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Description of key information

Based on a study with oral exposure for 28 and 90 days (Central Toxicology Laboratory, UK, 2004), the predominant effect of AKD is the induction of generalised tissue inflammation. The inflammations are most likely not a specific response to AKD, but a generic response of rats to administration of higher molecular weight hydrocarbons. The relevance of this effect for humans is questionable, although it would be prudent to assume that the toxicity is relevant to humans and therefore the highest NOAEL is selected as the key value.
The NOAEL for repeated oral uptake of AKD was established at 6.8 mg/kg bw/day in the 90-day rat feeding study.

The findings in the EOGRTS study (Charles River 2021), where male and female rats received oral doses up to 250 mg/kg bw during a 10 week pre-mating period showed very limited inflammatory responses in the adrenals and kidneys of the parental animals at the highest dose. These effects were not considered to be adverse and the NOAEL in this study was set at 250 mg/kg bw. In the F1 generation that was indirectly exposed during gestation and lactation and directly (by gavage) until day 97 post-natal, no adverse effects were noted at any of the dose levels.
The NOAEL for reproduction and developmental toxicity as well as the NOAEL for general toxicity was 250 mg/kg bw.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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.
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
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
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
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
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).
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.
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.
Critical effects observed:
not specified

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
other: generic inflammatory effects

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Summary of studies


 


In a study according to OECD 422 parental animals (Wistar rats) received 100, 350 and 1000 mg/kg bw in cornoil during 2 weeks premating. Males were dosed during mating and to termination on day 29/30 of the study. Females were dosed during mating, gestation and to day 4 of lactation. Litters were weighed and examined on days 1 and 5 postpartum. 


At a dose level of 1000 mg/kg bw in adults there were no mortalities, no clinical changes and no adverse effects on bodyweight or food consumption and no neurotoxic effects. There were no effects on breeding parameters with the exception of a reduced proportion of implantation sites compared to the numbers of corpora lutea. In both sexes there was an increase in total white blood cell count due to increases in large unstained cells, lymphocytes and monocytes and in females there was also an increase in basophils. There were increases in total bilirubin and cholesterol in both sexes and in albumin, total protein and plasma calcium in females. In addition there were increases in some enzymes in both sexes. In both sexes there was an increase in spleen weight and in liver, kidney and ovary weights in females. Macroscopic enlargement of the spleen and lymph nodes was seen in females. Microscopically, inflammatory changes were seen in a variety of tissues in both sexes but the overall incidence, severity and distribution was greater in females. There were no effects on pups.


At a dose level of 350 mg/kg bw in adults there was a reduced proportion of implantation sites


compared to the numbers of corpora lutea. In both sexes there was an increase in total white blood


cell count due to increases in large unstained cells, lymphocytes and monocytes and in females there


was also an increase in basophils. There was an increase in cholesterol, albumin, total protein and plasma calcium in females. In both sexes there was an increase in spleen weight and in liver, kidney and ovary weights in females. Macroscopic enlargement of the spleen and lymph nodes was seen in females. Microscopically, inflammatory changes were seen in a variety of tissues in both sexes but the overall incidence, severity and distribution was greater in females.


At a dose level of 100 mg/kg bw in adults there was a reduced proportion of implantation sites compared to the numbers of corpora lutea. In both sexes there was an increase in total white blood cell count due to increases in large unstained cells, lymphocytes and monocytes and in females there was an increase in basophils. There was an increase in spleen weight and in liver and kidney weights in females. Macroscopic enlargement of the spleen and lymph nodes was seen in females. Microscopically, inflammatory changes were seen in a variety of tissues in both sexes but the overall incidence, severity and distribution was greater in females.


Based on these findings it was concluded that no NOAEL for generic toxicity and effects on reproduction could be derived in this study. The NOAEL for developmental effects is set at 1000 mg/kg bw in absence of any effects on the offspring.


 


In a 90-day toxicity study rats (12/sex per dose) were fed diets containing 0 (control), 65 ,650 or 6500 ppm for at least 90 consecutive days (mean actual dose received ca 6.5, 65 and 650 mg/kg bw).


At a dose level of 650 mg/kg bw in the diet a reduction in bodyweights from weeks 8 and 6 in males and females respectively was reported. 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 aminotransferase and aspartate aminotransferase 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 a dose level of 65 mg/kg bw in the diet there were similar findings, but of lesser extent than in the high 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 mainly in the lymph nodes that were investigated with a reduction of severity of these changes compared to the top dose group.


