6-aminopenicillanic 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:

2009-09-04 to 2009-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted to current accepted guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

In addition to the blood biochemical parameters investigates, bile acids were also assessed. The mammary gland from all animals that died or were killed in extremis and all control and high dose animals were processed for histopathological examinations, e

GLP compliance:

yes (incl. QA statement)

Remarks:

The work described was performed in compliance with UK GLP standards (Schedule 1, GLP Regulations (SI 1999/3106 as amended by SI 2004/0994)) and the Swiss Ordinance relating to GLP, adopted May 18 2005 [SR 813.112.1]

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6-aminopenicillanic acid

EC Number:

208-993-4

EC Name:

6-aminopenicillanic acid

Cas Number:

551-16-6

Molecular formula:

C8H12N2O3S

IUPAC Name:

6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Details on test material:

- Name of test material : 6-aminopenicillanic acid
- Substance type: Off white powder
- Physical state: Solid
- Analytical purity: 98.7 %
- Impurities : Any other single impurity 0.07 %, Penicinic acid 0.17 %, Related substance and degradation products 0.85 %
- Purity test date: 29-05-2009
- Lot/batch No.: 09X10247
- Expiration date of the lot/batch:
- Storage condition of test material: Room temperature in the dark

Test animals

Species:

rat

Strain:

other: Wistar Han™: HsdRccHan™ WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Oxon, UK
- Age at study initiation: Approximately 6 to 8 weeks old
- Weight at study initiation: males - 170 to 191 g; females - 136 to 157 g
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet : Pelleted diet available ad libitum (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories UK Ltd ,Oxon, UK)
- Water: Mains drinking water available ad libitum provided by polycarbonate bottles.
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): At least 15 changes per hour
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were therefore prepared fortnightly and stored at approximately 4ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle : The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 1127-1831). Results from the previous study show the formulations to be stable for at least twenty days.
- Amount of vehicle (if gavage) and concentration in vehicle: Please refer to table 1

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of 6-Aminopenicillanic acid in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test material formulations were diluted with water to give a final, theoretical test material concentration of approximately 0.1 mg/mL. Standard solutions of test material were prepared in water at a nominal concentration of 0.1 mg/mL. Please refer to table 2 for HPLC conditions.

Duration of treatment / exposure:

A single exposure per day for 28 days

Frequency of treatment:

Test material was doses for 28 consecutive days.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 300 and 1000 mg/kg bw per day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females in each dose group (40 animals in total)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected on data from a previous toxicity study on 6-aminopenicillanic acid.

Positive control:

Not reported

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity ill-health or behavioural changes immediately before dosing, immediately post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends.


BODY WEIGHT: Yes
- Time schedule for examinations: Group mean weekly bodyweights were examined


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: Yes. Weekly food efficiencies were examined.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were performed on all animals from each test and control group at the end of the 28 days. Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture on day 29
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: No
- How many animals: All animals included in the study
- Parameters checked in table [No.3] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: No
- How many animals: All animals were investigated
- Parameters checked in table [No.4] were examined.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to treatment on days 7, 14, 21 and 25 all animals were observed for sign of functional/behavioural toxicity. Functional performance tests were aso performed during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All animals were examined
- Battery of functions tested: sensory activity, grip strength and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 5)
HISTOPATHOLOGY: Yes (see table 6)

Other examinations:

Stage of Oestrus
At termination, a vaginal smear was taken from all females and the stage of oestrus was recorded

Statistics:

Data were assessed to give group mean values and their corresponding standard deviations where deemed appropriate. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (P) are presented as follows:
P < 0.01 **
P < 0.05 *
p >= 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No unscheduled deaths occured during the study

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths occured during the study

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Description (incidence and severity):

Food and water consumption were also found to be unaffected

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 specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

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
No clinically observable signs of toxicity were detected. Animals of either sex treated with 1000 mg/kg/day had increased salivation immediately post dosing on Days 16, 19 (females only), 20 (females only) and 24 (males only). One male treated with 300 mg/kg/day and one female treated with 30 mg/kg/day also had increased salivation immediately after and one hour after dosing on Day 21 and Day 24 (respectively). This was considered to be of no toxicological significance as this is quite common with unpalatable or slightly irritant test material formulations.

