N,N''-(4-methyl-m-phenylene)bis[N',N'-dimethylurea]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a GLP-compliant study according to OECD 422 guideline including a 14-d dose range finding study, no significant treatment-related adverse effects have been reported up to the maximum recommended dose of 1000 mg/kg bw/day.

Link to relevant study records

Reference

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

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

Specific details on test material used for the study:

Sponsor's identification :TDI-Urone
Description : White powder
Chemical name :N,N”-(Methyl-1,3-phenylene)bis[N’,N’-dimethylurea]
Product name : Dyhard UR 500
Purity : >= 95%
pH : 9.2
Water solubility :20 g/L at 20 ºC
Batch number : 1268
Date received : 28 October 2011
Storage conditions :Room temperature in the dark
Expiry date : 01 June 2013

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.

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.

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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.

Dose descriptor:

NOAEL

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.

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

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

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.

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.

Dose descriptor:

NOAEL

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.

Key result

Reproductive effects observed:

not specified

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 partum. Haematology 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

good

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A GLP guideline study according to OECD 422 including a 14-d dose range finding study is available. No significant adverse effects have been reported.

Effects on developmental toxicity

Description of key information

A developmental toxicity study (OECD 414, GLP) and a combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test (OECD 422, GLP) conducted on rats are available for the test substance. No treatment related effects on developmental toxicity were observed in both studies up to the maximum recommended dose of 1000 mg/kg bw/day. Therefore, the NOAEL for maternal and developmental toxicity was greater than 1000 mg/kg bw/day in both the OECD 414 and OECD 422 study.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-11-13 to 2023-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted: 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of the Test Item in study report: TDI-Urone 80

SOURCE OF TEST MATERIAL
- Source: Alzchem Trostberg GmbH
- Batch number of test material: 022704
- Purity: 2,4-TDI Urone: 78.6 (% w/w), 2,6-TDI Urone: 20.8 (% w/w)
- Expiry Date: 2022-08-13
- Storage: tightly closed polyethylene container in a dry, cool, and well-ventilated place (18 - 28 °C).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Dose formulation was prepared by mixing the test item with distilled water at the desired concentrations. The required test item formulations were prepared approximately 30 minutes prior to the start of dosing of animals.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Bio-Technology (India) Pvt. Ltd. No. 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal District, Hyderabad, Telangana 500 078
- Age at study initiation: 12 - 13 weeks (at the start of acclimatization)
- Weight at study initiation: Males : 307.87 – 417.35 grams; Females : 217.75 – 282.86 grams (batch – 1) 207.66 – 295.12 grams (batch – 2)
- Fasting period before study: no
- Housing: clean, sterilized solid floored standard polypropylene rat cages covered with stainless steel grill with provisions for feed and water. Cages were placed on stainless steel racks. Cage rotation was done at weekly intervals. Clean and sterilized corn cob was used as bedding material. During acclimatization period, animals were housed in groups of two/three per
cage. After the acclimatization period, one female was housed with one male until the confirmation of mating (for a maximum period of 14 days) in cages equipped with stainless steel bottom grills with blotting paper underneath. After the confirmation of mating, the females were housed individually. Clean and sterile nesting materials (Nestlets-Sterilised Cotton Nesting Material Pad; Gusmer Enterprises, Inc; Lot no. 0224161000) was provided to all dams from gestation day (GD) 14 until scheduled sacrifice on GD 20. Male animals housed individually during the mating period, and females housed individually during the gestation period were provided with the enrichment devices (tunnels). Female animals for the study were allocated in two batches (each batch of 50 females). Phytoestrogen levels in bedding material was tested and was ensured to be within the acceptable limits (350 μg of genistein equivalents/gram of rodent laboratory feed or bedding material).
- Diet: ad libitum, laboratory rodent pellet feed (manufactured by Special Diet Services - England, supplied by Vivo Biotech Ltd., Hyderabad, India) Phytoestrogen levels in the feed was tested and was ensured to be within the acceptable limits (350 μg of genistein equivalents/gram of rodent laboratory feed or bedding material).
- Water: ad libitum, potable well water (IIBAT) processed through reverse osmosis
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 – 22.9
- Humidity (%): 47 - 60
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: Batch - 1 Females: From: 2021-11-26 To: 2021-12-11 until 2021-12-21;
Batch - 2 Females: From: 2021-11-26 To: 2021-12-11 until 2021-12-31

