Butanedioic acid, 1-[2-[(1-oxo-2-propen-1-yl)oxy]ethyl] ester

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:

11 April 2006 to 21 August 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Directive 96/54/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Date of inspection: 30 August 2005 Date of Signature: 21 November 2005)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

478-130-6

EC Name:

-

Cas Number:

50940-49-3

Molecular formula:

C9H12O6

IUPAC Name:

2-(acryloyloxy)ethyl hydrogen succinate

Details on test material:

- Name of test material (as cited in study report): MAES
- Substance type: Colourless turbid liquid.
- Physical state: Liquid.
- Lot/batch No.: J417708-A.
- Storage condition of test material: Room temperature in the dark.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, UK.
- Age at study initiation: 5 to 6 weeks old.
- Weight at study initiation: 211 - 238 g (male) and 143 - 180 g (female).
- Fasting period before study: Not reported.
- Housing: The animals were housed in groups of 5 by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet: Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK. Ad libitum.
- Water: Mains drinking water ad libitum.
- Acclimation period: 9 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC.
- Humidity (%): 55 ± 15%.
- Air changes (per hr): ≥ 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness.

IN-LIFE DATES: Day 0 (the day of dosing) up to Day 28.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was prepared at 3.75, 37.5 and 75 mg/ml, as a solution in polyethylene glycol 400. The stability and homogeneity of the test material formulations were determined at the test laboratory. The formulations to be stable for at least 14 days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.

DIET PREPARATION
Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The stability and homogeneity of the test material formulations were determined at the test laboratory. The samples used were test material extracted with acetonitrile with final theoretical concentration of 0.1 mg/ml (see 'Details on analytical verification of doses or concentrations'). The formulations were found to be stable for at least 14 days.
- Amount of vehicle (if gavage): 4 ml/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Summary; The concentration of MAES in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.
Samples; The test material formulations were diluted with acetonitrile to give a final theoretical test material concentration of 0.1 mg/ml.
Standards; Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml.
Procedure; The sample and standard solutions were analysed by HPLC.
Homogeneity determinations; The test material formulations were mixed throughly and samples were taken from the top, middle and bottom of the container, shaking between sampling in triplicate.
Stability determinations; The test material formulations were sampled and analysed initially and then after storage at 4ºC in the dark for 14 days.
Verification of test material formulation concentrations; The test material formulations were sampled and analysed within 3 days of preparation.
Results; Mean concentration found was in the range of 93 – 101% of nominal concentrations over the dosing period. The results showed the formulation to be stable for at least 14 days.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
15 mg/kg/day
Basis:
other: nominal in vehicle

Doses / concentrations 2

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
other: nominal in vehicle

Doses / concentrations 3

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

5 animals per sex per dose and the control group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of the range-finding study performed. See the summary of the range-finding study in 'any other information on materials and methods incl. tables'.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, immediately post dosing and 1 and 5 hours after dosing during the working week. Animals were observed immediately before dosing, and 1 hour after dosing at weekends and public holidays.
- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and weekly intervals thereafter. Bodyweights were also recorded prior to terminal kill.

FOOD CONSUMPTION: Yes
Weekly food consumption was recorded for each cage group.

FOOD EFFICIENCY: Yes
- Food efficiency (the ratio of bodyweight gain / food consumption) was calculated.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily for each cage group by visual inspection of the water bottles for any overt changes.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematological and blood chemical investigations were performed on Day 28. Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Anaesthetic used for blood collection: Not reported.
- Animals fasted: No.
- How many animals: All surviving animals.
- Parameters checked in Tables 10 and 11.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Prior to the start of treatment and on Days 3, 10, 17 and 25, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
- Dose groups that were examined:
All animals.

- Parameters examined:
Behavioural assessment: Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation.

Functional Performance Tests: Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The evaluation period was one hour for each animal. The time (seconds) each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex and finger approach.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All suriviving animals were killed by intravenous overdose of sodium pentobarbitone, followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ weights:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation.
Adrenals, liver, brain, ovaries, epididymides, spleen, heart, testes, kidneys and thymus.

HISTOPATHOLOGY: Yes
Samples of the following tissues were preserved from all animals and preserved in buffered 10% formalin.
Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi. Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (cervical and mesenteric), muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus.

All tissues were despatched to PPropath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, UK. The tissues (except aorta, bone & bone marrow, eyes, muscle, oesophagus, pancreas, pituitary, salivary glands and skin) from all control and 300 mg/kg/day group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 15 and 150 mg/kg/day dose group animals.

Since there were indications of treatment-related changes in the bone marrow, stomach and duodenum, examination was subsequently extended to include sections of these tissues from all animals in the remaining groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Other examinations:

No.

