Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-12 - 2012-06-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See section "Principles of method if other than guideline"
Principles of method if other than guideline:
The body weight range of male animals at the start of treatment on this study was 315-362 g which slightly exceeded the predicted range in the Study Plan (190 to 350g). All animals were of the corrected age specification and were considered acceptable for use on the study. This deviation from Study Plan was considered to have had no impact on the scientific integrity of the study.

The formulations of the Test Item used for pre-study chemistry were prepared in distilled water and not water obtained by reverse osmosis. For the purposes of formulations these was considered to be no practical difference between distilled water and water obtained by reverse osmosis, therefore this deviation from Study Plan was considered to have had no impact on the scientific integrity of the study.

Achieved concentration was scheduled to be measured on three occasions during the study. On the third occasion achieved concentration was lower than anticipated at the low dosage, although it was anticipated that this represented a sampling error (the sample being taken before the formulation had been completely mixed) rather than a problem with the formulation procedure. In view of this an addition sampling occasion was instigated to confirm the accuracy of the formulation procedure. It is considered that this deviation from Study Plan had no adverse impact on the scientific integrity of the study.

For animals 12, 13 and 14 the water residue was not recorded on Day 4 of gestation in error. Water residue was recorded on Day 5 and therefore it was possible to calculate the amount consumed by these animals during Day 3 and 4. As the water consumption data was presented over the period Day 0 to Day 7 of gestation there was no overall loss of data and there was considered to be no impact on the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Chemical name: N-methylpiperazine
Description: Clear colourless liquid
Purity: 99.9%
Batch number: 101124
Label: N-METHYLPIPERAZINE 101124 NMP F-5711
CAS number : 109-01-3
UN number: 2734
Date received: 19 August 2011
Storage conditions: Ambient temperature, in the dark, over silica gel
Expiry date: Not supplied
Specific details on test material used for the study:
- Chemical name: N-methylpiperazine
- Description: Clear colourless liquid
- Purity: 99.9%
- Batch number: 101124
- Label: N-METHYLPIPERAZINE 101124 NMP F-5711
- CAS number: 109-01-3
- UN number: 2734
- Date received: 2011-08-19
- Storage conditions: Ambient temperature, in the dark, over silica gel
- Expiry date: Not supplied

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for eight days during which time their health status was assessed. A total of ninety animals (fifty males and forty females) were accepted into the study. At the start of treatment the males weighed 315 to 362g (see Deviations from Study Plan), the females weighed 191 to 225g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the non-recovery dose group animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Recovery group animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Addendum 1. Mains drinking water was supplied from polycarbonate bottles attached to the cage on the day of arrival and during the treatment-free recovery period. Reverse osmosis water was supplied from seven days prior to the start of treatment and throughout the treatment period (either untreated or containing the required concentration of Test Item). The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK, Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system and print-outs of hourly temperatures and humidities are included in the study records. Study Plan target ranges for temperature and relative humidity 22 ± 3°C and 50 ± 20% respectively and there were no deviations from these target ranges.


Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
For the purpose of this study, the test item was prepared at the appropriate concentrations as a solution in water obtained by reverse osmosis. Prior to treatment in the preliminary study (Harlan Laboratories Ltd., Project Number 41102856) the pH of formulations containing concentrations of the test item between 1 and 15 mg/ml were investigated and found to be alkaline. After discussions with the sponsor it was decided that test item formulations used on the preliminary study and this main study would be adjusted to an approximate pH of 9. No adjustment of pH was made for the reverse osmosis water supplied to the control group or for tap water supplied to recovery animals during the treatment-free recovery period.

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in water obtained by reverse osmosis. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services (see Deviations from Study Plan). Results show the formulations were homogeneous and that formulations were to be stable for at least ten days at 4°C and at room temperature (during storage in the drinking bottle used to deliver water to the animals). Formulations were generally prepared on a weekly basis (or more frequently depending on the number of cages being used at certain points of the study) and stored at ambient temperature in the animal room in the dark.

Samples of test item formulations were taken on four occasions during the study and analysed for concentration of N-methylpiperazine at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 84 to 101% of nominal concentration indicating that the formulation procedure was sufficiently accurate for the purpose of this study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of N-methyl piperazine in the test item formulations was determined by gas chromatography (GC) using an external standard technique.
- Samples: The test item formulations were diluted with methanol to give a final, theoretical test item concentration of approximately 0.01 mg/ml.
- Standards: Standard solutions of test item were prepared in methanol at a nominal concentration of 0.01 mg/ml.
- Procedure: The standard and sample solutions were analysed by GC using the following conditions:

GC system Agilent Technologies 5890, incorporating autosampler and workstation
Column DB-5 (30 m x 0.53 mm id x 5 µm film)
Oven temperature program : initial 50 ºC for 1 mins
rate 10 ºC/min
final 260 ºC for 0 mins
Injection temperature :250 ºC
Flame ionisation detector temperature :250 ºC
Injection volume: 1 µl
Retention time : ~ 4.8 mins