At 6.5 mg/kg bw in the diet there were no changes of biological or toxicological significance


when compared to the controls.


The NOAEL in this study was considered equivalent to 6.5 mg/kg bw (6.3 and 6.8 mg/kg bw for males and females respectively).


 


In the EOGRTS study according to OECD 443 rats received 0, 10, 50 and 250 mg/kg bw in 1% CMC with 0.1% Tween 80 during 10 weeks pre-mating. Males were dosed during mating and females were dosed during mating, gestation and lactation. The F1 animals were dosed from weaning day 21 post-natal until day 89-97 (3 cohorts)


No adverse effects were observed up to the highest dose level tested in the F0 generation (250 mg/kg/day).


There were no treatment related effects on mortality/moribundity, clinical signs, food consumption, estrous cycle, haematology, clinical pathology including measurement of thyroid hormones (total TSH and T4 levels), urinalysis, gross necropsy findings, sperm analysis and organ weights.


Males at 10, 50 and 250 mg/kg/day showed a minor lower weight gain, the duration of which appeared dose-dependent, but the effects were limited to a decrease of <10% of controls and at the end of the treatment period overall weight gain was similar to controls. There were no changes in white blood cell count.


Possible test item-related microscopic findings consisted of increased incidences of inflammatory cell infiltrates in the kidneys of both sexes, adrenal gland of females and eyes of males at 50 and/or 250 mg/kg/day. These effects were seen at relatively low degrees, in few cases slight. Occurrence of the same findings in the concurrent control group and absence of any additional related degenerative or proliferative findings, led to the conclusion that these effects are non-adverse.


No test item-related changes were noted in any of the developmental parameters investigated up to the highest dose level tested (250 mg/kg/day) during the gestation and lactation phases.


There were no treatment related effects on pup mortality, gestation index and duration, post-implantation survival index, live birth, viability and weaning indices, parturition, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, pup clinical signs, pup body weight, anogenital distance, areola/nipple retention, thyroid hormone levels of pups (T4 of PND 4 pups,  T4 and TSH of PND 22 pups), macroscopic examination and brain, thymus and spleen weight of pups sacrificed at the end of the lactation period.


For the F1 as dosed after weaning no developmental toxicity was observed up to the highest dose level tested (250 mg/kg/day).


No treatment-related effects on F1-animals were recorded for mortality, clinical signs, body weight and food consumption, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrus, time between vaginal opening and first estrus, length and regularity of the estrous cycle, sperm motility, sperm concentration and morphology, haematology, coagulation parameters, clinical pathology, total T4 and TSH levels, urinalysis, splenic lymphocyte subpopulation data (other than NK-cell counts), follicular and corpora lutea count, macroscopy, organ weights and stage-dependent qualitative evaluation of spermatogenesis in the testis).


At 250 mg/kg/day, mean body weights of males and females were slightly lower between Weeks 3 and 6 or Weeks 2 and 7 for males and females, respectively. These changes were minor and body weights became similar to control values as treatment progressed.


Splenic lymphocyte subpopulation analysis recorded higher relative NK-cell counts for Cohort 1A males at 250 g/kg/day. Since the higher NK-cell counts occurred in absence of corroborative findings indicative of an affected immune system, this shift was considered not to represent an adverse immunological effect.


A treatment related increase in macrophage aggregates of the mesenteric lymph node was seen at 50 mg/kg bw in females and 250 mg/kg bw in both sexes, correlated with a higher lymph node weight in males at 250 mg/kg bw. The effect was considered non-adverse.


Based on the findings the NOAEL for generic toxicity (F0 and F1), reproduction toxicity and developmental toxicity is 250 mg/kg bw. Some (adverse) inflammatory changes were observed microscopically in a variety of tissues, but these occurred at a higher dose level than in previous


toxicity studies, which may be related to differences in study design and/or the choice of the vehicle.


 


Conclusion


 


Based on the effects seen in the 3 studies, a generic inflammatory response could be considered as a non-specific adverse effect of the substance. It cannot be fully excluded that this is an effect as is seen in rats after administration of higher molecular weight hydrocarbons[1]. An influence of the vehicle and the test design (gavage versus diet) on the study outcome can also not be excluded. However, the overall picture from the three studies shows inflammation is the critical effect. It is unclear whether this effect is relevant for humans.


