Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11/01/12 to 11/12/12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N''-(4-methyl-m-phenylene)bis[N',N'-dimethylurea]
EC Number:
241-523-6
EC Name:
N,N''-(4-methyl-m-phenylene)bis[N',N'-dimethylurea]
Cas Number:
17526-94-2
Molecular formula:
C13H20N4O2
IUPAC Name:
3-[3-(dimethylcarbamoylamino)-4-methylphenyl]-1,1-dimethylurea
Constituent 2
Chemical structure
Reference substance name:
3-[3-(dimethylcarbamoylamino)-2-methylphenyl]-1,1-dimethylurea
Cas Number:
17607-23-7
Molecular formula:
C13H20N4O2
IUPAC Name:
3-[3-(dimethylcarbamoylamino)-2-methylphenyl]-1,1-dimethylurea
impurity 1
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Water
impurity 2
Reference substance name:
unknown
IUPAC Name:
unknown
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for five days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 304 to 357g, the females weighed 189 to 222g, and were approximately twelve weeks old.

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

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. 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., Cheshire, UK) except for mated females during gestation and lactation.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK, Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system and print-outs of hourly temperatures and humidities are included in the study records. The temperatures and relative humidity controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively. Short term deviations from these targets were considered not to affect the purpose or integrity of the study.

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:
polyethylene glycol
Details on exposure:
Prior to preparation of the test item formulations, vehicle determination has been performed at Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Harlan Laboratories Ltd., Project Number: 41104305). Polyethylene glycol 400 (Sigma-Aldrich Company Ltd., Poole, UK) has been considered a suitable vehicle for use in this study. The test item was prepared at the appropriate concentrations as a suspension in the selected vehicle. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twenty one days. Formulations were prepared weekly or fortnightly and stored at 4ºC in the dark.

Samples of each test item formulation were taken and analysed for concentration of TDI-Urone at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 3% of the nominal concentration.


VEHICLE
Concentration in vehicle:
0, 25, 75 and 250 mg/ml

- Amount of vehicle (if gavage):
4 ml/kg

Details on mating procedure:
- M/F ratio per cage:
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

- Length of cohabitation:
Maximum of 14 days

- Proof of pregnancy:
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).

- After successful mating each pregnant female was caged:
Mated females were housed individually during the period of gestation and lactation.

- Any other deviations from standard protocol:
No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of TDI-Urone in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

The test item formulations were diluted with mobile phase to give a final, theoretical test item concentration of approximately 0.01 mg/ml.

Standard solutions of test item were prepared in mobile phase at a nominal concentration of 0.01 mg/ml.

The standard and sample solutions were analysed by HPLC using the following conditions:
HPLC: Agilent Technologies 1200, incorporating autosampler
and workstation
Column: Eclipse XDB C18 (150 x 4.6 mm id)
Mobile phase Acetonitrile :water (30:70 v/v)
Flow-rate 1 ml/min
UV detector wavelength: : 230 nm
Injection volume: 25 µl
Retention time : ~ 1.7 to 2.5 mins

The test item formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for twenty one days.

Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed within two days of preparation.
Duration of treatment / exposure:
Male dose groups: 43 days
Female dose groups: 5 days post-partum
Frequency of treatment:
Daily
Details on study schedule:
Groups of ten male and ten 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.

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

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

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.

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.

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.

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

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.

Blood samples were taken from five randomly selected females from each dose group at termination 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. One female which did not show positive evidence of mating was also killed and examined macroscopically on Day 25 post coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control (concurrent vehicle)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
intermediate dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
high dose
No. of animals per sex per dose:
10 animals per sex per dose (including control).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen based on the results of a preliminary range-finder (14 days) performed.

- Rationale for animal assignment (if not random):
Random.

Positive control:
Not applicable

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).


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

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 during the final week of treatment. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).

The following parameters were observed:
Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach


Body Weight
Individual body weights were recorded on Day 1 (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 measured daily throughout the study (with the exception of the pairing phase).
Oestrous cyclicity (parental animals):
Each pregnant female was observed at approximately 08:30, 12:30 and 16:30 hours and around the period of expected parturition. Observations were carried out at approximately 08:30 and 12:30 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
Sperm parameters (parental animals):
No data
Litter observations:
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)
Postmortem examinations (parental animals):
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 Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot. Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+) Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids

Pathology
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. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. The female which failed to mate was killed on Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964) (see section 8. References).
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed along with Cervix), Pituitary.