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulation was prepared by mixing the test item with distilled water at the desired concentrations. The required test item formulations were prepared approximately 30 minutes prior to the start of dosing of animals. The test item was administered at approximately the same time (±1 h) each day as a single dose orally by gavage using a stainless steel oral intubation needle fitted to a graduated syringe. The dose volume was maintained at 1 ml/100 g body weight (b.w.). Animals in all the groups were administered the same volume of either the test item formulation or vehicle. Vehicle control group received the vehicle in the highest amount used as in the low dose group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulation was subjected to homogeneity, stability and active ingredient analysis once prior to the initiation of dosing and once during the second week of the dosing period. The sampling was performed from the top, middle, and bottom of the container with dose formulations in duplicate preparations for three dose levels. The analysis was performed by validated analytical method (IIBAT study no. 21196) at the Analytical Chemistry Department of IIBAT. The mean recovery for the dose verification samples analyzed prior to the initiation of dosing (November 17, 2022) ranged between 93.93 and 106.59%, and the mean recovery for the dose verification samples analyzed during the second week of dosing period (December 09, 2022) ranged between 97.08 and 105.10%.

Details on mating procedure:

- If cohoused: 50 male and 50 female animals were randomly cohabited
- M/F ratio per cage: 1:1 ratio (male: female).
- Length of cohabitation: 7 days if vaginal plug or sperm is not observed.
- After .7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 (Gestation Day (GD) 0) of pregnancy

Duration of treatment / exposure:

from implantation (GD 5) to one day prior to expected parturition (GD19).

Frequency of treatment:

daily

Duration of test:

27-39 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 females/ test item treatment groups and 24 females for control group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses for the main study were selected based on the results of the sponsor study (Study no. 41104304).
- Time of day for (rat) dam blood sampling: gestation day (GD) 20

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
- Animals were observed for pertinent behavioral changes, and all signs of toxicity, including mortality, morbidity to recorded in terms of time of onset, degree and duration. During pregnancy, maternal animals were monitored for signs of abortion or premature delivery, if any.

BODY WEIGHT: Yes
- Time schedule for examinations: females were weighed on GD 0, 3, 5, 8, 11, 14, 17, and on GD 20 prior to terminal sacrifice. During mating period, body weight of the females was taken at weekly intervals.

FOOD CONSUMPTION: Yes
The feed consumption of the animals during the main study was calculated for gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20. The feed consumption was calculated from the feed input and leftover feed data and reported as g/rat/day. The feed consumption was recorded during the mating period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: GD 20
- Gross pathology: detailed examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. Evaluation of the dams during caesarean section and subsequent fetal analyses and sample collection were done randomly across available groups to avoid sampling bias.
- Organs examined: Thyroid gland (weight and histopathology)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Serum: Yes
- Blood samples was collected individually from all dams on gestation day 20 and all non-pregnant females at scheduled termination day (GD 20). The blood samples were drawn from the animals through cardiac puncture at necropsy before dissecting the animals, in vials free of anticoagulant.
Blood samples were analyzed for thyroid hormones triiodothyronine (T3), thyroxine (T4) and
thyroid stimulating hormone (TSH) using ELISA method

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [one half per litter]
- Skeletal examinations: Yes: [one half per litter]
- Head examinations: Yes: [half per litter]
- Anogenital distance of all live rodent pups: yes

Statistics:

Data from non-gravid females was excluded from calculation of means and comparative statistics.

