RD 14153

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 08 July 2008 and 10 March 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

This was a joint study and also run according to OECD 415 (one generation study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor's identification: THIXATROL MAX
Description: off white powder
Lot number: 1325J17001
Date received: 01 July 2008
Storage conditions: approximately 4°C in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
A sufficient number of male and female Wistar Han™:HsdRccHanTM:WIST strain rats were obtained from Harlan Laboratories UK Ltd, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for at least 6 days (Subgroup A) and 7 days (Subgroup B) during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study for Subgroup A and a total of eighty eight females were accepted onto the study for Subgroup B. At the start of treatment the Subgroup A males weighed 263 to 305 g, the Subgroup A females weighed 140 to 199 g, and were approximately ten weeks old. During week 8 of the study, Subgroup B females were introduced to the study. Subgroup B females weighed 169 to 213 g and were approximately ten weeks old at the start of treatment.

The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories UK 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).

ENVIRONMENTAL CONDITIONS
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 mean temperatures and humidities were included in the study records. The temperature and relative humidity controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively. Occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.

IN-LIFE DATES: From: Day 0 To: End of study

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory. Results showed the formulations to be stable for three hours. Formulations were prepared daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test material formulations were analysed for concentration of Thixatrol Max during the study period.
The concentration of Thixatrol Max in the test material formulations was determined by HPLC using an external standard technique.
The results indicate that the prepared formulations were within +/- 5% of the nominal concentration.

Duration of treatment / exposure:

For Subgroup A animals, the test material was administered daily, for one hundred and eight consecutive days

Frequency of treatment:

For Subgroup A animals, the test material was administered daily, for one hundred and eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.

For Subgroup B females, the test material was administrated daily for at least twenty-eight days prior to pairing, and throughout the mating, gestation and lactation phases of the study, up to Day 21 post parium.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
other: nominal

Doses / concentrations 3

Remarks:

Doses / Concentrations:
250 mg/kg/day
Basis:
other: nominal

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
other: nominal

No. of animals per sex per dose:

In sub group A 10 animals were tested per sex per dose

In sub group B only females were tested, 22 females were tested at each dose level

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: For Subgroup A animals, individual bodyweights were recorded on Day 1 and at weekly
intervals thereafter. Bodyweights were also recorded at terminal kill.

For Subgroup B females, individual bodyweights were recorded on Day 1 of treatment and at weekly intervals during the pre-mating phase. Mated females were weighed on Day 0,7, 14 and 21 post coitum and on Days 1, 4, 7, 14 and 21 of lactation

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes
For Subgroup A females, food consumption was recorded for each cage group at weekly intervals throughout the study

Dietary intake for Subgroup A males and Subgroup B females was recorded weekly for each cage group until pairing. Following confirmation of mating, dietary intake for Subgroup B females was recorded on Days 0 to 7, 7 to 14 and 14 to 21 post coitum and Days 1 to 4,4 to 7,7 to 14 and 14 to 21 of lactation.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water
bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all Subgroup A control and high dose animals were examined pre-treatment and during Week 10. Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% "Tropicamide" solution (Alcon Laboratories (UK) Ltd., Imperial Way, Watford, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

HAEMATOLOGY: Yes
Haematological and blood chemical investigations were performed on all Subgroup A animals from each test and control group during Week 10. Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to sampling.

The following parameters were measured on plasma from blood collected into tubes
containing lithium heparin anti-coagulant:
Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (AlG) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (CI-), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili)

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes
Urinalytical investigations were performed on all Subgroup A test and control group animals during Week 10. Urine samples were collected over night by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.

The following parameters were measured on collected urine:
Volume, Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Reducing Substances, Blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity, grip strength and motor activity
Sensory Reactivity:
Each Subgroup A animal was individually assessed for sensory reactivity to auditory,
visual and proprioceptive stimuli.

Grip strength:
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.

Motor Activity:
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Subgroup A animals of one sex were tested at each occasion and 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 one hour for each animal.

