Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th March to 15th July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

EC Number:

269-358-5

EC Name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Cas Number:

68227-46-3

Molecular formula:

C21H35N3O4.C3H6O3

IUPAC Name:

2-hydroxypropanoic acid 2-(dimethylamino)ethyl N-[5-({[(2-ethylhexyl)oxy]carbonyl}amino)-2-methylphenyl]carbamate

Details on test material:

- Name of test material (as cited in study report): WRS-2390TX
- Physical state: Vicous liquid
- Analytical purity: 71.2% in water
- Expiration date of the lot/batch: 15th November 2004
- Storage condition of test material: The test item was stored at ambient laboratory temperature in the dark for the duration of the study.
- Sponsor's information on this batch detailed in Notebook No. 02-181-126.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent on 09 March 2004.
- Age at study initiation: Approximately 4 weeks old
- Weight at study initiation: Males 81-94 g and females 71-79 g.
- Housing: The animals were housed singly per cage by sex and dose group in suspended polypropylene cages (overall dimensions 42 x 27 x 20 cm) with stainless steel grid tops, integral food hoppers and solid bottoms. Sterilised white wood shavings were used as bedding and were regularly analysed for significant contaminants. Each cage was supplied with a polycarbonate water bottle with DurethanTM cap and stainless steel nozzle.
Cages and hoppers were changed once every 2 weeks. Bottles and cage shavings were changed once every week during the course of the study.
Each day on completion of all work, floors were swept and then mopped with disinfectant solution (0.5% Tego 2000; supplied by Th. Goldschmidt and Co Ltd., Tego House, Victoria Road, Ruislip, Middlesex).
The entire room was washed with disinfectant solution once each study week.
- Diet (e.g. ad libitum): Rat and Mouse No. 1 Expanded SQC Diet supplied by Special Diets Services Ltd., Stepfield, Witham, Essex. The diet was available ad libitum except during urine collection. The diet was supplied with a batch analysis for major nutritive components and significant contaminants.
The food used was not considered to contain any additional substances in sufficient concentration to have any influence on the outcome of the study.
- Water (e.g. ad libitum): The animals had access to domestic mains water ad libitum except during urine collection. The water used by Inveresk Laboratories is analysed at 6 monthly intervals for dissolved materials, heavy metals, pesticide residues, pH, nitrates and nitrites.
The water used was not considered to contain any additional substances in sufficient concentration to have any influence on the outcome of the study.
- Acclimation period: The animals were allowed to acclimatise to the Rodent Toxicology Accommodation for a period of 17 days prior to the commencement of dosing.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 50% ± 20%
- Air changes (per hr): Minimum of 15 air changes per h.
- Photoperiod (hrs dark / hrs light): There was a 12 h light/dark cycle in operation, light hours being 0700-1900 h.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared by simple mixing of appropriately weighed quantities of test item to measured volumes of vehicle (water for irrigation).
Vehicle formulation (without the item under investigation) was prepared for the Control animals.
Formulations were prepared and dispensed daily for dosing.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Water for irrigation
- Concentration in vehicle: Individual animal doses were calculated using a daily weighing. A constant dose volume of 20 ml/kg body weight was used.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to study commencement, trial formulations of 0.1-200 mg.ml-1 WRS-2390TX were investigated for stability, concentration and homogeneity. This work was carried out the Toxicology Support laboratory under a separate protocol (Inveresk Study No. 421667, Method No. 2166).
Formulations were analysed on 2 occasions during the study treatment period, on Day 1 and during Week 4. Formulation samples were also taken during Weeks 2 and 3 of treatment and stored, deep frozen at approximately -20°C, however, these samples were not analysed.
On each occasion, triplicate samples were withdrawn from each formulation (including Control). These samples were analysed to provide data on concentration and homogeneity.
The analyses were undertaken by the Toxicology Support laboratory, using a method supplied by the Sponsor and previously validated in the Inveresk laboratory under a separate protocol (Inveresk Study No. 421667, Method No. 2166).