Continuous data variables (maternal body weights, gravid uterine weights, weight changes, feed consumption data, thyroid hormone data, fetal body weight (separately by sex, and combined), organ weights (absolute and relative), anogenital distance, and crown-to-rump length was analyzed for normality using Shapiro-Wilk test, and homogeneity of variance between groups was checked by Levene’s test. Where significant homogeneity is detected, a one-way analysis of variance (ANOVA) was carried out followed by parametric multiple comparison procedures using Dunnett's test for post hoc comparison. For non-normally distributed data, non-parametric ANOVA, Kruskal-Wallis test was used. If the results of the test are significant (p<0.05), the Mann-Whitney U test or Dunn’s test (pairwise comparison) was performed to identify significant group differences from control.
The group mean numbers of corpora lutea, implantation sites, viable fetuses, the mean litter proportions of prenatal data (% per litter of viable and nonviable fetuses, early/late resorptions, total resorptions, pre-/post-implantation loss and fetal sex distribution) was analysed using Kruskal-Wallis, non-parametric ANOVA to determine intergroup differences. If the results of the non-parametric ANOVA are significant (p<0.05), the Mann-Whitney-U test was performed to identify significant group differences from control.

The mean litter proportion (% per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particular external, visceral and skeletal malformation or variation was tabulated. The mean litter proportions of fetal malformations and
developmental variations was subjected to the Kruskal-Wallis non-parametric ANOVA test
followed by Mann-Whitney U test (if appropriate) as described above.
A p-value of <0.05 was considered statistically significant.

Indices:

Female Mating Index (%) = [(No. of vaginal plug or sperm positive females)/(Total no. of females cohabited with males)] x 100

Female Fertility Index (%) = [(No. of pregnant females)/(No. vaginal plug or sperm positive females)] x 100

Preimplantation loss (%) = [(number of corpora lutea – number of implantations)/(number of corpora lutea)] x 100

Postimplantation loss (%) = [(number of implantations – number of live fetuses)/(number of implantations)] x 100

Fetal death = resorptions + dead fetuses

AGD Index = AGD of fetus/Cube root of body weight

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity or behavioral changes was observed in any of the dams of treatment group or control groups throughout the experimental period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality/morbidity was observed in any of the animals of treatment or control group throughout the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related effects on body weight and body weight change observed during gestation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Feed consumption measured at various intervals during the gestation period (GDs 0-3, 3-5, 5-8, 8- 11, 11-14, 14-17 and 17-20) was comparable between the control and treatment groups, except there was a statistically significant decrease in the feed consumption in all the treatment groups of 250, 500, and 1000 mg/kg b.w. during GD 5-8. The decrease in the feed consumption was 8.48, 7.20 and 6.77% in the animals of 250, 500, and 1000 mg/kg b.w. respectively when compared with the control group. This decrease in the feed consumption was not having any correlation with the body weight of the animals as there were no changes in the body weight of treatment group animals during GD5 or GD8 when compared with the control group. Hence these changes in the feed consumption have no toxicological significance. The possible reason for the decrease in the feed consumption in the treatment group dams during GD 5-8 could be the animals adapting to the test item treatment during the initial days of dosing (dosing was initiated on GD5), which is evidenced by no change in the feed consumption data in the treatment group animals compared to the control animals for the subsequent gestation period (from GD 8 till GD 20).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone levels (T3, T4, TSH) on GD 20 were comparable between control and treatment groups, except there was a statistically significant increase in T3 (0.24 ± 0.05 ng/mL) and T4 (30.74 ± 4.93 ng/mL) in 1000 mg/kg b.w. group when compared with control group. The increase in T3 and T4 parameters in 1000 mg/kg b.w. animals was 27.58 and 16.23% respectively when compared with control. The absolute thyroid weight was comparable between the control and the treatment groups and there was no statistically significant effect observed between the control (0.0295 ±0.0065 g) and high dose (0.0260 ±0.0059 g) and no test item related histopathological alterations in the thyroid.
Based on the missing correlates in organ weight and histopathology, this finding was considered as incidental and of no toxicological significance (for more information, please refer to “Details on results”).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No clinical signs of toxicity or behavioral changes was observed in any of the dams of treatment group or control groups throughout the experimental period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and relative thyroid weight was comparable between the control and treatment groups, except there was a statistically significant decrease in the absolute (14.20%) and relative (11.97%) thyroid weight of 250 mg/kg b.w. animals, and in the relative (12.62%) thyroid weight of 1000 mg/kg b.w animals. These changes in the thyroid weight were not dose dependent as there were no changes in the absolute and relative thyroid weight in the 500 mg/kg b.w. animals, and no changes were observed in the absolute value in the 1000 mg/kg b.w. animals. Hence these changes were considered incidental and of no toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Detailed examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents had revealed no abnormalities. No gross lesions were observed in the uterus and endometrial area between each implants.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item related histopathological findings were observed in the thyroid of treatment group of animals.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No test item related histopathological findings were observed in the thyroid of treatment group of animals.