-Homogeneity Determinations: The test item formulations were assessed visually.
- Stability Determinations: The test item formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark and room temperature (ambient) for ten days. This storage was conducted in the same type of water bottles used to deliver the drinking water to the animals.
- Verification of Test Item Formulation Concentrations: The test item formulations were sampled and analysed within two days of preparation.
Duration of treatment / exposure:
Up to fifty-three consecutive days (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 500, 2500 and 10000 ppm. A control group of ten males and ten females received untreated drinking water (obtained by reverse osmosis) over the same treatment period. Two recovery groups, each of five males received via the drinking water the high dose (1000 ppm) or untreated water alone for forty-two consecutive days and then were maintained without treatment (tap water) for a further fourteen days.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Basis: nominal in water
Dose / conc.:
500 ppm
Remarks:
Basis:
other: Nominal in water (Mean dosages: Males - 44 mg/kg bw/day, Females - pre-pairing - 49 mg/kg bw/day, Females - gestation - 61 mg/kg bw/day, Females - lactation - 87 mg/kg bw/day)
Dose / conc.:
2 500 ppm
Remarks:
Basis:
other: Nominal in water (Mean dosages: Males - 190 mg/kg bw/day, Females - pre-pairing - 231 mg/kg bw/day, Females - gestation - 268 mg/kg bw/day, Females - lactation - 416 mg/kg bw/day)
Dose / conc.:
10 000 ppm
Remarks:
Basis:
other: Nominal in water (Mean dosages: Males - 466 mg/kg bw/day, Females - pre-pairing - 574 mg/kg bw/day, Females - gestation - 653 mg/kg bw/day, Females - lactation - 1055 mg/kg bw/day)
No. of animals per sex per dose:
10 animals per sex per dose (including control).
Control animals:
yes, concurrent no treatment
Details on study design:
The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
Clinical Observations
- All animals were examined for overt signs of toxicity, ill-health and behavioural change on a daily basis. During the treatment-free period, recovery animals were also observed daily. All observations were recorded.

Functional Observations
- Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each non-recovery dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioural Assessments
- Detailed individual clinical observations were performed for each non-recovery 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, Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.

Functional Performance Tests
- Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
- 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. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
- Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed: Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex , Tail pinch, Startle reflex, Finger approach

Body Weight
- Individual body weights were recorded on Day 1 and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

Food Consumption
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed for each cage of recovery group animals throughout the study period.
- Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for non-recovery males (except during the mating phase) and recovery group animals throughout the study period and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females, during gestation and lactation.

Water Consumption
- Water intake was measured daily throughout the study (with the exception of the pairing phase), including the first week prior to Test Item administration.

Laboratory Investigations
- Haematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group prior to termination (Day 42 for males and Day 4 post partum for females). In addition haematological and blood chemical investigations were performed on all recovery group animals after the fourteen day treatment-free period at termination (Day 56). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
- Haematology: The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), Total leucocyte count (WBC), Differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed
- Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).
- Blood Chemistry: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids
Sacrifice and pathology:
Pathology
- Adult non-recovery males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult non-recovery females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
- For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
- Recovery group animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 57.
- All adult animals and offspring, including those dying during the study, 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, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation)

Histopathology
- Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated: Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Oesophagus, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides•, Skin (hind limb), Eyes*, Spinal cord (cervical, mid-thoracic and lumbar), Gross lesions, Heart, Spleen, Ileum (including peyer’s patches), Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trachea, Liver, Testes•, Lungs (with bronchi) #, Thymus, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary gland , Uterus/Cervix, Muscle (skeletal), Vagina
- All tissues were despatched to the histology processing Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing. The tissues from five selected non-recovery control and 10000 ppm dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 10000 ppm and any animals which did not achieve a pregnancy, were also processed. In addition, sections of testes and epididymides from all control and 10000 ppm males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Since there were no indications of treatment-related changes, examination was not extended to include similarly prepared sections of animals from the low, intermediate and recovery groups. Microscopic examination was at AnaPath GmbH, Oberbuchsiten, Switzerland.
Statistics:
Due to the nature and quantity of this data please see section "any other information on material and methods including tables"

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs considered to be of any toxicological significance were apparent for adult animals during the study.
Yellow staining of the cage bedding was observed at 10000 ppm from Day 20 and 500 and 2500 ppm from Day 21. This was considered to reflect staining due to the test item and was considered to be of no toxicological significance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
At 10000 ppm, body weight gain of males was generally slightly lower than control throughout the treatment period, with differences occasionally attaining statistical significance. At the end of treatment period, overall body weight gain was approximately 80% of the control. Recovery of body weight gain was apparent during the treatment-free recovery period with overall gain being similar to control at the end of the study.