Duration of treatment / exposure:

28 days (4 weeks)

Frequency of treatment:

daily up to 24 h before terminal kill

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
7.5 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females (10 per dose in total)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were agreed with the Sponsor after evaluation of a preliminary study carried out under a separate protocol at Inveresk (Inveresk Project No. 456836: One Week Oral (Gavage) Dose Range Finding Study in Rats, Report No. 23668). Dose level selection took into account the maximum tolerated dose in the test model and other factors such as anticipated human exposure.
In Inveresk Project No. 456836: One Week Oral (Gavage) Dose Range Finding Study in Rats, dosing Sprague-Dawley rats at dose levels up to 1000 mg/kg/day resulted in body weight reduction and correlating food consumption performance in animals treated with as low as 30 mg/kg/day when compared to Controls.
- Rationale for animal assignment (if not random):
On arrival from the suppliers, the animals were introduced to cages on racks. Cages were ascribed to a treatment group by the use of random number sequences.
Each animal was ascribed a cage card which identified that animal by project number, cage number, animal number, sex and treatment group. Each cage card was colour coded according to treatment group.
Additionally, each animal was ascribed a second cage card which identified that animal by study number, neurotox identification, animal number and sex. This cage card was not colour coded.
Each animal received a subcutaneously implanted electronic identification chip which identified it individually within the study and which corresponded to that animal‟s number.
Extra animals were numbered as follows:
Males 41-42
Females 43-44
No replacements were required during the pretrial period and the extra animals were killed humanely during Week 3 of the study.


the following protocol devations were recorded. the deviations were determined not to affect the validity of the study:
- The protocol stated that a certificate of analysis would be supplied with the test item, however, this document was not included. The Sponsor did provide documentation stating that the test item contained 71% solid, propanoic acid, 2-hydroxy-, compd. with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) (4-methyl-1,3-phenylene) bis [carbatmate] (1:1), CAS number 68227-46-3 in water. The Sponsor also provided a Test Item Data Sheet (TIDS) which confirmed the purity, stability, safety instructions and other information relevant to the use and storage of the test item. This deviation from the protocol is not considered to have affected the outcome or integrity of the study.
- Due to a technical error, food consumption values were not recorded on Day 28 of treatment. This deviation from the protocol is not considered to have affected the outcome or integrity of the study.
- Water consumption was monitored by visual inspection of the water bottles on a weekly basis up to Week 3 of treatment. However, water consumption was not monitored during Week 4 of the study in error. After reviewing the in-life data, it was considered that this deviation from the protocol does not affect the outcome or integrity of the study.

Positive control:

nda

Examinations

Observations and examinations performed and frequency:

DETAILED FUNCTIONAL OBSERVATIONS: Yes
-Once during the pre-treatment week (Week -1) and once weekly thereafter, a more detailed examination was made of all animals. These examinations were conducted by a technician not involved in the dosing procedures or in the collection of the body weight or food consumption data, and was performed at an approximately standardised time of day with respect to the time of dosing. Before the independent technician entered the room on each occasion to perform the examinations, the cage card showing treatment group location was removed from each cage, leaving the second pre-prepared card as the cage identifier.

Cageside observations:
Posture/condition on first approach (animal undisturbed), checking for:
Prostration
Lethargy
Writhing
Circling
Breathing abnormalities
Gait abnormalities
Tremor
Fasciculation
Convulsions
Biting (of cage components or self mutilation)
Vocalisations
Piloerection
Ease of removal from cage

Body temperature:
This was taken and recorded from the implanted electronic identification chip.

Condition of the eyes, checking for:
Pupillary function (reaction to visual stimulus)
Miosis
Mydriasis
Exophthalmos
Encrustation
Lacrimation

Condition of the coat.
Presence of salivation.
Overall ease of handling.

