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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 June 2003 - 12 May 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed under GLP and accoding to internationally accepted guidelines. No Certificate of Analysis included in the report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agrochemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000. Test method: 90 days oral toxicity, code number 2-1-9.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-Oxetanone, 3-C12-16-alkyl-4-C13-17-alkylidene derivs.
EC Number:
284-932-5
EC Name:
2-Oxetanone, 3-C12-16-alkyl-4-C13-17-alkylidene derivs.
Cas Number:
84989-41-3
Molecular formula:
C32H60O2 / C34H64O2 / C36H68O2
IUPAC Name:
(4E)-4-(C13-C17)alkylidene-3-(C12-C16)alkyloxetan-2-one
Details on test material:
Name: Aquapel® 364
Source: Hercules Ltd., Salford, UK
Colour: Amber
Physical state: Waxy solid
Batch reference number: 3LP1456
CTL test substance reference number: Y09622/003
Purity (% w/w): 90.1% (This is given in:- HERCULES Technical Service Work Report: No. TSWR BLD 00-448
Dated 23 March 2001)
Storage conditions: Ambient temperature in the dark
Expiry: December 2006

No Certificate of Analysis included in the report.

Test animals

Species:
rat
Strain:
other: Alpk:APfSD (Wistar-derived)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rodent Breeding Unit, Alderley Park, Macclesfield, Cheshire, UK
- Age at study initiation: 4 - 5 weeks old
- Weight at study initiation: males 120 - 145g, females 100 - 125g
- Fasting period before study: not applicable
- Housing: up to 5 animals per sex per cage initionally and in fours in experimental groups.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70. The relative humidity exceeded the nominal (maximum 74%) on 4 days.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 12 June 2003 To: 12 May 2004

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): once
- Mixing appropriate amounts with (Type of food): The diets for groups 1 to 3 (control, 65 and 650 ppm diets) were prepared in 30 kg batches using 2x1kg premixes, each prepared by mixing an appropriate amount of test substance suspended in 150ml corn oil with milled CT1 diet. The test substance was suspended in corn oil (CTL Reference Number: Y00790/014), by warrning to a nominal 50°C in a water bath prior to inclusion in the dietary pre-mix. The amount of test substance in both premixes was made up to a total of 30kg diet. The 6500 ppm diet was prepared in 30kg batches using 3 premixes. Each premix was prepared by mixing 65g of test substance suspended in 100ml corn oil with 1000 g of milled CT1 diet. Each premix was divided into 3 aliquots. Each aliquot was mixed with a further 600 g of milled CT1 diet. The amount of test substance in all 9 aliquots was made up to a total of 30 kg diet.
- Storage temperature of food: The prepared diets were dispensed into glass jars, which were stored in trays. The test diets were stored in the freezer (approximately -20°C).


VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0.975 g/ 300g or 9.75 g / 300 g or 65.0 g /300 g = 0.325%, 3.25% or 21.67%
- Amount of vehicle (if gavage): not applicable
- Lot/batch no. (if required): Y00790/014
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from all dietary levels (including controls) were taken prior to the start of the study and on two occasions during the study and analysed quantitatively for the test substance. Prior to feeding the experimental diets, the homogeneity of the test substance in CT1 diet was determined by analysing samples from the low and high dose levels and the chemical stability of the test substance in diet was determined at these same dose levels over a period of up to 10 days at room temperature and up to 39 days in the freezer (approximately -20°C). Samples were analysed by HPLC.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
Continuously in the feed
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 (control), 65, 650 or 6500 mg/kg
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Mean values for males were: 6.3, 63.4 and 645.0; mean values for females were 6.8, 69.6 and 690.6 mg Aquapel/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: 28 day range finding study at the same laboratory (Report Number: CTL/RR0906/Regulatory/Report).
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): no data
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table were included: no data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: The bodyweight of each rat was recorded immediately before feeding of the experimental diets commenced, on days 2 to 8 and weekly thereafter throughout the study and on the day of termination.