Details on results:

Thyroid hormone results:
Analysis of literature had revealed that T3 and T4 values in GD 20 Wistar rats were ranging up to 3.92 ± 1.23 and 72.80 ± 30.04 ng/mL respectively (Deshmukh et al., 2021). GD 20 Long Evans rats had T3 and T4 values ranging up to 0.5366 ± 0.0543 ng/mL and 17.33 ± 1.96 ng/mL respectively (O’Shaughnessy et al., 2018). Moreover, in this study, there is no difference in the absolute thyroid weight of 1000 mg/kg b.w. animals and there is no test item related histopathological alterations in the thyroid. It is also evident that a statistically significant change in the thyroid hormone levels (T4) is common due to slight disturbances of the normal homeostasis that leads to hormonal fluctuations and are often not biologically relevant (Beekhuijzen et al., 2019). A prenatal developmental toxicity study in Wistar rats (study no. 21140) run in parallel to this study at IIBAT had the T3 and T4 values in the control animals ranging up to 0.58 ± 0.28 ng/mL (n=21) and 52.65 ± 4.54 ng/mL (n=21) respectively. Hence the difference in T3 and T4 values in the animals of 1000 mg/kg b.w. when compared with the control animals could be considered as incidental and is of no toxicological significance

Number of abortions:

no effects observed

Description (incidence and severity):

None of the dams showed any signs of abortion or premature delivery prior to the scheduled termination on GD 20.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean number of corpora lutea, implantations sites, and mean percent pre-and postimplantation losses were similar between control and treatment groups (for more information, please refer to Table 1 in section "Any other information on results incl. tables").

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

No late resorptions or dead fetuses were observed in any of the dams of treatment and control groups. Hence, the fetal death was accounted only by the early resorptions in the dams (Fetal death = resorptions + dead fetuses). The mean number of early resorptions, mean number of live fetuses and the percent viable fetus per litter were similar between treatment and control groups (for more information, please refer to Table 1 in section "Any other information on results incl. tables").

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses observed in any treatment group or control group (for more information, please refer to Table 1 in section "Any other information on results incl. tables").

Changes in pregnancy duration:

not specified

Description (incidence and severity):

No premature delivery was observed in the dams

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Out of 99 animals which showed the evidence of copulation, 96 were found to be pregnant, and 3 were non-pregnant (one in control group and two in 250 mg/kg b.w.).

Other effects:

no effects observed

Description (incidence and severity):

The mean gravid uterine weight of treatment and control groups were similar

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean values of fetal weight (either separated by sex, or combined) were similar between treatment and control groups (for more information, please refer to Tables 2 and 3 in section "Any other information on results incl. tables").

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The fetal sex was found to be evenly distributed among the control and treatment groups.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean values of anogenital distance and anogenital index were similar between treatment and control groups (for more information, please refer to Tables 2 and 3 in section "Any other information on results incl. tables").

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

Gross external examination of the fetus had revealed the following findings: subcutaneous hemorrhage in forelimbs of one female fetus (control; dam no. 2259, uterine position no. L3), blood clot above the left forelimbs of one female fetus (250 mg/kg b.w.; dam no. 2255, uterine position no. L2), subcutaneous hemorrhage in left pelvic region of one female fetus (500 mg/kg b.w; dam no. 2335, uterine position no. L2), and cleft palate with tongue protrusion in one male fetus (250 mg/kg b.w.; dam no. 2347, uterine position no. L6). All these findings were incidental and of no toxicological significance.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No test item related effects on the incidence of major or minor skeletal malformations were observed in the fetuses of treated or control dams. Cleft palate, which is classified as major malformation, was observed in one fetus of a dam belonging to the 250 mg/kg b.w. group, and wavy ribs, which is classified as minor malformation was observed in one fetus of the dam belonging to 1000 mg/kg b.w. (for more information, please refer to section “Details on embryotoxic/teratogenic effects”).

