Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 22 August 2012 and 04 March 2013. The in-life phase of the study was conducted between 05 September 2012 (first day of treatment) and 20 October 2012 (final necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD 422)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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:
There was a concentration error for the lowest dose group and the pituitary was lost during removal for three animals. See "Deviations from study plan" below for more information.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Paste, grease containing 17.29% Diurea 8 in medicinal white oil
Details on test material:
Description, Identification and Storage Conditions:

Sponsor's identification: Diurea thickener 8 base grease
Description: pale brown paste
Grease base: White mineral oil (Petroleum)
Thickener: 17.29%
Label: Diurea thickener 8 in medicinal white oil
Date received: 23 July 2012
Storage conditions: room temperature in the dark
Expiry date: not supplied

Description, Identification and Storage Conditions of Vehicle

Sponsor's identification: MOL WO M 46 Medicinal white oil
Description: clear colourless liquid
Batch number: 9037038
Label: MOL WO M 46 Medicinal white oil
Batch number: 9037038
Date received: 23 July 2012
Storage conditions: room temperature in the dark
Expiry date: Not supplied

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in MOL WO M 46 medicinal white oil. The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for at least twelve days. Formulations were therefore prepared weekly and stored at 4ºC in the dark.

Samples of each test item formulation were taken and analysed for concentration of Diurea thickener 8 base grease. The results indicate that the prepared formulations were within ± 4% of the nominal concentration.

Test animals

Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST
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. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for five days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 308 to 352g, the females weighed 195 to 219g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and 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.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. 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.) except for paired animals and mated females during gestation and lactation.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd. 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. The temperatures and relative humidity controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively. There were no deviations from these targets.

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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: MOL WO M 46 Medicinal white oil
Details on exposure:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg of MOL WO M 46 medicinal white oil.
Details on mating procedure:
During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper and were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.
Duration of treatment / exposure:
The male dose groups were killed and examined macroscopically on Day 43.

At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Frequency of treatment:
The test item was administered daily.
Details on study schedule:
Chronological Sequence of Study:

i) Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.

iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

v) On completion of the pre-pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.

vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.

ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
375 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Twelve male and twelve female rats per dose, plus a control group of twelve males and twelve females rats.
Control animals:
yes, concurrent vehicle
Details on study design:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

The dose levels were chosen based on the results of previous toxicity work. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item. The results of the study are believed to be of value in predicting the likely toxicity of the test item to man and to screen for potential adverse effects on reproduction.
Positive control:
No data

Examinations

Parental animals: Observations and examinations:
Clinical Observations

All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends (except for females during parturition where applicable). All observations were recorded.

Functional Observations:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each 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 animal using a purpose built arena. The following parameters were observed:

Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behaviour
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper/Hypothermia
Skin colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation

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
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Blink reflex
Startle reflex

Body Weight:
Individual body weights were recorded on Day 1 (prior to dosing) 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.

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.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) 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 observed daily by visual inspection of water bottles for any overt changes.

Intergroup differences did not indicate any need for more formal gravimetric measurements.


Reproduction Screening

Pregnancy and Parturition:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:

i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Laboratory Investigations:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). 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
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
Sperm parameters (parental animals):
No data
Litter observations:
Litter Data:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development:
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum.

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).

All adult animals, 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 were dissected free from fat and weighed before fixaton from five selected males and females from each dose group. Tissues shown with a ♣ were weighed from all remaining animals:

Adrenals
Brain
Epididymides♣
Heart
Kidneys
Liver
Ovaries♣
Pituitary (post fixation)♣
Prostate♣
Seminal vesicles♣
Spleen
Testes♣
Thymus
Thyroid (weighed post-fixation with Parathyroid)♣
Uterus (weighed with Cervix)♣

Histopathology:
Samples of the following tissues were removed from five selected males and females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown with a ♣ were preserved from all remaining animals:

Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland♣
Colon
Duodenum
Epididymides• ♣
Eyes*
Heart
Ileum (including peyer’s patches)
Jejunum
Kidneys
Liver
Lungs (with bronchi) #
Lymph nodes (mandibular and mesenteric)
Mammary gland♣
Muscle (skeletal)
Ovaries♣
Pancreas
Pituitary♣
Prostate♣
Oesophagus
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles♣
Skin (hind limb)
Spinal cord (cervical, mid-thoracic and Gross lesions lumbar)
Spleen
Stomach
Thyroid/parathyroid
Trachea
Testes•♣
Thymus
Urinary bladder
Uterus/Cervix♣
Vagina♣

All tissues were despatched to the histology processing Test Site for processing. The tissues from five selected control and 1500 mg/kg bw/day 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 1500 mg/kg bw/day animals were also processed. In addition, sections of testes and epididymides from all control and 1500 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Microscopic examination was conducted by the Study Pathologist.

* = eyes fixed in Davidson’s fluid.
• = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later.
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative.
Postmortem examinations (offspring):
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Reproductive indices:
Mating Performance and Fertility:
The following parameters were calculated from the individual data during the mating period of the parental generation:

i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:

Mating Index (%) = (number of animals mated/number of animals paired) x 100

Pregnancy Index (%) = (number of pregnant females/number of animals mated) x 100

Gestation and Parturition Data:
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:

Parturition Index (%) = (number of females delivering live offspring/number of pregnant females) x 100
Offspring viability indices:
Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
 
i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
 
% pre–implantation loss = (number of corpora lutea - number of implantati on sites/number of corpora lutea) x100
% post–implantation loss = (number of implantati on sites - total number of offspring born/number of implantati on sites) x 100
 
ii) Live Birth and Viability Indices:
The following indices were calculated for each litter as follows:
 
Live Birth Index (%) = (number of offspring alive on Day 1/number of offspring) x 100
Viability Index (%) = (number of offspring alive on Day 4/number of offspring alive on Day 1) x 100
 
iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula: (number of male offspring/total number of offspring) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

Adult Responses

Mortality:
There were no unscheduled deaths.

Clinical Observations:
No toxicologically significant clinical observations were detected.

One female treated with 1500 mg/kg bw/day and one male treated with 375 mg/kg bw/day had red/brown staining around the eyes on Days 16 and Day 36 respectively. In isolation and in the absence of a true dose related response these findings were considered to be of no toxicological importance.

Functional Observations

Behavioural Assessments:
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.

All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Functional Performance Tests:
There were no toxicologically significant changes in functional performance.

Females from all treatment groups showed a statistically significant reduction in the final 20% of motor activity. A statistically significant reduction in overall motor activity was also evident in females treated with 750 or 375 mg/kg bw/day and females treated with 375 mg/kg bw/day also showed a statistically significant reduction in overall mobile activity. In absence of true dose related responses or any supporting clinical observations to suggest a neurotoxic effect, the intergroup differences were considered to be of no toxicological importance.

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

All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Body Weight:
There were no treatment related effects in body weight development.

Statistical analysis of the data did not reveal any significant intergroup differences.

Food Consumption:
No adverse effect on food consumption or food efficiency was detected in treated animals.

Females treated with 1500 or 750 mg/kg bw/day showed a statistically significant increase in food consumption during the final week of gestation. An increase in this parameter is considered not to be an adverse effect of treatment and therefore the intergroup differences were considered not to be toxicologically significant.

Water Consumption:
No adverse effect on water consumption was detected.

Daily visual inspection of water bottles revealed no intergroup differences.

Reproductive Performance

Mating:
There were no treatment-related effects on mating or in conception rates for treated animals.

Fertility:
There were no treatment-related effects on fertility.

Gestation Length:
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. Gestation lengths were between 22 and 23½ days.

Laboratory Investigations

Haematology:
No toxicologically significant effects were detected in the haematological parameters examined.

Males from all treatment groups showed a statistically significant reduction in neutrophil and eosinophil counts. In the absence of a true dose related response or any histology correlates the intergroup differences were considered not to be of toxicological significance. Animals of either sex treated with 1500 mg/kg bw/day showed a statistically significant increase in clotting time. The majority of individual values were within the normal range for rats of the strain and age used therefore the intergroup differences were considered not to be of toxicological significance.