-Observations in a standardised arena (2 min observation period):
Latency (time to first locomotory movement).
Level of mobility (each time animal crossed a line between 2 sectors).
Rearing (number of times animal reared on its hindlegs).
Grooming (record of each grooming episode).
Urination/defecation.
Arousal (level of alertness).
Posture, tremor/convulsions, vocalisation, piloerection - recorded as for Cageside observations.
Palpebral closure.
Gait abnormalities.
Stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Reaction to sudden sound (click above the head noting the reaction to the auditory startle stimulus).
Reaction to touch on the rump with a blunt probe.

Grip strength:
This was measured using a method derived from that of Meyer O A, Tilson H A, Byrd W C and Riley M T (1979): Neurobehavioral Toxicology 1, 233-236.. A strain gauge was used, to which was attached a wire pull-bar. Once the animal gripped the bar, the body was pulled until its grasp was broken; the strain gauge records the force required. The procedure was repeated 3 times for the forelimbs and 3 times for the hindlimbs, and the mean fore and hind grip strengths calculated.

Pain perception:
This was assessed by measurement of the tail flick response, using a technique based on the method devised by D'Amour and Smith (1941): J Pharmac Exp Therapy 72, 74. The apparatus used shined a calibrated infra-red heat source onto the tail and automatically measures the reaction time of the animal (approximate to 0.1 s). The test was conducted twice, at different sites within a ca 2 cm length of the tail. The mean of the two pain response values was calculated. Where a pain response was greater than 10 s, a value of 10 s was used in the calculation of the mean. It was ensured that no visible injury to the tail was caused by this test.

Landing Foot Splay:
Maize oil was applied to the hind paws of each animal. The animal was then held in a horizontal, prone position with the nose ca 32 cm above bench surface covered with absorbent paper. When the animal was calm, it was dropped. The distance between the prints of the central footpads was measured. The procedure was repeated 3 times. The mean of the three foot splay values was recorded. If the rat did not land on its feet, it was recorded.

Motor activity:
Each animal was placed in an individual monitoring cage, scanned by a motion sensor utilising infrared pyroelectric detectors. Movement was detected in 3 dimensions anywhere in the cage, and was differentiated into large and small movements. Each animal was monitored for one session of 0.5 h, activity counts were recorded over successive periods of 2 min each.

VIABILITY AND CLINICAL SIGNS: Yes
Time schedule: All animals were checked early in the morning and as late as possible each day for viability and at frequent intervals throughout wach day for any signs of ill health or reaction to treatment. The onset, intensity and duration of any clinical sign was recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta. Once during the pre-treatment week (Week -1) and during Week 4 of treatment, the following additional functional assessments were performed. Again, these assessments were performed at an approximately standardised time of day with respect to the time of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded twice during the week prior to the start of treatment. From the start of treatment, individual body weights were recorded daily in order to determine the correct dose volume to be administered. For reporting of group mean and individual figures, only twice weekly weights are presented. Animals showing weight loss or deterioration in condition were weighed more frequently as necessary.

FOOD CONSUMPTION:
The quantity of food consumed by each animal was measured and recorded twice each week commencing one week prior to the start of treatment up until the end of the study. Due to a technical error, food consumption values were not recorded on Day 28 of treatment. This deviation from the protocol is not considered to have affected the outcome or integrity of the study.