OTHER: Reprodcuctive performance, the following was investigated:
Oestrus Cycle Assessments
Mating
Pregnancy and Parturition
Litter Data
Physical Development
Pathology
Semen Assessment
Organ Weights
Histopathology

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Oestrus Cycle Assessments
Mating
Pregnancy and Parturition
Litter Data
Physical Development
Pathology
Semen Assessment
Organ Weights
Histopathology

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

no adverse effects

Mortality:

no mortality observed

Description (incidence):

no adverse effects

Body weight and weight changes:

no effects observed

Description (incidence and severity):

no adverse effects

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

no adverse effects

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

Description (incidence and severity):

no toxicological significant effects

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

no treatment related effects

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

no toxicological significant effects

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

no treatment related effects

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

no treatment related effects

Details on results:

The administration of Thixatrol Max by oral gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of 50, 250 or 1000 mg/kg/day.

There were no toxicologically significant changes in haematology or blood chemistry. Subgroup A males showed an reduction in left cauda epididymal spermatid counts and an increase in cauda epididymal weights following the post-mortem procedures. No treatment-related effects were observed following semen assessments or histopathological examinations and an effect on fertility was not indicated.

Effect levels

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 treatment related effects were seen, therefore the NOAEL has been determined to be 1000 mg/kg bw/day for both systemic and reproductive toxicity

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Results:

Mortality: There were no unscheduled deaths during the study in both Subgroup A or Subgroup B.

Clinical observations: No clinically observable signs of toxicity were detected throughout the treatment period for both Subgroup A and Subgroup B.

Behavioural assessments: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Functional performance tests: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Sensory reactivity assessments: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Bodyweight: No adverse effect on bodyweight development was detected for Subgroup A animals throughout the treatment period, or for Subgroup B females prior to mating, or during the gestation and lactation phases of the study.

Food consumption: No adverse effects on dietary intake or food efficiency were detected for Subgroup A animals during the treatment period, or for Subgroup B females during the pre-mating, gestation and lactation phases of the study.

Water consumption: No intergroup differences were detected for treated animals from both Subgroup A and Subgroup B when compared to their concurrent controls.

Ophthalmoscopy: No ocular effects were detected for high dose Subgroup A animals.

Urinalysis: There were no treatment-related effects detected in the urinalytical parameters investigated for Subgroup A animals.

Haematology: No toxicologically significant effects were detected for treated Subgroup A animals when compared to concurrent controls.

Blood chemistry: No toxicologically significant effects were detected for treated Subgroup A animals when compared to concurrent controls.

Organ weights: No toxicologically significant intergroup differences were detected for both Subgroup A animals and Subgroup B females.

Necropsy: No treatment-related macroscopic abnormalities were detected for both Subgroup A and Subgroup B animals at terminal kill.

Histopathology: No treatment-related microscopic abnormalities were detected for animals from both Subgroup A and Subgroup B.

Applicant's summary and conclusion

Conclusions:

Oral administration of Thixatrol Max by gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight days prior to mating and through the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of up to 1000 mg/kg/day. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 1000 mg/kg/day for both systemic and reproductive toxicity.

Executive summary:

Introduction:

The study was designed to investigate the effects of the test material when administered throughout the reproductive cycle of the rat and complies with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983). The study was also designed to assess subchronic exposure to the test material to the rat and is based on the OECD Guidelines for Testing of Chemicals No 408 "Subchronic Oral Toxicity - Rodent" (Adopted 21 September 1998). This study was also designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 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:

For the Subchronic phase (Subgroup A), the test material was administered by gavage to three groups, each of ten male and ten female Wistar Han™: HsdRccHan™: WIST strain rats, for one hundred and eight consecutive days, at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, bodyweight development, dietary intake and water consumption were monitored during the study. Haematology, blood chemistry and urinalytical investigations were undertaken for all Subgroup A animals during Week 10. Ophthalmoscopic examination was also performed on Subgroup A control and high dose animals once prior to the start of treatment and during Week 10. During Week 11, sensory reactivity, grip strength and motor activity assessments were performed.