Visceral malformations:

no effects observed

Description (incidence and severity):

No remarkable soft tissue alterations were observed in any of the fetuses of the treated dams or control dams.

Details on embryotoxic / teratogenic effects:

Skeletal malformations/variations:
Overall incidence of these major and minor malformations occurring as single observations among the fetuses of their respective groups was very low, with 0.66% for cleft palate in the 250 mg/kg b.w. group and 0.58% for the occurrence of wavy ribs in the 1000 mg/kg b.w. group, respectively. Moreover, these incidences were compared with the literature (Spontaneous Malformations in Laboratory Animals: Frequency of External, Internal and Skeletal Malformations in Rats (Mortial,1987; Cong. Anom., 27: 147-206, 1987). As a result, these malformations were considered spontaneous and not treatment related. There are normal variants observed in the skeletal examinations of the fetuses of the treatment and control groups. These normal variants were spontaneous and not due to test item treatment and had no toxicological significance. The incidence of malformations (major/minor) and normal variants in the fetuses were statistically similar between treatment and control groups.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Summary of animal uterine content data

Group No.	No. of. Corpora lutea	No. of. Implantation sites	Pre-implantation loss (%)	No. of. Early resorption	No. of. Late resorption	Total resorptions	No. of. Live fetus	No. of. Dead fetus	Total no. of fetus	Fetal death	Percent viable fetus per litter	Percent non-viable fetus per litter	Post-implantation loss (%)	^#Gravid uterine weight (g)
Group: G1 (Vehicle Control)	14.70	14.70	0.00	0.91	0.00	0.91	13.78	0.00	13.78	0.91	100.00	0.00	6.47	82.15
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	1.61	1.61	0.00	1.28	0.00	1.28	2.28	0.00	2.28	1.28	0.00	0.00	9.09	20.59
Group: G2 (250 mg/kg b.w)	14.78	14.78	0.00	1.48	0.00	1.48	13.30	0.00	13.30	1.48	100.00	0.00	9.60	81.07
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	2.41	2.41	0.00	1.62	0.00	1.62	2.36	0.00	2.36	1.62	0.00	0.00	9.53	12.80
% Change from Control	0.59	0.59	-	61.90	-	61.90	-3.47	-	-3.47	61.90	0.00	-	48.47	-1.31
Group: G3 (500 mg/kg b.w)	14.48	14.48	0.00	1.80	0.00	1.80	12.68	0.00	12.68	1.80	100.00	0.00	11.96	80.00
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	2.55	2.55	0.00	2.40	0.00	2.40	3.04	0.00	3.04	2.40	0.00	0.00	15.44	16.93
% Change from Control	-1.47	-1.47	-	97.14	-	97.14	-8.00	-	-8.00	97.14	0.00	-	84.99	-2.62
Group: G4 (1000 mg/kg b.w)	14.88	14.88	0.00	1.24	0.00	1.24	13.64	0.00	13.64	1.24	100.00	0.00	8.36	86.32
	±	±	±	±	±	±	±	±	±	±	±	±	±	±
	1.83	1.83	0.00	2.22	0.00	2.22	2.83	0.00	2.83	2.22	0.00	0.00	14.94	16.39
% Change from Control	1.25	1.25	-	35.81	-	35.81	-1.03	-	-1.03	35.81	0.00	-	29.23	5.07
Data from the non-pregnant animal(s) is excluded from the calculation of mean and SD and from the statistical analysis; n=23 for G1 and G2; n=25 for G3 and G4; Values are not statistically different from control (P > 0.05) Type of analysis - Kruskal Wallis at 0.05 level of significance

 

Table 2 Summary of fetal data (male)

 

