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: Guideline study with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2001

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: 7-AMCA
Chemical name: 7-Amino-3-methoxy-cefalosporanic acid
Molecular formula: C9H12N2O4S
CAS No.: 24701-69-7
Supplier: Sponsor
Batch No.: 36423006
Certificate of analysis: Available
Purity: 90.1 % on anhydrous basis (HPLC)
Water content: 0.3 %
Solubility: In water 0.1 g/L at 20 °C
In methanol 0.05 g/L
Appearance: Yellowish to yellow powder
Conditions of storage: Refrigerator, in the dark. Protected from moisture
Stability: Stable over ca. 6 months under conditions of storage. Stable over several weeks at room temperature
Date of expiry: October 2000

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga. Sulzfeld. Germany.
- Age at study initiation: Ca. 6 w.
- Weight at study initiation: mean of ca. 120 g for males; mean of ca. 100 g for females.
- Housing: Single caging in Makrolon cages
- Bedding material: Aspen wood chips
- Diet: Altromin 1324 forte ad libitum
- Water: Tap water, acidified to pH 3 with HCl, ad libitum
- Acclimation period: 13 d.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 22
- Humidity (%): ca. 44
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 %
Details on oral exposure:
The test substance was administered as a suspension in 0.5 % CMC. Dose volume: 10 mL/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the test substance solutions
Determinations of the test substance in some selected preparations for stability, concentration and homogeneity were performed by HPLC using the following conditions:

System: Thermo Separation Instruments.
Column: Hypersil CPS 3 µm 150*3 mm Code No. NCN31503, produced by Forschungszentrum Seibersdorf, Austria.
Column temperature: 30 °C.
Flow: 0.4 mL/min.
Mobile phase: 50 % 0.05 % trifluoroacetic acid in water LiChrosolv, 50 % acetonitril LiChrosolv.
Solvent (for standards): 0.2 % Na-acetate and/or deionised water.
Injection volume 10 µL.
Detection: UV detector set at 290 nm.

The concentrations of the test substance were determined by relating the areas under the curve to standards.
The following analyses were performed:
Stability: The stability of the test substance in suspension (100 mg and 1000 mg in 10 mL 0.5 % CMC) was investigated once before the start of the study.
Analyses were performed immediately after and 4 hours after the preparation. The difference between the concentrations was used for estimating the stability of the test substance. The stability was considered to be sufficient, if the loss in concentration was less than 5 % within 4 hours.
Conditions for the solutions before analysis:
• Ambient temperature.
• Ambient light.
• Preparations were kept in closed glass bottles.

Concentration and homogeneity: Investigations were performed on Day 2. Three samples of about 2 mL each were taken from each preparation. Concentration was considered as sufficient, if the mean of the test substance concentrations of the 3 samples taken was within ± 10 % of the target concentration.
Homogeneity was considered to be sufficient, if the test substance concentrations of the 3 samples taken did not exceed ± 10 % of their mean.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week.
Day 1 was the first day of dosing the test substance.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 - 100 - 316 - 1000 mg/kg bw (groups K-A-B-C)
Basis:
other: nominal
Remarks:
Doses / Concentrations:
0 - 1000 mg/kg bw (groups KR - CR)
Basis:
other: nominal, for the satellite recovery groups.
No. of animals per sex per dose:
5 males and 5 females in each treated groups and in the control group.
Additional 5 males and 5 females in each of the recovery group (1000 mg/kg bw) and the recovery control group.
Control animals:
yes, concurrent vehicle
Details on study design:
The dose selection was based on a dose range finding study.
Additional control and high dosed recovery groups were observed for further 14 days without test substance administration.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
Daily observations
All animals were carefully observed for general signs and the health status once a day and additionally checked once a day for viability.
Detailed clinical observations
More detailed clinical observations were made while the animals were either in the standard arena or in the hand of the animal technician on:
• Days -1, 6, 13, 20, 27: all animals of all groups.
• Days 34, 41: all animals of the recovery groups.
Special emphasis was put on skin, fur, eyes, visible mucous membranes, incisors, secretion and excretion, body odour, autonomous activities (e.g. lacrimation, piloerection, pupillar size, abnormal breathing, body surface temperature), vocalisation, abnormal locomotion, movements and posture, presence of convulsions or paralysis, stereotypes, bizarre behaviour, visible or palpable tissue masses.
Functional observations
Assessment of the behaviour, of the motor activities, and of the sensory reactivity to different stimuli (acoustic, tactile, visual and proprioceptive) was performed outside the home cage in a standard arena. The eyelid and auricular reflexes (tactile and proprioceptive) were tested by slightly touching of cornea and pinna with a nylon cord of approximately 0.6 mm diameter. Acoustic reactivity was tested by reaction to a sudden moderate sound (clapping of the hands). Visual reactivity was derived from the reaction to a white sheet, brought near to the animal without any physical contact.
For the testing of the righting reflex the animals were held between the front- and hindlegs, turned on their backs and dropped from a height of 30 cm into the bedding material of the standard arena. A landing on all four legs is a normal reaction. This and a measurement of the forelimb grip strength were conducted in all animals on Day 27.