WATER CONSUMPTION:
Water consumption was monitored by visual inspection of the water bottles on a weekly basis up to Week 3 of treatment. However, water consumption was not monitored during Week 4 of the study in error. After reviewing the in-life data, it was considered that this deviation from the protocol does not affect the outcome or integrity of the study.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes were examined using an indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl®). Anterior, lenticular and fundic regions were evaluated.
- Dose groups that were examined: An ophthalmoscopic examination was carried out on all animals during Pretrial, and on all Control and High dose animals during Week 4 of dosing.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples were obtained from all animals in each study group during Week 4 of treatment.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: The animals were not deprived of food overnight prior to sampling.
- Haematology Parameters:
Haemoglobin
Red Blood Cell Count
Haematocrit
White Blood Cell Count
Mean Cell Volume
Mean Cell Haemoglobin
Mean Cell Haemoglobin Concentration
Platelets

Differential White Blood Cell Count:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Large Unclassified Cells


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples were obtained from all animals in each study group during Week 4 of treatment.
- Animals fasted: The animals were not deprived of food overnight prior to sampling.
- Clinical Chemistry Parameters:
Urea
Glucose
Aspartate Aminotransferase
Alanine Aminotransferase
Sodium
Potassium
Chloride
Total Protein
Albumin
Globulin
AG Ratio
Creatinine
Calcium
Phosphate
Total Bilirubin
Cholesterol
Alkaline Phosphatase
- Coagulation Parameters:
Prothrombin Time
Activated Partial Thromboplastin Time


URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from all animals in each study group during Week 4.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: The samples were collected over a 4 h period of food and water deprivation.
- Urinalysis Parameters:
Appearance
pH
Specific Gravity
Volume
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood Pigments
Microscopy of the Spun Deposit


NEUROBEHAVIOURAL EXAMINATION: Yes

Sacrifice and pathology:

GROSS PATHOLOGY/HISTOPATHOLOGY: Yes

The scheduled terminations occurred after completion of 4 weeks of treatment. The animals were killed by exposure to carbon dioxide and had their terminal body weight recorded, followed by exsanguination.
Each animal was subjected to a detailed necropsy performed under the guidance of a veterinary pathologist.
The necropsy consisted of an external and internal examination and recording of observations for all animals. All gross lesions were recorded in descriptive terms, including location(s), size (in mm), shape, colour, consistency and number.
The organs marked “x” in the “Weighed” column in the table below were weighed from all animals before sampling and preservation. Paired organs were weighed separately and the sum of the individual organs was used for reporting purposes. Terminal body weights were used for organ weight analysis. Representative samples of the tissues detailed were taken from all animals and fixed in 10% neutral buffered formalin unless otherwise stated. Carcasses were discarded following tissue sampling.
Tissues marked “x” in the “Examined” column in the table below were processed from all Control and High dose animals. Tissues marked “xa” in the “Examined” column were processed from all male animals. Sections were cut 4-6 μm thick, stained with haematoxylin and eosin (H&E) and evaluated by a pathologist.

Tissues Collected Weighed Examined Comments
Abnormal Tissue - x Included local lymph nodes to masses.
Adrenal x 2 x x -
Aortic Arch - x -
Brain x x Forebrain, midbrain and cerebellum and pons.
Epididymis x 2 x xa -
Eye x 2 - x Both eyes were fixed in Davidson's fluid. One only was
processed and examined histologically.

Gastro-intestinal Tract: Opened at necropsy and mucosa examined. Peyers patches were examined from small intestine.