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined pre-experimentally and of the control and high dietary concentration group during the week prior to termination.
- Dose groups that were examined: see above

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (identity), all rats were killed by over exposure to halothane Ph.Eur. vapour.
- Animals fasted: No
- How many animals: all animals
- Parameters examined: haemoglobin, haematocrit, red blood cell count, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration platelet count, total white cell count, differential white cell count

Clotting measurements consisting of prothrombin time and activated partial thromboplastin time with kaolin were made on samples of blood collected in tubes containing trisodium citrate as an anticoagulant. Blood cell morphology, including a differential white blood cell count was assessed by automated methods for all animals. Manual blood films were prepared and analysed as necessary.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: No
- How many animals: all animals
- Parameters examined: The following measurements were made on the plasma from blood samples collected into tubes containing lithium heparin as an anticoagulant: urea, creatinine, glucose, albumin, total protein, cholesterol, triglycerides, total bilirubin, creatine kinase activity, alkaline phosphatase activity, aspart ate aminotransferase activity, alanine aminotransferase activity, gamma-glutamyl transferase activity, calcium phosphorus (as phosphate), sodium, potassium, chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: one week prior to termination. Samples were collected over a period of 16- 18 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume, colour, appearance, specific gravity, pH, glucose, ketones, protein, bilirubin, blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in week 13
- Dose groups that were examined: all animals
- Battery of functions tested:
a) Assessment of signs of autonomic functions, e.g. lachrymation, salivation, piloerection, exophthalmus, urinary incontinence, diarrhoea, pupillary response to light and ptosis.
b) Description, incidence and severity of any convulsions, tremors or abnormal motor movements, both in the home cage and standard (open) arena.
c) Ranking by severity, the subject's reactivity to general stimuli such as removal from the cage or handling.
d) Ranking by severity, the subject's arousal level or state of alertness during observations of the unperturbed subject in the standard (open) arena.
e) Descriptions and incidence of posture and gait abnormalities observed in the home cage and in the standard (open) arena.
f) Assessment of audition by response to a sudden sound (e.g. clicking fingers).
g) Description and incidence of any unusual or abnorrnal behaviours, excessive or repetitive actions (stereotypes), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose or mouth, and any other observations that may facilitate interpretation of the data.

Locomotor activity was monitored by an automated activity recording apparatus.

OTHER: none
Sacrifice and pathology:
All rats were killed by over exposure to halothane Ph.Eur. vapour followed by exsanguination by cardiac puncture.

GROSS PATHOLOGY: Yes
All animals were examinedpost mortem. This involved an external observation and a detailed internal examination of all organs and structures.

HISTOPATHOLOGY: Yes

The following tissues were examined in situ, removed and examined and fixed in an appropriate fixative:
gross lesions including masses
adrenal gland
aorta
brain (cerebrum, cerebellum and brainstem)
bone marrow (femur)
caecurn
colon
duodenum
epididymis
eyes (retina, optic nerve)
heart
ileum
j ej unum
kidney
larynx
liver
lung
lymph node - cervical
lymph node - hepatic
lymph node - mesenteric
lymph node - pancreatic
mammary gland (females only)
nerve - sciatic
nose
oesophagus
ovary
oviduct
Peyer's patches
pancreas
parathyroid gland
pharynx
pituitary gland
prostate gland
rectum
salivary gland
seminal vesicle
spinal cord (cervical, thoracic, lumbar)
skin
spleen
sternum
stomach
testis
thymus
thyroid gland
trachea
urinary bladder
uterus (including cervix)
voluntary muscle

An additional sample of liver was taken from each animal and stored frozen at -80°C, pending examination for lipid droplets. Tissues for histology were routinely processed, embedded in paraffin wax, sectioned at 5µm and stained with haematoxylin and eosin. The additional liver samples were sectioned at 5µm and stained with Oil Red O.