Body weight
Body weight was determined of all animals on Day -6, Days 1, 8, 15, 22, 28 and on Days 29, 35 and 43 in all animals of the recovery groups.

Feed consumption
Determined for Days -6 to 1, 1-7, 7-14, 14-21, 21-28 of all animals and for Days 29-35 and 35-43 of all animals of the recovery groups.

Sacrifice and pathology:
Necropsy
Animals were killed by CO2-asphyxia and subjected to a necropsy including a gross pathological examination on
• Day 29: groups K, A, B and C.
• Day 44: groups KR and CR.
Spontaneously died animals were necropsied as soon as possible.
The following organs / tissues were taken from all animals and fixed in Bouin's solution:
• gross lesions, tissue masses or tumours • ovaries • adrenal glands • pancreas* • aorta* • pituitary * • brain • prostate • caecum* • rectum* • coagulating glands * • salivary glands * • colon • seminal vesicles * • duodenum • sciatic nerve • epididymes * • skeletal muscle (thigh) • eyes* • skin and mammary gland * • femur bone with joint * • spinal cord (cervical, thoracal, lumbar) • heart • spleen • ileum • sternum • jejunum • stomach • kidneys • testes • lacrimal glands (extraorbital) * • thymus • liver • thyroid and parathyroid glands • lungs • trachea • lymph nodes (mandibular, mesenteric) • urinary bladder • oesophagus • uterus
*:not included in the routine histopathology of groups K and C.

Organ weights
Weights of the following organs were determined of all animals:
• adrenal glands • kidneys (both together) • brain * • spleen • epididymis (both together) • testes (both together) • heart • thymus • liver
*: weighed after fixation.
Relative organ weights were calculated by relating the absolute organ weights to the last determined individual body weight and to the brain weight.

Histopathology
Histopathological examination was performed of all fixed organs or tissues listed above, except for those labelled with a "*" in all animals of groups K and C. In groups KR, A, B and CR the spleens were examined in both males and females, and the kidneys in the females only.
Appropriate parts of organs or tissues were embedded in paraffin. Sections of about 5 µm were stained with haematoxylin and eosin. Evaluation of slides was performed using a light microscope Leica-DMRB. To describe the severity of lesions, the following grades were applied, if appropriate: minimal (1), mild (2), moderate (3), marked (4), severe (5). The term "focal" together with a higher degree of severity also stands for "multifocal ".
Other examinations:
Haematology
Blood samples were taken from the retrobulbar vein plexus of the left eye each in slight ether anaesthesia. Feed was withdrawn in the evening of the day before at about 5 p.m. and offered again immediately after the end of the blood sampling. Blood was taken in the morning of Day 29 from all animals of groups K, A, B and C between 7 and 8:30 a.m.
Parameters determined:
• Red blood cell count (RBC)
• Haemoglobin concentration (Hb)
• Haematocrit (Hct)
• Mean corpuscular haemoglobin (MCH)
• Mean corpuscular haemoglobin concentration (MCHC)
• White blood cell count (WBC)
• Mean cell volume (MCV)
• Platelet count (PL T)
• Differential white blood cell count (Leucocyte count, % of the different cell species)
• Prothrombin time (Quick) as indicator of blood clotting capacity.
Haematological examination of the blood samples was performed using Coulter STKS - Instrumentation Laboratory and for the determination of clotting time Roche STA Compact (Stago). Baseline data (control data, gained under comparable conditions from the same strain of rats) are available in the test facility. As no significant differences were present on Day 29, no blood samples were taken at the end of the recovery period.

Clinical biochemistry
Blood samples were taken from the same animals and at the same time as for haematology. Serum was obtained by centrifugation of the blood samples after incubation at 37 °C for about 1 hour.
Parameters determined:
• alanine aminotransferase (ALT, GPT)
• albumin (ALB)
• alkaline phosphatase (ALP)
• aspartate aminotransferase (AST, GOT)
• cholesterol (CHO)
• creatinine (CREA)
• glucose (GLU)
• potassium (K+)
• sodium (Na +)
• total protein (TP)
• urea (BUN)
Clinical chemical examination of the serum samples was performed using a Roche Hitachi 917 for the chemical parameters and an ion-selective electrode for electrolyte determinations. Baseline data (control data, gained under comparable conditions from the same strain of rats) are available in the test facility. As no significant differences were present on Day 29, no blood samples were taken at the end of the recovery period.