Statistics:

All data was summarised in tabular form. 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) were calculated as follows:
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One female treated with 300 mg/kg/day was found dead on Day 4 and showed no adverse clinical signs prior to death. Clinidal signs were considered to be related to the irritancy of the test substance administered. Animals of either sex treated with 300 mg/kg/day showed increased salivation around the time of dosing from Day 3 (males) and 7 (females) onwards. Animals of either sex treated with 150 and 15 mg/kg/day showed incidents of increased salivation around the time of dosing during the final two weeks of treatment. Observations of this nature are often reported following oral administration of an unpalatable or slightly irritant test material and, in isolation, are considered not to be indicative of systemic toxicity.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality: One female treated with 300 mg/kg/day was found dead on Day 4 and showed no adverse clinical signs prior to death. There were no other unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 300 mg/kg/day showed a slight reduction in bodyweight gain during Week 4, however, statistical significance was not achieved. No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 300 mg/kg/day showed a reduction in food efficiency during Week 4, no effects were seen in the lower dose groups.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 300 mg/kg/day showed slight reductions in haemoglobin, haematocrit and erythrocyte count and a statistically significant reduction in mean cell haemoglobin concentration. No such effects were seen at the lower doses.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One male and four females treated with 300 mg/kg/day together with two males and one female treated with 150 mg/kg/day showed a raised limiting ridge in the stomach. No such effects were detected in animals of either sex treated with 15 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A slightly lower incidence of higher grades of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment for females treated with 300 mg/kg/day.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS
See Table 1 for the results, as attached.
An isolated incident of noisy respiration was evident in one male and one female treated with 300 mg/kg/day. Red/brown staining around the mouth/snout was also evident in two animals treated with 300 mg/kg/day. One male treated with 150 mg/kg/day showed generalised fur loss and scab formation from Day 18 onwards. The animal that died during the study showed no adverse clinical signs prior to death.

BODY WEIGHT AND WEIGHT GAIN
See tables 6 and 7 for the results, as attached..

FOOD CONSUMPTION AND FOOD EFFICIENCY
See tables 8 and 9 for the results, as attached. Animals of either sex treated with 300 mg/kg/day showed a reduction in food efficiency during Week 4.
No such effects were detected in food efficiency for animals of either sex treated with 150 or 15 mg/kg/day or in food consumption for animals of either sex treated with 300, 150 or 15 g/kg/day.

WATER CONSUMPTION
Daily visual inspection of water bottles revealed no intergroup differences.

HAEMATOLOGY
See Table 10 for the results, as attached.
Animals of either sex treated with 300 mg/kg/day showed slight reductions in haemoglobin, haematocrit and erythrocyte count (statistical significance was however not always achieved). Associated changes for males also included a statistically significant reduction in mean cell haemoglobin concentration. Females from all treatment groups showed a statistically significant reduction in eosinophil count. In the absence of a dose related response these intergroup differences were considered not to be toxicologically significant.

CLINICAL CHEMISTRY
See Table 11 for the results, as attached..
There were no toxicologically significant changes in the blood chemical parameters measured.
Males treated with 300 and 150 mg/kg/day showed a statistically significant increase in plasma urea. All individual values were within the normal ranges for rats of the strain and age used, and in the absence of any histopathological evidence to suggest renal dysfunction, the increase was considered to be of no toxicological importance. Females from all treatment groups showed a statistically significant reduction in alanine aminotransferase. Reductions in plasma levels of this enzyme are unlikely to be of any toxicological significance, and with the majority of individual values within the normal range for rats of the strain and age used, the intergroup differences were considered to be of no toxicological importance.

NEUROBEHAVIOUR
See tables 2 - 5 for the results, as attached..
Behavioural Assessments: There were no treatment-related changes in the behavioural parameters measured.
All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Functional Performance Tests
There were no treatment-related changes in the functional performance parameters measured.
Statistical analysis revealed no significant intergroup differences.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used, and was of no toxicological importance.

ORGAN WEIGHTS
See Table 12 for the results, as attached..
There were no treatment related changes in the organ weights measured.
Statistical analysis revealed no significant intergroup differences.

GROSS PATHOLOGY
See Table 13 for the results, as attached..
The female treated with 300 mg/kg/day that was found dead on Day 4 showed a dark liver and a raised limiting ridge in the stomach together with a pale and sloughing glandular gastric epithelium. One male treated with 300 mg/kg/day incurred a damaged eye during removal at necropsy. This was a physical injury occurring after termination and was considered unrelated to treatment. One female treated with 15 mg/kg/day showed red lungs at necropsy. In the absence of any histopathology correlates this intergroup difference was considered of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following treatment-related microscopic changes were detected: See Table 14 for the results, as attached..
BONE MARROW: A lower incidence of higher grades of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment for females treated with 300 mg/kg/day. This was a marginal effect that was dependent upon three animals at the high dose level having lower grades of severity. There was no convincing effect at any other dose level, or among males at any dose level.

STOMACH: Gastric changes characterised by acanthosis/hyperkeratosis of the forestomach and limiting ridge, mucosal hypertrophy, and mucosal basophilia were seen among animals of either sex treated with 300 mg/kg/day or at 150 mg/kg/day, but not at 15 mg/kg/day.