All processed tissues from the control and high dose groups were examined by light microscopy. In addition, due to findings in the high dose group the following tissues were processed and examined from the low and intermediate dose groups.
adrenal gland
heart
ileum
jejunum
kidney
liver
lung
lymph node - cervical
lymph node - hepatic
lymph node - mesenteric
lymph node - pancreatic
nerve - sciatic
Peyer's patches
pancreas
salivary gland
spleen
stomach
uterus (including cervix)
voluntary muscle
Other examinations:
From all animals at scheduled termination, the following organs were removed, trimmed free of extraneous tissue and weighed:
adrenal glands
brain
epididymides
heart
kidneys
liver
ovanes
spleen
testes
thymus
uterus/cervix
Paired organs were weighed together.
Statistics:
All data were evaluated using analysis of variance andlor analysis of covariance for each specified parameter using the MIXED procedure in SAS (1999).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no test substance related clinical signs in either male or female animals.

BODY WEIGHT AND WEIGHT GAIN
There was an apparent dose related reduction in the bodyweights of males from week 8, with maximum effects of 1%, 8% and 6% at 6500,650 and 65 ppm respectively. However, the control group had mean bodyweights greater than the historical control data from week 6 to 13 whereas all treated groups had mean bodyweight values within the historical control range. Females in the top dose group had consistently statistically significantly lower bodyweights with respect to the controls from week 6 (maximum effect approximately 7%), there were no adverse effects in females at lower doses.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was generally lower in all male treated groups, with a maximum effect of 14% lower in males in the 6500ppm group. However, as for bodyweights, the control group had mean food consumption values greater than the mean historical control data from weeks 2 to 11 inclusive (except weeks 5 and 10) and all treated groups generally had mean food consumption values within the historical control range. There were no consistent effects in females, though females in the 6500ppm group had lower food consumption during some weeks.

FOOD EFFICIENCY
Food utilisation in males was significantly reduced in the 6500ppm group during weeks 5-8, 9- 13 and overall (weeks 1 - 13) and in the group receiving diet containing 650ppm. Aquapel® 364 in the diet, food utilisation was reduced during weeks 5-8 when compared to the controls. Females in the 6500ppm group had a reduced food utilisation in weeks 1 to 4 only.

OPHTHALMOSCOPIC EXAMINATION
No test substance related ophthalmoscopic observations were noted either pre-dose or during the week prior to termination in male or female animals.

HAEMATOLOGY
In males and females fed the diet containing 6500 ppm Aquapel® 364 there were statistically significant reductions in haemoglobin, haematocrit and mean cell haemoglobin. Haemoglobin was also significantly reduced in females fed the diet containing 650ppm Aquapel® 364 and haematocrit was also reduced in this group, but not significantly. Reductions in mean cell volume and platelet count were seen in males in the top dose group and reductions in red blood cell count and mean cell haemoglobin concentration were seen in females at the top dose level. The decrease in red blood cells was slight in both males and females at 6500ppm and was reflected in the small decrease in haemoglobin and associated parameters. The decrease in prothrombin time in the top dose group in males and females is of no pathological significance. The white blood cell, lymphocyte, neutrophil, monocyte, basophil, and large unstained cell counts were significantly increased in both sexes at 6500ppm Aquapel® 364 in the diet, with an increase in monocyte, basophil and large unstained cell counts also seen in females at 650ppm. The rise in white blood cell count was largely due to a rise in lymphocytes, although the majority of the granulocyte series was also raised together with the mononuclear cells.

CLINICAL CHEMISTRY
In males and females administered Aquapel® 364 in the diet at 6500ppm there was a significant increase in alkaline phosphatase, gamma-glutamyl transferase, alanine aminotransferase and aspartate aminotransferase levels. The levels of alanine aminotransferase and aspartate aminotransferase in some animals in the 650ppm group were also affected to a lesser extent. In males there was a slight reduction in triglycerides and total bilirubin in the top dose group. However, the concurrent control value for total bilirubin was exaggerated by a single high value and the mean values in the top dose group were within the range of historical control values. In males there was a slight increase in sodium and chloride levels in the top dose group. Chloride levels in a few males was also slightly higher in the 650ppm group.
In females there was an increase in total protein, cholesterol, creatine kinase, potassium and calcium at the 6500ppm group, with cholesterol also being increased in the 650ppm group and calcium increased at all doses. The mean concurrent control value for total protein was lower than historical controls and mean values from the top dose group were within the historical control range. The potassium levels in the 65 and 650ppm group were within the range of mean values for historical controls.
The changes seen in triglycerides, cholesterol, bilirubin, total protein, creatine kinase, sodium, chloride, potassium and calcium were not consistent between males and females. These findings are not considered to be of pathological significance.