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Oesophagus, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides•, Skin (hind limb), Eyes*, Spinal cord (cervical, mid-thoracic and lumbar), Gross lesions, Heart, Spleen,
Ileum (including peyer’s patches), Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trachea, Liver, Testes•, Lungs (with bronchi) #, Thymus, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary gland, Uterus/Cervix, Muscle (skeletal), Vagina

All tissues were despatched to the histology processing Test Site. The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy 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 1000 mg/kg bw/day animals were also processed. In addition, sections of testes and epididymides from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Since there were indications of treatment-related changes in the liver of high dose males, examination was subsequently extended to include similarly prepared sections of the liver from all ten animals per sex from the low and intermediate groups.

Postmortem examinations (offspring):
Not examined
Statistics:
Please refer to section below "Any other information on materials and methods"
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:
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).

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 implantation sites) / Number of corpora lutea] x100

Post–implantation loss = [(Number of implantations ites - Total number of offspring born)/ Number ofimplanattion sites] x 100


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 born) x 100

Viability Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100


Sex Ratio (% males)
Sex ratio was calculated for each litter 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 generation)

General toxicity (P0)

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Mortality
There were no unscheduled deaths during the study.


Clinical Observations
Incidents of increased salivation were recorded in animals of either sex treated with 1000 mg/kg bw/day from Day 16 onwards. This finding is commonly observed following the oral administration of an unpalatable and/or locally irritant test item formulation and, in isolation, is considered not toxicologically significant.

No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.


Functional Observations
Behavioural Assessments
There were no treatment related effects detected in the behavioural parameters measured.

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 the functional parameters measured.

At 1000 mg/kg bw/day males showed statistically significant increase in the overall mobility (p<0.01) and females showed statistically significant reduction in overall activity (p<0.05) and statistically significant increase in hind limb grip strength measurement (p<0.05) when compared to control values. Increased mean values in overall mobility detected in males and reduced in overall activity recorded in females were within normal ranges for Functional Performance Assessments in the rats of the age and strain employed. Increase in grip strength measurement evident in females was recorded in one test out of three repetitions only. In view of the facts above and in the absence of similar findings detected in other functional observation parameters measured, in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be deemed to irregular control values or fortuitous and, therefore, of no toxicological significance.

No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.


Sensory Reactivity Assessments
There were no treatment related changes in sensory reactivity scores for treated animals when compared to controls.

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.


Body Weight
There were no obvious adverse effects on body weight development detected for treated animals in comparison to controls.

Females treated with 1000 or 300 mg/kg bw/day showed statistically significant reduction in body weight gains (p<0.01) during Week 2 of maturation when compared to control values. This effect was not present for the remaining phases of the study hence of limited significance.
No such effects were detected in all treated males and females treated with 100 mg/kg bw/day.


Food Consumption
No obvious adverse effect on dietary intake was detected in all treated animals when compared to controls throughout the treatment period.

Food efficiency values were slightly lower in females treated with 1000 or 300 mg/kg bw/day during Week 2 of maturation phase when compared to control values. This reduction occurred as a consequence of body weight gains reduction during this period of the study.

No such effects were detected in all treated males and females treated with 100 mg/kg bw/day.


Water Consumption
No adverse effect on water consumption was detected.

Females treated with 300 mg/kg bw/day showed statistically significant (p<0.05 to p<0.001 respectively) reduction in water consumption between Weeks 1 to 3 of gestation. In the absence of dose related response or similar effects in animals treated with 1000 mg/kg bw/day this finding was considered of no toxicological importance.

No such effects were detected in animals of either sex treated with 1000 or 100 mg/kg bw/day and in males treated with 300 mg/kg bw/day.


Reproductive Performance

Mating
There were no treatment related effects on mating performance at any of the dose levels investigated. The majority of animals mated within the first five days of pairing (i.e. at the first oestrus opportunity).