DUODENUM: Mucosal hypertrophy was seen in relation to treatment for males only treated with 300 mg/kg/day, 150 mg/kg/day, and at 15 mg/kg/day with an apparent dose response in terms of severity grades. However there were no associated degenerative changes. Females were not similarly affected.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

The one animal that died at 300 mg/kg/day showed no adverse clinical signs prior to death or any microscopic indication as to the cause of death. Other findings could be associated with gastric irritancy and are often reported following oral administration of an unpalatable or slightly irritant test material formulation. The potential for gastric irritancy was supported macroscopically and microscopically at this dose level. The anaemic response and microscopic changes detected in the bone marrow at 300 mg/kg/day were marginal.

Significant effects due to the corrosivity of MAES were also observed in the 150 mg/kg/day dose group and identified as acanthosis/ hyperkeratosis of the forestomach and limiting ridge, mucosal basophilia and mucosal hypertrophy of the stomach and duodenum (males only).

Changes detected at 15 mg/kg/day were confined to minimal non-degenerative duodenum changes which were considered not to represent an adverse health effect.

Applicant's summary and conclusion

Conclusions:

The oral administration of MAES to rats for a period of up to twenty-eight consecutive days at dose levels of 15, 150, and 300 mg/kg/day resulted in minor but occasionally significant treatment-related effects in animals of either sex treated with 300 or 150 mg/kg/day. These effects can be considered not to represent "serious damage" to health as defined by the criteria given in the EC labelling guide of Commission Directive 200l/59/EC. The dose of 15 mg/kg/day can be regarded as a "No Observed Adverse Effect Level" (NOAEL) for local effects in both sexes.

Executive summary:

Introduction.The study was designed to investigate the systemic toxicity of the test material.It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (Adopted 27 July 1995).

Methods.The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to twenty-eight consecutive days, at dose levels of 15, 150 and 300 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Mortality.One female treated with 300 mg/kg/day was found dead post dosing on Day 4. There were no other unscheduled deaths.

 

Clinical Observations.Animals of either sex treated with 300 mg/kg/day showed episodes of increased salivation around the time of dosing, noisy respiration and/or red/brown staining around the mouth/snout. One male treated with 150 mg/kg/day showed generalised fur loss and scab formation from Day 18 onwards and animals of either sex treated with 150 and 15 mg/kg/day showed incidents of increased salivation around the time of dosing during the final two weeks of treatment. The animal that died during the study showed no adverse clinical signs prior to death.

 

Behavioural Assessment.There were no treatment-related changes in the behavioural parameters measured.

 

Functional Performance Tests.There were no treatment-related changes in the functional performance parameters measured.

 

Sensory Reactivity Assessments.There were no treatment-related changes in sensory reactivity.

 

Bodyweight.Animals of either sex treated with 300 mg/kg/day showed a reduction in bodyweight gain during Week 4 only; statistical significance however was not achieved. No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

 

Food Consumption.Animals of either sex treated with 300 mg/kg/day showed a reduction in food efficiency during Week 4. No such effects were detected in food efficiency for animals of either sex treated with 150 or 15 mg/kg/day or in food consumption for animals of either sex treated with 300, 150 or 15 mg/kg/day.

 

Water Consumption.No intergroup differences were detected.

 

Haematology.Animals of either sex treated with 300 mg/kg/day showed slight reductions in group mean haemoglobin, haematocrit and erythrocyte count. Associated changes for males also included a statistically significant reduction in mean cell haemoglobin concentration. No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

Blood Chemistry.No toxicological significant changes were detected.

Organ Weights.No treatment-related effects were detected.

Necropsy.The female treated with 300 mg/kg/day that was found dead on Day 4 showed a dark liver and a raised limiting ridge in the stomach together with a pale and sloughing glandular gastric epithelium. One male and four females treated with 300 mg/kg/day together with two males and one female treated with 150 mg/kg/day showed a raised limiting ridge in the stomach. No such effects were detected in animals of either sex treated with 15 mg/kg/day.

 

Histopathology.The following treatment-related microscopic changes were detected:

 

BONE MARROW: A lower incidence of higher grades of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment for females treated with 300 mg/kg/day. This was a marginal effect that was dependent upon three animals at the high dose level having lower grades of severity. There was no convincing effect at any other dose level, or among males at any dose level.

 

STOMACH:Gastric changes characterised by acanthosis/hyperkeratosis of the forestomach and limiting ridge, mucosal hypertrophy, and mucosal basophilia were seen among animals of either sex treated with 300 mg/kg/day or at 150 mg/kg/day, but not at 15 mg/kg/day.

 

DUODENUM:Mucosal hypertrophy was seen in relation to treatment for males only treated with 300 mg/kg/day, 150 mg/kg/day, and at 15 mg/kg/day with an apparent dose response in terms of severity grades. However there were no associated degenerative changes. Females were not similarly affected.

 

Conclusion.The oral administration of MAES to rats for a period of up to twenty-eight consecutive days at dose levels of 15, 150, and 300 mg/kg/day resulted in minor but occasionally significant treatment-related effects in animals of either sex treated with 300 or 150 mg/kg/day. These effects can be considered not to represent "serious damage" to health as defined by the criteria given in the EC labelling guide of Commission Directive 200l/59/EC. The dose of 15 mg/kg/day can be regarded as a "No Observed Adverse Effect Level" (NOAEL) for both sexes.