URINALYSIS
There was an increase in urine volume and a decrease in urine specific gravity in both sexes at 6500ppm. In addition urine pH was higher in females at 6500ppm. The small changes in urine parameters are not considered to be of pathological significance.

NEUROBEHAVIOUR
No effects on any of the functional observation battery parameters or motor activity were noted in any of the dose groups (65, 650 and 6500ppm) when compared to the control diet.

ORGAN WEIGHTS
In both sexes at 6500ppm Aquapel® 364 in the diet the weight of the spleen and liver were significantly increased, with an increase in these organ weights in females at 650ppm. The kidneys were also increased in females at 6500 and 650ppm.

GROSS PATHOLOGY
A small number of common spontaneous lesions were observed, non of which were considered to be related to the administration of Aquapel® 364 in the diet.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment related inflammatory changes, were observed in a variety of tissues in animals at 6500 and 650ppm Aquapel® 364 in the diet. These changes included foci of inflammation and histiocytosis. The inflammation consisted predominantly of mononuclear cells, which in the liver, was invariably associated with histiocytic infiltration. Foci of histiocytes were also often seen within the liver sinusoids, unaccompanied by other inflammatory cells. The histocytosis was characterised by accumulation of foamy macrophages of varying sizes, often coalescing.
In males and females administered Aquapel® 364 in the diet at 6500ppm histiocytosis was seen in the lymph nodes (mesenteric, hepatic and pancreatic, spread throughout the cortex and medulla), liver, spleen, jejunum, ileum and Peyer's patch. Inflammation was observed in the liver (hepatitis), lung, kidney (nephritis), heart (degenerative cardiomyopathy), pancreas (pancreatitis), muscle (myositis and mononuclear cell infiltration), adrenal gland, sciatic nerve and stomach. The Oil Red O stain of liver samples showed no differences between the group administered Aquapel® 364 in the diet at a concentration of 6500ppm and the control group.
In males and females administered Aquapel@ 364 in the diet at 650ppm histiocytosis was seen in the mesenteric, cervical, hepatic and pancreatic lymph nodes and Peyer's patch and inflammation was seen in the stomach. In males inflammation was seen in the sciatic nerve and adrenal gland. Effects were more predominantly seen in females, with histiocytosis in the liver, jejunum and spleen and inflammation in the liver (hepatitis), heart, kidney, pancreas and lung. In all other tissues the incidence and severity of any inflammation was similar to the control group.
In the group administered a low dose of Aquapel® 364 in the diet (65ppm) the small number of changes were of similar incidence and severity as those seen in the control group and were considered not to be related to the administration of Aquapel® 364 in the diet.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
6.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No treatment related effects seen at low dose level
Dose descriptor:
NOAEL
Effect level:
6.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
63.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Toxicity characterised by lower bodyweights, food consumption and red blood cell parameters and increases in white blood cell counts, liver enzymes changes and spleen and liver weights and inflammation and histiocytosis were seen in a number of tissues.
Dose descriptor:
LOAEL
Effect level:
69.6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Toxicity characterised by lower bodyweights, food consumption and red blood cell parameters and increases in white blood cell counts, liver enzymes changes and spleen and liver weights and inflammation and histiocytosis were seen in a number of tissues.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The mean achieved concentrations of Aquapel® 364 in each dietary preparation were within 8% of the nominal concentration with the exception of the 65ppm sample prepared on the 21th August 2003 which was within 26% of the nominal. The homogeneity of Aquapel® 364 in diet preparations at concentrations of 65 and 6500ppm, (for batch sizes of 30 and 31kg respectively), was determined and considered satisfactory; The chemical stability of Aquapel® 364 in the experimental diets determined at concentrations of 65ppm and 6500ppm when stored in a freezer, was shown to be acceptable for 32 days and 38 days respectively, covering the period of storage prior to dosing.