Stomach - x
Duodenum - x
Jejunum - x
Ileum - x
Caecum - x
Colon - x
Rectum - x

Heart x x -
Implants - - For identification purposes.
Kidney x 2 x x -
Liver x x Three lobes fixed with 2 lobes examined.
Lung x x Inflated after weighing; all lobes examined.
Marrow Smear (femur) - - Smear air-dried and fixed in methanol.
Mesenteric Lymph Node - x -
Oesophagus - x -
Optic Nerve x 2 - x Fixed in Davidson's fluid. One only was examined.
Ovary x 2 x x -
Pancreas - x -
Pituitary x x -
Prostate x x -
Rib - - Including costochondral junction.
Sciatic Nerve - x -
Seminal Vesicles and Coagulating Glands - x -
Skin + Mammary Gland - x -
Spinal Cord - x Sections from cervical, midthoracic and lumbar regions.
Spleen x x -
Sternum - x Including bone marrow.
Submandibular Lymph Node - x -
Submaxillary (Mandibular) Salivary Gland x 2 x x -
Testis x 2 x xa Weighed individually, then fixed in Bouin‟s fluid, trimmed
after ca 18-24 h then returned to Bouin‟s for a further
3-4 h. Trimmed tissue then transferred to alcohol, prior
to processing for examination.
Thigh Muscle - x -
Thymus x x -
Thyroid with Parathyroid x 2 x x Weighed together after fixation.
Tongue - x -
Trachea - x -
Urinary Bladder - x Contracted bladders distended with fixative; epithelial
surface in animals which underwent histological
evaluation was examined after fixation.
Uterus (including oviducts and cervix) x x -
Vagina - x -

Other examinations:

nda

Statistics:

Body weight, food consumption, haematology, clinical chemistry, selected urinalysis data and selected neurotoxicity data were statistically analysed for homogeneity of variance using the 'F-max' test, where appropriate. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a non-parametric test, such as Kruskal-Wallis ANOVA, was used and pairwise comparisons were made using chi squared protection (via z-tests, the non-parametric equivalent of Student's t-test).
Organ weights were also analysed as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate.
The following pairwise comparisons were performed:
Control Group vs Low Dose
Control Group vs Intermediate Dose
Control Group vs High Dose

All statistical tests were two-sided and performed at the 5% significance level. Males and females were analysed separately.
Histological incidence data were analysed using Fisher's Exact Probability Test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation

Mortality:

mortality observed, treatment-related

Description (incidence):

Salivation

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There was a premature decedent (Animal 8, Group 2 male) which was killed shortly after bleeds for laboratory investigations (Day 28 of treatment). At necropsy, the animal had an enlarged and reddened right eye and small epididymides. Retrobulbar haemorrhage was present in the right eye. This finding correlated with the necropsy findings of a reddened and enlarged right eye. Retrobulbar haemorrhage secondary to orbital venipuncture was considered to be the cause of death and not related to treatment.
Isolated incidences of excess salivation were observed (mainly at Day 11 of treatment) in 2 males and 4 females treated at 30 mg/kg/day. There were no other treatment-related clinical signs.


BODY WEIGHT AND WEIGHT GAIN
A reduction in body weight gain was observed throughout the study in both sexes treated at 7.5 mg/kg/day. This reduction in group mean body weight gain was statistically significant compared to Controls. A slight decrease in body weight was also noted throughout treatment. The decrease was not statistically significant. There was a statistically significant decrease in body weight gain during the first week of treatment, however from Day 10 onwards body weight performance at 30 mg/kg/day was comparable with Controls throughout the remainder of the study.


FOOD CONSUMPTION
From Day 0 to Day 3 of treatment, a statistically significant decrease in food consumption was observed in both sexes treated at 30 mg/kg/day. Decreased food consumption was also noted in both sexes by Day 7 of treatment, but not statistically significant. From Days 10 to 24 of the study, food consumption in all animals treated at 30 mg/kg/day was comparable with Controls.
In both sexes treated at 7.5 mg/kg/day, food consumption was lower than Controls throughout the study. Statistically significant decreases were noted sporadically from Day 3 up to Day 21 of treatment.
Food consumption performance in animals treated at 2 mg/kg/day was comparable to Controls.


WATER CONSUMPTION
Visual assessment revealed no notable intergroup differences in either sex.


OPHTHALMOSCOPIC EXAMINATION
There were no intergroup differences noted in either sex.