For females at 10000 ppm, body weight gains during the two week pre-pairing phase were slightly lower than control but, these differences from control failed to attain statistical significance and, at the level observed, may represent normal biological variation. However, subsequent body weight gains during gestation and lactation were clearly lower than control with both body weight and body weight gain frequently attaining statistical significance. The differences from control for body weight gain during gestation could not be attributed to differences in litter size for the pregnant females and appeared to represent an underlying effect on maternal body weight gain. Supporting this mean body weight on Day 1 of lactation was statistically significantly lower than control.

There was no adverse effect of treatment on body weight performance of either sex at 500 or 2500 ppm, including for females the gestation and lactation phases of the study.

See section 'Any other information on results ' for details
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse effects of treatment on food consumption for males observed during the study at 500, 2500 or 10000 ppm.
At 10000 ppm there was a suggestion of slightly inferior food conversion efficiency during the first week of the study but subsequent food utilisation was similar to control for the remainder of the study. There were no obvious effects of treatment on food conversion efficiency for males observed during the study at 500 or 2500 ppm.

There was no adverse effect of treatment on food consumption or food conversion efficiency of females during the pre-pairing phase of the study at 500, 2500 or 10000 ppm.

At 10000 ppm, food consumption of females was lower than control during gestation and lactation; differences from control were most marked during lactation, a period of high physiological demand on the female due to the demands of the litter.

There was no obvious effect of treatment on food intake of females at 500 or 2500 ppm during gestation and lactation.
Food efficiency:
no effects observed
Description (incidence and severity):
There were no obvious effects of treatment on food conversion efficiency for males observed during the study at 500 or 2500 ppm.
There was no adverse effect of treatment on food consumption or food conversion efficiency of females during the pre-pairing phase of the study at 500, 2500 or 10000 ppm.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
At 10000 ppm, water intake for both sexes was noticeably lower than control (and also from previous consumption prior to treatment) throughout the treatment period, and including for females the gestation and lactation phases of the study.

At 500 and 2500 ppm there was no clear effect of treatment on water intake for either sex.

See section 'Any other information on results' for details
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
For males at 2500 and 10000 ppm, lower group mean eosinophil count at the end of treatment attained statistical significance when compared to control; however group mean values at these dosages were close to that of the historical control data. Additionally, values for treated animals were all within the historical control range, while one control value exceeded it, and there was no corresponding statistically significant difference in overall total leucocyte count for males at these dosages, compared to control. In view of this, and in the absence of any histopathological correlates, this finding was considered incidental and of no toxicological significance.

No statistically significant differences from control for haematology parameters were apparent for males at 500 ppm at the end of treatment or for males at 10000 ppm at the end of the two week treatment-free recovery period.

For females at 10000 ppm, total leucocyte count was lower than control, principally due to lower numbers of neutrophils and lymphocytes; differences for all these parameters attained statistical significance. Values for lymphocytes were lower than the historical control range for two animals at 10000 ppm but only one of these animals showed a total leucocyte count lower than the historical control range. For the control group, neutrophil and total leucocyte count for one female exceeded the historical control. Overall, in the absence of any histopathological correlates, the decrease in these haematology parameters was considered unlikely to be of any toxicological significance.

No statistically significant differences from control for haematology parameters were apparent for females at 500 or 2500 ppm.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
For males at 2500 and 10000 ppm, lower mean total cholesterol levels attained statistical significance when compared to control. Group mean values for the treated animals were close to the historical control mean, while the mean control value exceeded the historical control range; the differences observed were, therefore, considered to be incidental and reflect atypically high control values rather than any effect of treatment.
At 10000 ppm, higher blood chloride levels for males attained statistical significance when compared to control; all individual values at 10000 ppm were within the historical control range and the mean value at 10000 ppm was close to the historical control mean. In isolation, and in the absence of any histopathological correlates, this finding was considered to be of no toxicological significance.

For males at all dosages, inorganic phosphorus levels were lower than control but, although differences attained statistical significance, there was no dosage relationship. Values for all treated animals were within the historical control range and the group mean values were close to the historical control mean. In the absence of any supporting histopathological findings, this finding was considered to be unrelated to treatment and to be of no toxicological significance.

No statistically significant differences from control for blood chemistry parameters were apparent for recovery males at 10000 ppm.
For females at all dosages, mean billirubin levels were lower than control but, although differences attained statistical significance, there was no dosage relationship. The mean values for all groups were higher than the historical control mean but the control mean also exceeded the historical control range. A decrease in billirubin levels, in the absence of any effects on erythrocyte parameters, is unlikely to indicate an adverse effect of treatment. This finding was, therefore, considered to be of no toxicological significance and to reflect atypically high control values.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Assessment of the animals in an open arena did not reveal any adverse effects of treatment at 500, 2500 or 10000 ppm.