One control female (No. 14) failed to mate with control male partner (No. 4) following fourteen days of co-housing. In the absence of treatment this finding is considered to represent normally occurring biological variance.


Fertility
There were no treatment related effects detected on fertility indices and pregnancy rate in treated animals when compared to controls.


Gestation Length
There were no treatment related effects detected in the length of gestation between control and treated groups. All females showed a gestation length between 22 and 23½ days.

Statistical analysis of the gestation length data did not show any significant intergroup differences.


5.4 Laboratory Investigations
5.4.1 Haematology
Group mean values and standard deviations for test and control group animals are given in Table 18 (statistically significant differences are indicated). Individual data are given in Appendix 17 and Appendix 18.
There were no toxicologically significant effects detected in the haematological parameters investigated.
Males treated with 1000 mg/kg bw/day showed a statistically significant increase in platelet count (p<0.05) when compared to controls. The significance achieved (probability value) was minimal and in the absence of dose related response and similar findings detected in females this increase was considered not toxicologically significant.
5.4.2 Blood Chemistry
Group mean values and standard deviations for test and control group animals are given in Table 19 (statistically significant differences are indicated). Individual data are given in Appendices 19 and 20.
Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in urea concentration (p<0.01) and a statistically significant increase in total cholesterol concentration (p<0.01) when compared to controls. The importance of these findings is ambiguous as there are no correlations present i.e. with histopathological changes.
No toxicologically significant effects were detected in males treated with 1000 mg/kg bw/day and animals of either sex treated with 300 or 100 mg/kg bw/day.
Males from all treatment groups showed statistically significant reductions in urea (p<0.05) and total bilirubin concentration (p<0.05 at 300 and 100 mg/kg bw/day and p<0.01 at 1000 mg/kg bw/day). The majority of individual values and mean group values were within the normal ranges for the strain and age of the rats used. In the absence of dose relationship (urea findings) and in the view of the fact that control values were at the high end of the normal ranges due to naturally occurring biological variation, these findings were considered to represent limited toxicological importance.
Females treated with 1000 mg/kg bw/day showed statistically significant reductions in potassium (p<0.01), calcium (p<0.05) and inorganic phosphorus (p<0.05) concentration when compared with control values. However, the majority of individual values and mean group values were within normally expected range and differences recorded for potassium and calcium concentration can be attributable to high control values. These control values are at the high end of the historical control data range due to naturally occurring biological variation. These reductions were therefore considered to be of no 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.

Adults
There were no treatment related macroscopic abnormalities detected.

One male (No. 26) treated with 100 mg/kg bw/day had small testes and epididymides at necropsy. In the absence of dose related response or any treatment related histopathological changes evident for this male, these macroscopic findings were considered to have arisen incidentally and were considered to be unrelated to treatment.

One control male had reddened lungs (No. 5) and a further control male (No. 6) had increased pelvic space of the right kidney at necropsy. In the absence of treatment these findings are considered to have arisen incidentally.

No such findings were detected in all treated females and in males treated with 300 or 1000 mg/kg bw/day.


Organ Weights
Males treated with 1000 or 300 mg/kg bw/day showed statistically significant increase in liver weight (p<0.01 and p<0.05 respectively), both absolute and relative to terminal body weight. This finding was correlated to histopathological changes consisting of centrilobular hepatocellular hypertrophy of the liver.

No toxicologically significant effects were detected in females treated with 1000 or 300 mg/kg bw/day and in animals of either sex treated with 100 mg/kg bw/day.

Males treated with 1000 mg/kg bw/day showed a statistically significant increase in adrenals weight (p<0.05) and reductions in heart and pituitary weight (p<0.05) whilst females treated with 300 mg/kg bw/day showed statistically significant reduction in thyroid weight (p<0.01), both absolute and relative to terminal body weight. In the view of the fact that the majority of individual values were within normal ranges for rats of the strain and age used or in the absence of either dose relationship (heart and thyroid findings) or any histopathological correlates, it is considered of no toxicological importance.


Histopathology
The following treatment related microscopic findings were detected:

LIVER: centrilobular hepatocellular hypertrophy at a minimal or slight severity was detected in males treated with 300 or 1000 mg/kg bw/day.