The inflammation and effects on white blood cells as seen at 65 mg/kg bw in the 90-day study were minor. The dosing regimen selected in the OECD 422 study did not lead to a NOAEL for generic toxicity and inflammatory effects were seen at the lowest dose, 100 mg/kg bw. In the EOGRTS study no effects were seen any of the dose levels, although at 250 mg/kg bw non-adverse effects related to inflammation were seen.


It has to be noted that the 90-day study is performed with non-pregnant females, while in the EOGRTS and repeated dose reproduction screening study the females had been pregnant before necropsy and assessment of the critical parameters. It is generally accepted that pregnant females are likely to be more susceptible to effects of substances than non-pregnant females. The additional information from the F1 animals as treated in the EOGRTS that were never pregnant is helpful. No adverse effects were noted in these animals up to 250 mg/kg bw. This is considered to be indicative for minor differences in reaction to the substance between previously pregnant and never pregnant rats.


As stated before the uptake of the substance or its hydrolysis products (see toxicokinetic assessment) could be altered due to the use of different vehicles. In general an aquatic vehicle is preferred for toxicity testing (see OECD 443 guideline) and in the older two studies there have been considerations to deviate from this preferred vehicle (not being a technical or analytical issue).


As the exposure duration in the OECD 422 study is relatively short, this study is not further considered in the derivation of a NOAEL. Both other studies have comparable exposure durations.


Summarizing (see table) we can state that no effects were observed at 50 mg/kg bw, while at 65 mg/kg bw minor inflammatory effects were seen. The difference in the design of both studies (OECD 408 and 443) makes a comparison complicated. There is a clear tendency of adverse effects at and above 65 mg/kg bw, but nothing is reported below that dose level. Therefore in a weight of evidence approach it is proposed to set the NOAEL for the substance at 50 mg/kg bw based on sub-chronic exposure.


 


 






























































































































Dose mg/kg bw



6.5$



10



50



65$



100



250



350



650$



1000



BW



M ↓6%



 



 



M ↓8%



 



M ↓6% (F0)


 



 



M ↓11%


F ↓7%



M ↓5%


M(BWG)


↓11%



Effects on white blood cells



 



 



 



F  WBC↑ ns



WBC↑ >25 %*



 



WBC↑ >35 %*



WBC↑>80%*



WBC↑ >35 %*


(mainly LYMPH)



Lymph nodes histiocytosis/macrophage infiltrate



 



 



(F#)



M+F



M+F



(M+F#)



M+F



M+F



M+F



Kidneys inflammatory cell infiltrate



 



 



 



F



 



M+F (minimal)



M+F ?



F



M+F ?



Liver inflammatory cell infiltrate



 



 



 



 



F



 



M+F



M+F



M+F



Adrenals inflammatory cell infiltrate



 



 



 



 



 



F



 



M+F



 



Study type



90 day diet



OECD 443


Gavage



OECD 443 gavage



90-day diet



OECD 422 gavage



OECD 433 gavage



OECD 422 gavage



90-day diet (once)



OECD 422 Gavage



Study duration



13 wks



10 wks



10 wks



13 wks



Ca 4 wks



10 wks



Ca 4 wks



13 wks



Ca 4 wks



Vehicle



Corn oil



CMC



CMC



Corn oil



Corn oil



CMC



Corn oil



Corn oil



Corn oil



$ females never been pregnant


*effect in females much stronger


# effect in F1, considered non adverse


 


 


[1] It would appear that the generalised inflammations are a specific response to administration of AKDs, but a review of pertinent toxicology literature (Baldwin et al., 1992, Firrioli et al., 1995, Smith et al., 1996, Nash et al., 1996, Halladay, et al., 2002, Scotter et al., 2003) suggests that these represent a generic response of rats to administration of higher molecular weight hydrocarbons, i.e. mineral hydrocarbon oils and waxes. Depending on the viscosity and chain length of the paraffins the observed effects included organ weight changes of the mesenteric lymph nodes, spleen, liver and kidney as well as lesions of the mesenteric lymph nodes, liver and spleen (these findings being more marked in females). For AKDs inflammatory changes were seen in these organs, together with increased splenic extramedullary haematopoesis in the studies using corn oil as a vehicle but to a lesser extend when using an aqueous vehicle. Hydrocarbons distribute primarily to the liver, spleen, mesenteric lymph nodes and fat pads.


 

Justification for classification or non-classification

Based on the information available the substance is not classified for toxicity after repeated dosing according to CLP.