Group/Dose	AGD (mm)	AGD Index	Fetal weight (g)	Crown Rump (mm)
G1 (Vehicle Control)	2.29	1.43	4.0703	37.32
	±	±	±	±
	0.72	0.45	0.3956	2.81
G2 (Low dose) - 250 mg/kg b.w.	2.18	1.37	3.9543	36.88
	±	±	±	±
	0.73	0.45	0.4642	3.28
% Change from Control	-4.66	-3.92	-2.85	-1.16
G3 (Intermediate dose) - 500 mg/kg b.w.	2.09	1.31	4.0044	37.14
	±	±	±	±
	0.70	0.43	0.5083	2.78
% Change from Control	-8.79	-8.30	-1.62	-0.47
G4 (High dose) - 1000 mg/kg b.w.	2.09	1.31	4.0321	37.28
	±	±	±	±
	0.71	0.43	0.5021	2.80
% Change from Control	-8.59	-8.33	-0.94	-0.10
Data are expressed as Mean ± S.D Values are not statistically significant (P>0.05) Kruskal Wallis at 0.05 level of significance

 

Table 3 Summary of fetal data (female)

Group/Dose	AGD (mm)	AGD Index	Fetal weight (g)	Crown Rump (mm)
G1 (Vehicle Control)	2.11	1.33	3.9932	37.25
	±	±	±	±
	0.74	0.45	0.4179	2.58
G2 (Low dose) - 250 mg/kg b.w.	2.18	1.38	3.9669	36.99
	±	±	±	±
	0.73	0.45	0.4197	2.66
% Change from Control	3.39	3.66	-0.66	-0.69
G3 (Intermediate dose) - 500 mg/kg b.w.	2.08	1.31	4.0032	37.13
	±	±	±	±
	0.70	0.43	0.5086	2.78
% Change from Control	-1.26	-1.22	0.25	-0.31
G4 (High dose) - 1000 mg/kg b.w.	2.09	1.31	4.0322	37.28
	±	±	±	±
	0.71	0.43	0.5014	2.80
% Change from Control	-1.12	-1.33	0.98	0.09
Data are expressed as Mean ± S.D Values are not statistically significant (P>0.05) Kruskal Wallis at 0.05 level of significance

 

Conclusions:

In a developmental toxicity study (OECD 414, GLP) conducted in rats, TDI-Uron 80 did not cause maternal or developmental toxicity up to the maximum recommended dose of 1000 mg/kg b.w./day. The NOAEL for maternal and developmental effects for TDI-Urone 80 is greater than 1000 mg/kg b.w./day.

Executive summary:

In a developmental toxicity study (OECD 414, GLP) TDI-Urone 80 was administered in distilled water to 24 (control group) or 25 (test item treatment groups) female Wistar (Crl:WI) rats/dose by oral gavage at dose levels of 0, 250, 500, or 1000 mg/kg bw/day from days 5 through 19 of gestation.

There were no treatment-related effects in mortality, clinical signs, body weight or food consumption in maternal animals. Moreover, exposure to TDI-Urone 80 did not cause effects on gravid uterine weights, number of corpora lutea, number of implantations sites, and mean percent pre-and post-implantation losses. The mean number of early resorptions, mean number of live fetuses and the percent viable fetus per litter were similar between treatment and control groups. The maternal NOAEL is greater than 1000 mg/kg bw/day.

There were no treatment-related effects in developmental parameters. The mean values of fetal weight (either separated by sex, or combined), anogenital distance, anogenital index, and crown-to-rump length were similar between treatment and control groups. No effects of treatment with TDI-Urone 80 were observed upon visceral and skeletal fetal examinations. The developmental NOAEL is greater than 1000 mg/kg bw/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.

Reference 2

Endpoint:

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

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Sponsor's identification :TDI-Urone
Description : White powder
Chemical name :N,N”-(Methyl-1,3-phenylene)bis[N’,N’-dimethylurea]
Product name : Dyhard UR 500
Purity : >= 95%
pH : 9.2
Water solubility :20 g/L at 20 ºC
Batch number : 1268
Date received : 28 October 2011
Storage conditions :Room temperature in the dark
Expiry date : 01 June 2013

Species:

rat

Strain:

Wistar

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 (see Appendix 7 for Day 1 body weight values).

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. A certificate of analysis of the batch of diet used is given in Addendum 1. Mains drinking water was supplied from polycarbonate bottles attached to the cage (for Typical Water Quality Characteristics see Addendum 9). 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.