Statistics:
Statistical method:
Analysis of variance followed by the Scheffe-test: all data with means and standard deviations determined, comparison of more than two groups
t-test: all data with means and standard deviations determined, for comparison of two groups only
H-test of Kruskal and Wallis followed by the test of Nemenyi: counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled
Chi2 -test: counted events
Fisher's exact test: counted events, if the Chi2 -Test was not applicable

Results were analysed separately for males and females. P = 0.05 was chosen in each test. Two tailed test were used. Groups K and KR and C and CR were treated separately for statistical analysis.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals survived until the scheduled terminal sacrifice. Soft faeces was detected at the daily observation. No test substance related effects were observed in the detailed clinical observation and the functional observation.
Mortality:
mortality observed, treatment-related
Description (incidence):
All animals survived until the scheduled terminal sacrifice. Soft faeces was detected at the daily observation. No test substance related effects were observed in the detailed clinical observation and the functional observation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dosed recovery males had sign. lower body weights as the controls in the first week after end of test substance administration. This may have been caused by the test substance, but is considered of low relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the high dosed recovery group males, a significantly decreased feed consumption was noted in the first week of dosing (Day 1 to 7). The identically treated high dosed group had no corresponding trend.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
See also above under clinical signs.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weights were increased in both sexes in the high dosed recovery group. Kidney weight was increased in the high dosed female recovery group. Heart and thymus weight was reduced in the high dosed female recovery group.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No test substance related lesions were noted at histopathological examination. Only alterations, interpreted as spontaneous changes, were found in some animals without a dose relationship.
Details on results:
Clinical observations:
No mortality. Soft faeces in all dosed groups. Scattered
reduction of body weight and feed consumption in the high
dosed male recovery group.

Laboratory findings:
No relevant differences in haematological and biochemical
parameters were detected.

Effects in organs:
Liver weights were increased in both sexes in the high dosed
recovery group. Kidney weight was increased in the high
dosed female recovery group. Heart and thymus weight was
reduced in the high dosed female recovery group.

No other gross pathological or histopathological relevant
findings were obtained.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
316 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
other: soft faeces

Target system / organ toxicity

Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: unspecific: body weight, organ weights, soft faeces
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Stability of the test substance preparations:

The loss of test substance in solution was less than the chosen limit of 5 % within 4 hours for both the highest and the lowest concentration used in this study. The test substance solutions were therefore considered to be sufficiently stable.

Concentrations and homogeneity of the test substance preparations:

The deviations from the target concentrations and the deviations from the means were within the chosen limits of ±10 %. The concentration and the homogeneity of the test substance preparations were therefore acceptable.

Applicant's summary and conclusion

Conclusions:
The NOAEL is 316 mg/kg bw.
Executive summary:

The toxicity of 7-AMCA was investigated after a repeated oral administration to rats, according to the EC-method B.7. and to the OECD Guideline 407, "Repeated Dose 28-day Oral Toxicity Study in Rodents".

Methods

Test substance administration:

The test substance was administered as a suspension in 0.5 % aqueous Na-CMC orally by gavage to 3 groups of 5 male and 5 female F344 rats each, once a day on 7 days per week for 28 consecutive days (Day 1 to 28). Doses used:

• Group A (low dose): 100 mg/kg bw and day

• Group B (mid dose): 316 mg/kg bw and day

• Group C (high dose): 1000 mg/kg bw and day

An equally sized negative control group (group K) received the vehicle of the test substance.

The dose volume was 10 mL/kg bw and day.

In addition, two groups of 5 males and 5 females each, i.e. one high dose recovery group (group CR) and one control recovery group (group KR), were treated in the same way as their corresponding groups, but were kept for further 14 days without test substance administration in an attempt to observe the reversibility or persistence of test substance induced lesions.

Investigations:

• Animal observations: once a day (plus a daily check for viability).

• Detailed clinical observations: once a week.

• Functional observations: once in the last week of the dosing period.

• Body weight: once a week.

• Feed consumption: for each week.

• Haematology: on Day 29.

• Clinical biochemistry: on Day 29.

• Necropsy with gross pathological examination: on Day 29 and on Day 44.

• Organ weight determination.

• Histopathological examination.

Results

• Mortality, observations in life: All animals survived until the scheduled sacrifice. The only test substance related finding in life was soft faeces in all dosed groups.

• Body weights: Males, high dose recovery group: Reduced on Day 35.

• Feed consumption: Reduced: Males, high dosed recovery group, Days 1 to 7. Elevated: Females, low dosed group, Days 14 to 21.

• Haematology and clinical chemistry: No significant differences were found between a dosed group and the controls.

• Organ weight determination: Liver weights were increased in both sexes in the high dosed recovery group. Kidney weight was increased in the high dosed female recovery group. Heart and thymus weight was reduced in the high dosed female recovery group.

• Necropsy with gross pathological examination and histopathology: No test substance related alterations were observed.

• Recovery groups: Significant body weight / organ weight differences were present after the end of the recovery period. This might be due to a delayed onset of test substance effects.

The oral NOAEL is 316 mg/kg bw.