HAEMATOLOGY
Statistically significant increases in monocyte levels were noted in all treated male groups. However, this finding was not seen in females, was within its historical background range and with no histological findings to correlate this change, it was not considered to be related to administration with WRS-2390TX.
Decreased mean cell haemoglobin levels achieved statistical significance in all treated female groups. Statistically significant decreases in haemoglobin and haematocrit levels were also noted in females treated at 30 mg/kg/day. Again, these findings were not seen in males, were within their historical background ranges and with no histological findings to support these changes, they were not considered to be related to administration with WRS-2390TX.
A slight increase in large unclassified cells was noted in both sexes treated at 30 mg/kg/day. With no histological evidence (for example, in the liver or kidneys) to support this change, it was considered unlikely to be related to treatment with WRS-2390TX.


CLINICAL CHEMISTRY
Increases in aspartate aminotransferase and alanine aminotransferase levels were noted in both sexes treated at 30 mg/kg/day. However, these findings were both within their historical background ranges and there was no histological evidence to support the changes (inflammatory cell foci on liver were noted in one or two animals treated at 30 mg/kg/day and Controls).
Decreased creatinine levels were noted in all treated male groups. Calcium levels were also lower in males achieving statistical significance in animals treated at 7.5 or 30 mg/kg/day. A statistically significant decrease in total protein was noted in males treated at 30 mg/kg/day. However, these findings were not seen in females, were within their historical background ranges and with no histological findings (for example, in the liver or kidneys) to correlate the changes, they were not considered to be related to administration with WRS-2390TX.


URINALYSIS
There were no intergroup differences noted in either sex.


NEUROBEHAVIOUR
All clinical signs observed were those commonly noted during this type of study, and there were no significant differences in group difference or between sexes.
A statically significant increase in fore grip values were noted in females treated at 30 mg/kg/day during Week 4 of the study, however, this finding was not considered to be related to treatment. There were no other notable intergroup differences in detailed functional observations in either sex during the treatment period.


ORGAN WEIGHTS
Absolute thymus weights in females treated at 7.5 mg/kg/day were statistically significantly lower than Controls. After adjustment for body weight, a statistically significant decrease in thymus weights was still apparent. As this finding was not seen in females treated at 30 mg/kg/day, was not seen in males and there are no histological findings to support the decrease in thymus weight, it is not considered to be related to treatment with WRS-2390TX.
There were no other intergroup differences in either sex that were considered to be attributable to administration of WRS-2390TX due to lack of a dose related effect and/or no statistical significance.


GROSS PATHOLOGY
Small testes were found in two males given 30 mg/kg/day (Animals 16 and 20) and one male given 2 mg/kg/day (Animal 8): both testes were affected in Animal 8 and 16, only one testis was affected in Animal 20.
Both epididymides were found to be small in Animal 8, treated at 2 mg/kg/day.
All other necropsy findings were typical of spontaneously arising background findings in rats of this age and strain, on this kind of study at Inveresk.


HISTOPATHOLOGY: NON-NEOPLASTIC
Testes:
Moderate or minimal tubular atrophy was seen in 3 males given 30 mg/kg/day and marked tubular atrophy was seen in one male given 2 mg/kg/day. This finding correlated with the necropsy finding of small testes. Apart from one of the males given 30 mg/kg/day, all these animals showed mild or moderate diffuse interstitial cell hyperplasia.
Spermatid retention was found in one other male given 2 mg/kg/day.

Epididymides:
Oligospermia was found in 1 male given 2 mg/kg/day (Animal 8). Oligospermia and sloughing of spermatogenic cells were noted in 1 male given 30 mg/kg/day (Animal 16). Sloughing of spermatogenic cells was also found in another male given 2 mg/kg/day (Animal 9) and in another male given 30 mg/kg/day (Animal 19). Unilateral agenesis was found in one male given 30 mg/kg/day (Animal 20).
All other histology findings were typical of spontaneously arising background findings in rats of this strain and age, on this kind of study at Inveresk.

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 mg/kg bw/day (actual dose received)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

There was 1 premature decedent during the study. Retrobulbar haemorrhage secondary to orbital venipuncture was considered to be the cause of death and was not considered to be related to treatment with WRS-2390TX.