Blood Chemistry:
No toxicologically significant effects were detected in the blood chemical parameters examined.

Males treated with 1500 mg/kg bw/day showed a statistically significant reduction in creatinine. Females from this treatment group showed a statistically significant increase in glucose and a statistically significant reduction in albumin and albumin/globulin ratio. The majority of individual values were within the normal range for rats of the strain and age used and in the absence of any histology correlates the intergroup differences were considered not to be of toxicological significance. Females treated with 375 mg/kg bw/day showed a statistically significant increase in aspartate aminotransferase. In the absence of a true dose related response or any histology correlates the intergroup difference was considered not to be of toxicological significance.

Necropsy:

Adults - No toxicologically significant macroscopic abnormalities were detected.

One male treated with 375 mg/kg bw/day had increased pelvic space in the right kidney. Another male from this treatment group had a small right seminal vesicle. One female treated with 375 mg/kg bw/day had reddened lungs at necropsy. In the absence of any true dose related responses or any associated histopathological changes, the incidental findings were considered not to be of toxicological significance.

Organ Weights:
No toxicologically significant effects were detected in the organ weights measured.

Males treated with 1500 or 750 mg/kg bw/day showed a statistically significant increase in adrenal weight, both absolute and relative to terminal body weight. Males from all treatment groups showed a statistically significant increase in thyroid weight, both absolute and relative to terminal body weight whilst females treated with 375 mg/kg bw/day showed a statistically significant reduction in absolute and relative thyroid weight. In the absence of any true dose related responses or any supporting histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Histopathology:
No treatment related microscopic abnormalities were detected.

There was a higher degree of alveolar macrophages containing foamy cytoplasm in the lungs of control animals, occasionally accompanied by interstitial inflammation, that was indicative for aspiration of the vehicle (Medicinal White Oil). In high dose animals, the foamy alveolar macrophages were present however at a lesser incidence and severity grade. Therefore, the test item did not exacerbate this finding in the lungs when inspired.

All remaining microscopic findings recorded were within the range of normal background lesions which may be recorded in this study type and in the animals of this strain and age.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 500 mg/kg bw/day
Based on:
test mat.
Remarks:
Grease containing 17.29% Diurea 8 in medicinal white oil
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A statistically significant increase in body weight on Day 1 post partum was evident in female offspring from litters treated with 1500 and 750 mg/kg bw/day. Male offspring from litters treated with 750 mg/kg bw/day also showed a statistically significant increase in body weight on Day 1 post partum. An increase in body weight is considered not to be an adverse effect of treatment therefore the intergroup differences were considered not to be of toxicological importance.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Litter Responses:
In total twelve females from the control, 375, 750 and 1500 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability:
Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum were comparable to controls.

Females treated with 1500 mg/kg bw/day showed a statistically significant increase in post-implantation loss when compared to control females. Only one of the individual values were outside of the normal range and in absence of any associated changes in the number of implantation sites or the number of offspring born, the intergroup difference was considered not to be toxicologically significant.

Offspring Growth and Development:
There were no toxicologically significant effects detected.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, no milk in stomach, physical injury, found dead or missing, were considered to be low incidence findings commonly observed in offspring in studies of this type and were considered unrelated to test item toxicity.

A statistically significant increase in body weight on Day 1 post partum was evident in female offspring from litters treated with 1500 and 750 mg/kg bw/day. Male offspring from litters treated with 750 mg/kg bw/day also showed a statistically significant increase in body weight on Day 1 post partum. An increase in body weight is considered not to be an adverse effect of treatment therefore the intergroup differences were considered not to be of toxicological importance.

Pathology

Necropsy:

Offspring - No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
body weight and weight gain
gross pathology

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of Diurea thickener 8 base grease to rats by gavage, at dose levels of 375, 750 and 1500 mg/kg bw/day did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1500 mg/kg bw/day.

The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 1500 mg/kg bw/day.