During Week 8, females for the reproductive phase (Subgroup B) were introduced to the study. The test material was administered to gavage to three groups, each of twenty-two female Wistar HanTM: HsdRccHanTM: WIST strain rats,for at least twenty-eight days prior to mating and throughout the mating, gestation and lactation phases of the study at dose levels of 50, 250 and 1000 mg/kg/day. A control group of twenty-two females were dosed with vehicle alone (Arachis oil BP). Clinical signs, bodyweight development, dietary intake and water consumption were monitored for Subgroup B females. Oestrus cycle assessments were also undertaken from the start of treatment until pairing.

After Week 11 of the study, Subgroup A males were paired with Subgroup B females on a one male: one female basis within each dose group, for a maximum of twenty-one days. Pairing was continued until all twenty-two females from each group had mated with a Subgroup A male from the same dose group. On completion of the mating phase, all Subgroup A animals, were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

All pregnant Subgroup B females were allowed to girth birth and maintain their offspring until Day 21 post parium. Offspring development was observed during this time. On Day 21 post parium, females and their offspring were subjected to a gross necropsy examination and histopathological examinations of reproductive tissues from high dose and control females was performed.

Results.

Mortality: There were no unscheduled deaths during the study in both Subgroup A or Subgroup B.

Clinical observations: No clinical:y observable signs of toxicity were detected throughout the treatment period for both Subgroup A and Subgroup B.

Behavioural assessments: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Functional performance tests: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Sensory reactivity assessments: No treatment-related effects were detected for treated Subgroup A animals when compared to Subgroup A controls.

Bodyweight: No adverse effect on bodyweight development was detected for Subgroup A animals throughout the treatment period, or for Subgroup B females prior to mating, or during the gestation and lactation phases of the study.

Food consumption: No adverse effects on dietary intake or food efficiency were detected for Subgroup A animals during the treatment period, or for Subgroup B females during the pre-mating, gestation and lactation phases of the study.

Water consumption: No intergroup differences were detected for treated animals from both Subgroup A and Subgroup B when compared to their concurrent controls.

Ophthalmoscopy:No ocular effects were detected for high dose Subgroup A animals.

Urinalysis: There were no treatment-related effects detected in the urinalytical parameters investigated for Subgroup A animals.

Haematology:No toxicologically significant effects were detected for treated Subgroup A animals when compared to concurrent controls.

Blood chemistry: No toxicologically significant effects were detected for treated Subgroup A animals when compared to concurrent controls.

Oestrus cycle assessments: No significant differences in oestrus cycles were detected for treated Subgroup B females when compared to their concurrent controls.

Mating performance: There were no treatment-related differences in mating performance.

Fertility & pregnancy: There were no treatment-related effects detected on fertility or pregnancy status for treated animals when compared to controls.

Gestation length: No treatment-related effects on gestation lengths were detected for treated Subgroup B females when compared to controls.

Litter responses: There were no treatment-related effects detected for litters from treated animals when compared to controls. There were no differences in corpora lutea or pre/post implantation losses for treated animals when compared to controls.

No treatment-related effects were detected for offspring during the daily observations, litter weights or offspring bodyweights. Reflexological assessments did not reveal any significant differences between litters from treated and control dams. No treatmentrelated macroscopic abnormalities were detected at termination.

Semen assessment: No treatment-related effects were detected in sperm motility, morphology or homogenisation resistant spermatid counts from treated Subgroup A males when compared to concurrent controls.

Organ weights: No toxicologically significant intergroup differences were detected for both Subgroup A animals and Subgroup B females.

Necropsy: No treatment-related macroscopic abnormalities were detected for both Subgroup A and Subgroup B animals at terminal kill.

Histopathology: No treatment-related microscopic abnormalities were detected for animals from both Subgroup A and Subgroup B.

Conclusion:

Oral administration of Thixatrol Max by gavage to rats for either a period of one hundred and eight consecutive days or at least twenty-eight days prior to mating and through the gestation and lactation phases of the reproductive cycle for female rats, did not result in toxicologically significant effects at dose levels of up to 1000 mg/kg/day. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 1000 mg/kg/day for both systemic and reproductive toxicity.