungs, thus the effects described are no specific toxicological effects caused by the test item. This conclusion futher is supported by the fact that no other effects at all were being observed at dose level 200 mg/kd/d.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 50 mg/kgl/ay: No treatment-related changes noted.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
900 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; food consumption; haematology; clinical chemistry; gross pathology; histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
At the treatment levels used, a No Observed Effect Level of 50 mg/kg/day was established. Furthermore the dose levels 200 mg/kg/d and 900 mg/kg/d are to be considered as NOAEL and LOAEL, respectively. The substance tehrefore is not classified as to its repeated dose toxicity properties.