Daily oral dosing with WRS-2390TX for 4 consecutive weeks resulted in an overall reduction in body weight, body weight gain and food consumption performance in both sexes treated at 7.5 mg/kg/day. Decreased body weight gain and food consumption were also observed up to Day 7 of treatment in both sexes dosed at 30 mg/kg/day. However from Day 10 of treatment onwards, group mean body weight and food consumption in animals treated at 30 mg/kg/day were comparable with Controls. Although the toxicological implications of reduced body weight and food consumption performance in animals treated at 7.5 mg/kg/day in this study are unclear, these effects may be related to treatment.

Isolated incidences of excess salivation were noted throughout the study in a number of animals treated at 30 mg/kg/day. There were no treatment related effects in neurotoxicity clinical observations, motor activity, functional observations, water consumption, haematology and clinical chemistry evaluations, urinalysis and organ weights.

Tubular atrophy was found in the testes of 3 animals given 30 mg/kg/day. In Animal 20 the tubular atrophy was unilateral and was accompanied by unilateral agenesis of the epididymis. As a spontaneously occurring finding, unilateral agenesis of the epididymis would be expected to result in tubular atrophy of the testis on the same side. In the 2 other High dose group animals with bilateral tubular atrophy in the testes, sloughing of spermatogenic cells was found in the epididymides.

There were no findings in testes and epididymides of animals treated at 7.5 mg/kg/day.

Spermatid retention in the testes and sloughing of spermatogenic cells in the epididymides were observed in one male treated at 2 mg/kg/day. Marked tubular atrophy in the testes and oligospermia in the epididymis was observed in another male treated at 2 mg/kg/day. Available organ weight data confirm histology data.

However there were no histological findings seen in the testes and epididymides of animals treated at 2 or 7.5 mg/kg/day in the reproductive screening test (as reported in section 7.8.1). due to the lack of the similar effects in testes and epididymides in the reproductive screening test and the lack of a clear dose response relationship in this study, the findings seen in the testes and epididymides in animals given 2 mg/kg/day may not be related to treatment

In conclusion, daily oral treatment with WRS-2390TX for 4 consecutive weeks resulted in an overall body weight and food consumption reduction at 7.5 mg/kg/day and body weight and food consumption reduction during the first week of treatment at 30 mg/kg/day. There was some evidence of an effect on testes and epididymides of animals given 30 mg/kg/day. The effects on testes and epididymides observed in animals treated with 2 mg/kg/day may not be related to treatment.

 

Based on the results of this study the No Adverse Effect Level (NOAEL) for both sexes was considered to be 2 mg/kg/day although

the effects seen are not considered to be systemic effects that are indicative of serious damage to health of the organism and thus the substance has not been classified as a result of the findings of this study.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study the No Adverse Effect Level (NOAEL) for both sexes was considered to be 2 mg/kg/day. The effects seen are not considered to be systemic effects that are indicative of serious damage to health of the organism and thus the substance has not been classified as a result of the findings of this study. The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability & repeatability).

Executive summary:

In a 4 Week Toxicity Study Including Neurotoxicity Screening in Rats with Administration by Gavage (Inveresk report number: 24231) the No Adverse Effect Level (NOAEL) for both sexes was considered to be 2 mg/kg/day for the test material.

Daily oral treatment with WRS-2390TX for 4 consecutive weeks resulted in an overall body weight and food consumption reduction at 7.5 mg/kg/day and body weight and food consumption reduction during the first week of treatment at 30 mg/kg/day. There was some evidence of an effect on testes and epididymides of animals given 30 mg/kg/day. The effects on testes and epididymides observed in animals treated with 2 mg/kg/day may not be related to treatment.

The study was designed to be acceptable to the guidelines of the EU and US authorities, in accordance with the guidelines detailed in OECD 407 (adopted by the council on 27 July 1995) and conducted under conditions of Good Laboratory Practice.