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 method used for analysis of formulations and the results obtained are given in Appendix 26. The results indicate that the prepared formulations were within ± 3% of the nominal concentration.


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

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

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.

Procedure

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.

The test item formulations were sampled and analysed within two days of preparation.

Details on mating procedure:

- M/F ratio per cage:
1/1 (Animals were paired on a 1 male: 1 female basis within each dose group)

- 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 the oestrous cycle 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.

Duration of treatment / exposure:

Male dose groups: 43 days
Female dose groups: 5 days post-partum

Frequency of treatment:

Daily

Duration of test:

Male dose groups: 43 days
Female dose groups: 5 days post-partum

No. of animals per sex per dose:

10 animals per sex per dose (including control).

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen based on the results of a preliminary range-finder (14-days) performed.

Maternal 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.


3.3.2.1 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.

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) (see section 8. References).
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 a
ccurately calculated for females during gestation and lactation.


Water Consumption
Water intake was measured daily throughout the study (with the exception of the pairing phase) and data are presented in Appendix 10.


Reproduction Screening

Mating - Procedure
Animals 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.


Pregnancy and Parturition
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


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 (Propath UK Ltd, Hereford, UK) for processing. 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.

Examinations included:
- Gravid uterus weight: No

- Number of corpora lutea: Yes

- Number of implantations: Yes

- Number of early resorptions: No

- Number of late resorptions: No

Fetal examinations:

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)

Statistics:

Please refer to the section below "Any other information on materials and methods"

Indices:

Not applicable.

Historical control data:

Not applicable.

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

Description (incidence and severity):

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

Water consumption and compound intake (if drinking water study):

no effects observed

Behaviour (functional findings):

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

"Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Please refer to the "Any other information on results" section below

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: other:

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Please refer to the "Any other information on results" section below

Key result

Dose descriptor:

NOEL

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.

Key result

Dose descriptor:

NOAEL

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.

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

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.

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.

Laboratory Investigations

Haematology

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.

Blood Chemistry

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.

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 partum. Haematology 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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-compliant developmental toxicity study (OECD 414) and combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test (OECD 422) conducted according to the relevant guidelines.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A developmental toxicity study (OECD 414, GLP) and a combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test (OECD 422, GLP) conducted on rats available for the test substance.

In a developmental toxicity study (OECD 414, GLP) TDI-Urone 80 was administered to 23 or 25 females Wistar (Crl:WI) rats/dose by gavage at dose levels of 0, 250, 500, or 1000 mg/kg bw/day from days 5 through 19 of gestation. There were no treatment-related effects in mortality, clinical signs, body weight, food consumption in maternal animals. Moreover, exposure to TDI-Urone 80 did not cause effects on gravid uterine weights, number of corpora lutea, number of implantations sites, and mean percent pre-and post-implantation losses. The mean number of early resorptions, mean number of live fetuses and the percent viable fetus per litter were similar between treatment and control groups. The maternal NOAEL was greater than 1000 mg/kg bw/day.  There were no treatment-related effects in developmental parameters. The mean values of fetal weight (either separated by sex, or combined), anogenital distance, anogenital index, and crown-to-rump length were similar between treatment and control groups. No effects of the treatment with TDI-Urone 80 were observed upon visceral and skeletal fetal examinations. The developmental NOAEL was greater than 1000 mg/kg bw/day

No effects in developmental toxicity were observed after exposure to TDI-Uron 80 at doses of 100, 300 or 1000 mg/kg bw/day in a combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test (OECD 422, GLP) conducted on Wistar rats. No differences in sex ratio, litter size or viability assessment was evident for offspring from treated litters when compared to those from the controls. No treatment related effects were detected in offspring growth and development. The NOAEL was established at 1000 mg/kg bw/day.

Justification for classification or non-classification

No significant adverse effects have been reported in GLP-compliant studies according to the relevant OECD 422 and OECD 414 guidelines up to the maximum recommended dose of 1000 mg/kg bw/day. Therefore classification according to Regulation (EC) No 1272/2008 is not warranted.