(6R-trans)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

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:

January 25 - February 21, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD Guideline 407 and EU method B.7 under GLP conditions. No relevant deviations reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Remarks:

only non-relevant deviations reported (i.e. inadequate food supply, minor deviation in observation schedule)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Remarks:

only non-relevant deviations reported (i.e. inadequate food supply, minor deviation in observation schedule)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

22352-43-3

IUPAC Name:

22352-43-3

Constituent 2

Reference substance name:

7-aminodesacetoxycephalosporanic acid

IUPAC Name:

7-aminodesacetoxycephalosporanic acid

Constituent 3

Reference substance name:

(6R-trans)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

EC Number:

244-870-1

EC Name:

(6R-trans)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Cas Number:

22252-43-3

Molecular formula:

C8H10N2O3S

IUPAC Name:

7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Details on test material:

- Name of test material (as cited in study report): ZOR-F
- Substance type: white to off-white powder
- Physical state: solid
- Analytical purity: 99.2%
- Impurities (identity and concentrations): delta-2-7-ADCA 0.13%, 6-APA 7 mg/kg
- Lot/batch No.: D210058
- Expiration date of the lot/batch: 01-12-2003
- Storage condition of test material: store beIow 15 degrees C, protected from light and moisture.
- Other: note that test substance was stored in refrigerator protected from light, not meeting the above proposed conditions.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: not reported
- Fasting period before study: not reported
- Housing: 5 animals per sex in stainless steel cages
- Diet (e.g. ad libitum): ad libitum laboratory animal diet
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: preparation daily, max. 4 hours prior to dosing

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at Notox

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of week 4 formulations were controlled for homogeneity, accuracy of preparation, and stability during 4 hours in vehicle. Analytical method of Notox project 194579 was used.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 300, 900, 2700 mg/kg/day
Basis:
other: oral gavage

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on earlier projects (342337, 194568, 302941)

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: 1, 11, 15, 22, and 28 days

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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 data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: Yes (isoflurana)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [see page 11] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: Yes (isoflurana)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [see page 12] were examined.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see page 13)
HISTOPATHOLOGY: Yes (see page 13/14)

Other examinations:

none

Statistics:

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 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.

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) in the summary tables. No statistical analysis was performed on motor activity data. 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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

not of toxicological significance

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:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

not of toxicological significance

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

minor decrease alanine aminotransferase activity at 900 and 2700 mg/kg/day

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

not of toxicological significance

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

not of toxicological significance

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.
There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment.
Alopecia of the back and/or neck is commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. Lethargy was incidentally shown by one control and one group 3 male. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted among group 2 and 4 animals.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals were considered not to have been affected by treatment with the test substance.
Body weight of group 4 males appeared slightly reduced on day 8 but body weight gain was normal. Body weight gain of high dose females showed a statistically significant reduction on days 8 and 22. However, body weight values were similar to values of other similar studies and the reduction was not consistent over time. Therefore, these changes were considered to be of no toxicological relevance.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.
Mean white blood cell counts of group 3 and 4 females were reduced when compared to controls. However, no dose-related response was obtained and microscopic suppport for this change was absent. Changes in mean values of other parameters attaining a level of statistical significance were not related to the dose and/or means were comparable to controls of similar studies. Individual increases of neutrophil counts with concurrently reduced lymphocyte counts were noted among the dose groups without a treatment related distribution. This shift in type of white blood cells was considered to be a secondary non-specific response to stress and to be of no toxicological significance. Plasma samples of one group 3 male (no. 11) and one group 2 female (no. 29) appeared haemolytic. This finding was not related to the dose given and had occurred incidentally. These findings were considered to be of no toxicological importance.

CLINICAL CHEMISTRY
Alanine aminotransferase activity values were decreased among group 3 females and among group 4 animals of both sexes.
Slightly high mean bilirubin values were measured for all groups, while albumin values appeared slightly low for all female dose groups when compared to other studies. Differences of the mean values of these parameters between the groups were not reiated to the dose and microscopic support was absent. No explanation could be given for these changes that were slight in nature. Some high individual glucose values were obtained when compared to values of similar studies (upper level of reference range male 8.72 mmol/l (n=137), female 8.46 mmol/I (n=139)). The Incidence of these individual increases was not related to the dose. Other mean values achieving a level of statistical significance when compared to controls, occurred in the absence of a treatment-related distribution and were comparable to means of similar studies. No toxicological significance was ascribed to these findings.

ORGAN WEIGHTS
Organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals.
Mean absolute and relative spleen weights of all dose groups were considered to be slightly high when compared to mean values of similar studies. Mean adrenal weights of group 1 and 3 females were slightly high based on similar studies, but were considered to be normal when corrected for body weight. These changes were considered to be of no toxicological importance.

GROSS PATHOLOGY
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
Extramedullary hemopoiesis (primarily erythropoiesis) in the spleen was recorded in all dose groups at slight or moderate degree in males and at minimal to moderate degrees in females. All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:

NOAEL

Remarks:

definitive

Effect level:

2 700 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:

Wistar rats were treated daily during 28 days with 0, 300, 900, 2700 mg/kg/day of ZOR-F. A definitive NOEL of 300 mg/kg/day was established, since no effect was observed at that concentration. A NOAEL of 2700 mg/kg/day for ZOR-F is set since a slight decrease of plasma alanine aminotransferase activity is the only effect observed at 900 and 2700 mg/kg/day, which is considered to be not of toxicological relevance.