BODY WEIGHT AND WEIGHT GAIN
There were no toxicologically significant effects detected in bodyweight development. Females treated with 1000 and 300 mg/kg/day and males treated with 300 mg/kg/day showed a statistically significant reduction in bodyweight gain during the first week of treatment. Recovery was evident thereafter and in the absence of a true dose related response the intergroup differences were considered to be of no toxicological significance.

FOOD CONSUMPTION & FOOD EFFICIENCY
There was no adverse effect on food consumption or food efficiency during the study period.

WATER CONSUMPTION
There were no toxicologically significant effects detected in water consumption. Animals from all treatment groups did show an increase in overall water consumption when compared to control animals however the dose related response was unconvincing and the intergroup differences were considered to be of no toxicological importance.

HAEMATOLOGY
There were no toxicologically significant effects detected in the haematological parameters measured. Females treated with 1000 and 300 mg/kg/day showed a statistically significant reduction in erythrocyte count. Females treated with 1000 mg/kg/day also showed a statistically significant increase in neutrophil count. In the absence of any associated changes the intergroup differences were considered to be of no toxicological importance.

CLINICAL CHEMISTRY
There were no toxicologically significant effects detected in the blood chemical parameters measured. Females from all treatment groups showed a statistically significant increase in urea and a statistically significant reduction in alanine aminotransferase. The majority of the individual values were within the normal ranges expected for rats of the strain and age used and in the absence of a true dose related response the intergroup differences were considered to be of no toxicological significance. Animals of either sex treated with 1000 mg/kg/day and males treated with 300 mg/kg/day showed a statistically significant increase in bilirubin. Males treated with 1000 mg/kg/day also showed a statistically significant reduction in calcium. All individual values were within the normal ranges expected for rats of the strain and age used and as such the intergroup differences were considered to be of no toxicological importance. Females from all treatment groups and males treated with 1000 or 30 mg/kg/day showed a statistically significant reduction in bile acids. In the absence of a dose related response in males or any associated histological correlates the intergroup differences were considered to be of no toxicological importance.

NEUROBEHAVIOUR
There were no treatment-related changes in the behavioural parameters measured. There were no toxicologically significant effects detected in the functional performance parameters measured. Males from all treatment groups showed a statistically significant reduction in overall mobility. In the absence of any clinical signs of neurotoxicity the intergroup differences were considered to be of no toxicological importance. Females treated with 1000 and 300 mg/kg/day showed a statistically significant increase in mean forelimb grip strength. These intergroup differences were confined to one out of the three tests for each parameter and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the findings were considered to be of no toxicological significance. There were no treatment-related changes in sensory reactivity. All inter group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

ORGAN WEIGHTS
There were no toxicologically significant effects detected in the organ weights measured. Males treated with 300 mg/kg/day showed a statistically significant increase in thymus weight both absolute and relative to terminal bodyweight. Females treated with 30 mg/kg/day showed a statistically significant increase in absolute and relative kidney weight. In the absence of a dose related response or any histology correlates the intergroup differences were considered to be of no toxicological significance.

GROSS PATHOLOGY
There were no treatment-related macroscopic abnormalities detected.

HISTOPATHOLOGY
There were no treatment related microscopic abnormalities detected. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

STAGE OF OESTRUS
Assessment of stage of oestrus for females at termination showed no significant differences between treated and control groups

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: With the exception of excess salivation due to the bitter taste, no adverse effects were noted throughout the study.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of 6-aminopenicillanic acid at dose levels of 30, 300 and 1000 mg/kg/day for a period of twenty eight consecutive days did not result in any toxicologically significant effects in animals of either sex treated with 30, 300 or 1000 mg/kg/day. The "No Observed Adverse Effect Level" *NOAEL) was therefore considered to be 1000 mg/kg/day.