Functional Performance Tests: Functional performance, as assessed by measurement of grip strength and motor activity did not indicate any adverse effect of treatment at 500, 2500 or 10000 ppm.
At 10000 ppm, higher male fore limb grip strength during test 1 attained statistical significance compared with control. No further statistically significance differences were observed during the remainder of the test and, in isolation this finding was considered incidental and of no toxicological significance.

Sensory Reactivity Assessments: Sensory reactivity to different stimuli (auditory, visual and proprioceptive) did not indicate any adverse effect of treatment at 500, 2500 or 10000 ppm.


Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
At 10000 ppm, lower absolute and body weight relative liver weights for both sexes attained statistical significance when compared with control. No similar statistically significant decrease was apparent for males at this dosage following the two week recovery period and, in the absence of any evidence of histopathological change, this finding was considered to be of no toxicological significance. Additionally for females at 10000 ppm, higher absolute and body weight relative kidney weights attained statistical significance compared with control; again there was no accompanying histopathological change observed and this finding was therefore considered to be of no toxicological significance.

For males at 10000 ppm and females at all dosages, lower spleen weights attained statistical significance compared with control. There was no statistically significant decrease apparent for recovery males at 10000 ppm at the end of the treatment-free recovery period and, in the absence of any evidence of histopathological change, this finding was considered to be of no toxicological significance.

For males at all dosages, lower absolute and body weight relative prostate and seminal vesicle weights attained statistical significance compared with control. No statistically significant decrease was apparent for recovery males at 10000 ppm at the end of the treatment-free recovery period. In the absence of any evidence of histopathological change and no adverse effect of fertility indicating any functional deficit, this finding was considered to be of no toxicological significance.

For males at 2500 ppm, lower absolute and body weight relative epididymal weights attained statistical significance compared with control, but no similar decrease was apparent males at 10000 ppm, and this finding was considered incidental and unrelated to treatment.

For recovery males at 10000 ppm following at the end of the two week treatment-free period, absolute and body weight relative thymus and thyroid weights were statistically significantly lower than control. No significant decrease for these organ weights or evidence of histopathological change for these organs were apparent for males at the end of treatment. The lower thymus and thyroid weights for recovery males were therefore considered incidental and unrelated to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Necropsy of adult animals did not indicate any obvious adverse effect of treatment at 500, 2500 or 10000 ppm.
One male and one female at 500 ppm, one male and two females at 2500 ppm and five females at 10000 ppm showed reddened lungs at necropsy following the end of treatment. No similar necropsy findings were apparent for males at 10000 ppm at the end of treatment, although a similar finding was apparent for one male at this dosage following the two week recovery period. While the aetiology of this finding is uncertain, there was no evidence of any histopathological change in the tissues for these animals and, therefore, this finding was considered to be of no toxicological significance.
The incidence of other findings observed for other animals was unremarkable and considered to be of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The test item produced no histological evidence of toxicological properties in the organs and tissues examined.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
2 500 ppm
Based on:
dissolved
Sex:
male/female
Basis for effect level:
water consumption and compound intake

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Body weight gain

   days 1 -43  days 43 -57  days 1 -57
 control  90.1  12.0  97.6
 500 ppm  93.4 (104)  -  -
 2500 ppm  82.5 (92)  -  -
 10000 ppm  72.6** (81) 26.2* (218)   103.8 (106)

 ( ) = % Control

Water consumption (g/rat/day)

    week -1   week 1  week 2  week 5  week 6
 males  control  31.6  34.1  35.2  37.0  34.7
   500 ppm

 29.7 (94)

 32.0 (94)

 31.8 (91)      

 36.4 (98)

 35.4 (102)

 

 2500 ppm

 31.4 (99)

 28.3 (83)

 26.0 (74)

 31.0 (84)

 30.1 (87)

 

 10000 ppm

 28.5 (90)

 18.5 (54)          

 17.1 (49)

 17.5 (47)   

 16.3 (47)

 females

 control

 21.1

  20.5   

 20.4            

 

 

 

 500 ppm

 20.0 (95)   

 21.1 (103)  

 20.9 (103)       

 

 

 

 2500 ppm

  24.3 (115)

 20.7 (101)

  18.7 (92)       

 

 

 

 10000 ppm

 21.1 (100)    

 12.9 (63)  

  11.4 (56)             

 

 

          ( ) = % Control

Applicant's summary and conclusion

Conclusions:
Most findings observed at 10000 ppm were considered to be of little toxicological significance and were probably influenced, at least in part, by the marked reduction in water consumption due to palatability. However, clear effects on bodyweight gain and food consumption during lactation and gestation preclude this dosage from being a no observed adverse effect level (NOAEL) for the adult animal. The NOAEL for adult toxicity was therefore considered to be 2500 ppm.