Dose rates (based on nominal dietary levels of Aquapel® 364) were calculated in terms of mg Aquapel ® 364kg bodyweight. Mean values for males were: 6.3, 63.4 and 645.0; mean values for females were 6.8, 69.6 and 690.6mg Aquapel ® 364/kg/day.

Applicant's summary and conclusion

Conclusions:
Oral administration of Aquapel® 364 at dietary levels of 6500 and 650 ppm for at least 90 consecutive days resulted in toxicity characterised by lower bodyweights, food consumption and red blood cell parameters and increases in white blood cell counts, liver enzymes changes and spleen and liver weights and inflammation and histiocytosis were seen in a number of tissues. There were no test substance related findings at a dietary level of 65 ppm.
The no observed adverse effect level (NOAEL) for this study was considered to be 65 ppm Aquapel® 364, equivalent to 6.3 and 6.8 mg/kg bodyweight for males and females respectively.
Executive summary:

Study design:

Groups of twelve male and twelve female Alpk:APfSD (Wistar-derived) rats were fed diets containing 0 (control), 65 ,650 or 6500 ppm Aquapel® 364 for at least 90 consecutive days. Clinical observations, bodyweights and food consumption were measured throughout the study. An ophthalmoscopic examination was performed on all animals pre-study and on the control and 6500ppm group in week 12. A functional observation battery, including clinical assessments, measurements of grip strength, time to tail flick, landing foot splay and motor activity were conducted during the last week of the study. Urine samples collected during week 13 were analysed. At the end of the study, the rats were killed and examined post mortem. Cardiac blood samples were taken for clinical pathology, selected organs were weighed and specified tissues were taken for histopathological examination.

Results:

Administration of Aquapel® 364 in the diet at 6500ppm resulted in a reduction in bodyweights from weeks 8 and 6 in males and females respectively. Food consumption and utilisation were reduced in both sexes. Red blood cell parameters were reduced and white blood cell counts, alkaline phosphatase, gamma-glutamyl transferase, alanine aminotrans ferase and asp artate aminotrans ferase levels were increased in both sexes and cholesterol was increased in females when compared to controls. Spleen and liver weights were increased in both sexes and kidney weight increased in females. Treatment related inflammatory changes, were observed in a variety of tissues. These changes included foci of

inflammation and histiocytosis. At an administration level of 650ppm Aquapel® 364 in the diet there were similar findings, but of lesser extent to the 6500ppm dose group. In females, a number of red blood cell parameters were reduced and white blood cell counts, cholesterol levels and liver, spleen and kidney weights were increased. In both sexes, there was an increase in alanine aminotransferase, aspartate aminotransferase levels. In males, there were reductions in bodyweight, food consumption and food utilisation in some weeks. Inflammation and histiocytosis were seen there but there was a reduction in the incidence and severity of these changes compared to the top dose group. When Aquapel® 364 was administered in the diet at 65ppm there were no changes of biological or toxicological significance when compared to the controls.

Conclusion:

Oral administration of Aquapel® 364 at dietary levels of 6500 and 650 ppm for at least 90 consecutive days resulted in toxicity characterised by lower bodyweights, food consumption and red blood cell parameters and increases in white blood cell counts, liver enzymes changes and spleen and liver weights and inflammation and histiocytosis were seen in a number of tissues. There were no test substance related findings at a dietary level of 65 ppm.

The no observed adverse effect level (NOAEL) for this study was considered to be 65 ppm Aquapel® 364, equivalent to 6.3 and 6.8 mg/kg bodyweight for males and females respectively.