The remaining microscopic findings were those commonly observed in laboratory maintained rats of the age and strain employed and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at the limit dose of 1000 mg/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Litter Responses
In total nine females from control and in each case ten females from 100, 300 and 1000 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 of 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
There were no significant differences in the number of corpora lutea or implantation sites recorded for treated females when compared to controls.
The percentage of pre- and post-implantation losses for treated females was comparable to controls.

No differences in sex ratio or litter size were evident for offspring from treated litters when compared to those from the controls.

There were no treatment related effects detected in live birth index and viability index when compared to control values.
Statistical analysis of the data did not show any significant intergroup differences.


Offspring Growth and Development
No significant differences in litter weights or mean offspring weights were evident in the treated groups when compared to controls.

Surface righting assessments did not show any significant differences between litters from treated females when compared to controls.

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

Clinical signs consisted of small, weak, damaged limb, no milk in stomach, missing or found dead for offspring within litters from the control and treated groups. These effects are commonly observed amongst offspring of Wistar Rats used in this type of study and the incidence of these findings do not suggest a direct effect of treatment.


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)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at the limit dose of 1000 mg/kg bw/day.

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of TDI-Urone to rats by gavage, at dose levels of 100, 300 or 1000 mg/kg bw/day, resulted in treatment related effects recorded in animals of either sex treated with 300 or 1000 mg/kg bw/day. The treatment related effects were considered to be adaptive changes and therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was established at 1000 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction.The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with the Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods.The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male : one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partumHaematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5post partum. The female which did not show positive evidence of mating and did not produce a pregnancy was terminated after Day 25post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality.

There were no unscheduled deaths during the study.

Clinical Observations.

There were no toxicologically significant signs of toxicity detected.

Behavioural Assessment.

There were no treatment related effects detected during the behavioural assessment measurements.

Functional Performance Tests.

There were no toxicologically significant changes in the functional parameters measured.

Sensory Reactivity Assessments.

There were no treatment related changes in sensory reactivity scores.

Body Weight.

There were no obvious adverse effects on body weight development.

Food Consumption.

No obvious adverse effect on dietary intake was detected in all treated animals when compared to controls throughout the treatment period.

Water Consumption.

No adverse effect on water consumption was detected.

Reproductive Performance:

Mating.

There were no treatment related effects on mating performance at any of the dose levels investigated. The majority of animals mated within the first five days of pairing (i.e. at the first oestrus opportunity).

Fertility.

There were no treatment related effects detected on fertility in treated animals when compared to controls.

Gestation Lengths.

There were no treatment related effects detected in the length of gestation between control and treated groups. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.

No differences in sex ratio, litter size or viability assessment was evident for offspring from treated litters when compared to those from the controls.

Offspring Growth and Development.

No treatment related effects were detected in offspring growth and development.

Laboratory Investigations:

Haematology.

There were no toxicologically significant effects detected in the haematological parameters investigated.

Blood Chemistry.

Females treated with 1000 mg/kg bw/day showed a reduction in urea concentration and increase in total cholesterol concentration when compared to controls.

No toxicologically significant effects were detected in males treated with 1000 mg/kg bw/day and animals of either sex treated with 300 or 100 mg/kg bw/day.

Pathology:

Necropsy.

There were no treatment related macroscopic abnormalities detected.

Organ Weights.

Males treated with 1000 or 300 mg/kg bw/day showed an increase in liver weight, both absolute and relative to terminal body weight.

No toxicologically significant effects were detected in females treated with 1000 or 300 mg/kg bw/day and in animals of either sex treated with 100 mg/kg bw/day.

Histopathology.

The following treatment related microscopic findings were detected:

LIVER:centrilobular hepatocellular hypertrophy at a minimal or slight severity was recorded in males treated with 1000 or 300 mg/kg bw/day.

Conclusion.

The oral administration of TDI-Urone to rats by gavage, at dose levels of 100, 300 or 1000 mg/kg bw/day, resulted in treatment related effects recorded in animals of either sex treated with 300 or 1000 mg/kg bw/day. The treatment related effects were considered to be adaptive changes and therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was established at 1000 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.