Carbonohydrazide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The in-life phase of the study was conducted between 29 June 2011 (first day of treatment) and 22 August 2011 (final necropsy).

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 not effect the quality of the relevant results.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Carbohydrazide

IUPAC Name:

Carbohydrazide

Test material form:

other: solid

Details on test material:

Sponsor's identification: Carbohydrazide
Identification number: TIS I0352
CAS number : 497-18-7
Description : white solid
Chemical name: Carbohydrazide
Batch number : OJ24-21M
Date received : 04 April 2011
Expiry date : 24 January 2013
Storage conditions: room temperature in the dark over silica

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: male and female Wistar Han:RccHan:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., UK
- Age at study initiation: approximately twelve weeks old
- Weight at study initiation: At the start of treatment the males weighed 299 to 355g, the females weighed 191 to 216g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating 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.
- Diet (e.g. ad libitum): The animals were allowed free access to food. A pelleted diet was used.
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: The animals were acclimatised for thirteen days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperatures controls were set to achieve target values of 22 ± 3°C. Achieved ranges were 20-22°C.
- Humidity (%): The relative humidity controls were set to achieve target values of 55 ± 15%. Achieved ranges were 31-71% RH.
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
the test item was prepared at the appropriate concentrations as a solution in distilled water. The stability and homogeneity of the test item formulations were previously determined in a 14-day range finding study. Results from the previous study showed the formulations to be stable at 4°C in the dark for at least twenty three days. Formulations were therefore prepared in batches of no greater than two weeks duration and divided in to daily aliquots, which were stored at 4ºC in the dark until their use.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg bw/day of distilled water.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analysed for concentration of Carbohydrazide

The concentration of Carbohydrazide in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

The results indicate that the prepared formulations were within 93-115% of the nominal concentration and were considered acceptable for the purpose of this study.

Duration of treatment / exposure:

The test item was administered for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females).

Frequency of treatment:

The test item was administered daily.

Doses / concentrations

Remarks:

Doses / Concentrations:
initial dose levels of 30, 75 and 150 mg/kg bw/day. Following adverse toxicity the high dosage was reduced from 150 mg/kg bw/day to 100 mg/kg bw/day from Day 9 of the study.
Basis:
actual ingested

No. of animals per sex per dose:

ten males and ten females per dose (plus control)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of a 14 day range finding study, which gave the below results.
The oral administration of the test item, Carbohydrazide, to rats for a period of seven consecutive days at 250 mg/kg bw/day resulted in notable treatment-related effects in animals of either sex. Administration of the test item, at 50 and 100 mg/kg bw/day for fourteen consecutive days was much better tolerated. Based on these findings, a high dosage for further investigation of toxicity should probably be greater than 100 mg/kg bw/day but not exceed 150 mg/kg bw/day.

Chronological Sequence of Study
i) 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.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.
ix) 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, females and surviving offspring were killed and examined macroscopically.

Positive control:

No positive control.

Examinations

Observations and examinations performed and frequency:

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

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, 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. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex , Tail pinch, Startle reflex, Finger approach.

BODY WEIGHT:
Individual body weights were recorded for males on Day 1 and then weekly until termination. Individual body weights will also be recorded at terminal kill.
For females, individual body weights were recorded on Day 1 and then weekly until pairing. During the pairing phase, females were weighed daily until mating was confirmed (these data are retained with the raw data for the study but have only been reported when part of the profile of gestation body weights). Mated females were weighed on Day 0, 7, 14 and 20 post coitum and body weights for females observed to give birth were recorded on Days 1 and 4 post partum. Body weights will also be recorded at terminal kill.

FOOD CONSUMPTION:
During the maturation 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-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION:
Gravimetric measurement of water consumption was conducted for both sexes during the pre-pairing period. As no treatment related effects were suspected no further measurement of water intake was performed during the remainder of the study.

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 (Bile)

Sacrifice and pathology:

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. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 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.

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 with Cervix)


HISTOPATHOLOGY:
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin.
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Pituitary
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 brochi)
Thymus
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cevix
Muscle (skeletal)
Vagina

All tissues were despatched to the Test Site for processing. All tissues from five selected control and 150/100 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. In addition testes and epididymides from these animals were also stained with Periodic Acid-Schiff (PAS) stain and examined. Additionally reproductive tissues were similarly processed and examined for the remaining control and 150/100 mg/kg bw/day animals and any other animal with suspect fertility (i.e. pairing did not result in a pregnancy).

Other examinations:

Reproductive Performance:
- Mating.
- Fertility.
- Gestation Lengths.

Litter Responses:
- Offspring Litter Size and Viability.
- Offspring Growth and Development

Statistics:

The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Body weight and body weight change
Food consumption during gestation and lactation
Pre-coital interval and gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Haematology, blood chemistry, urinalysis volume and specific gravity, adult absolute and body weight-relative organ weights

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

The high dosage on this study was reduced from 150 mg/kg bw/day to 100 mg/kg bw/day on Day 9 of the study. For reporting purposes, the dosage for the high dosage group is expressed as 150/100 mg/kg bw/day (except where the observations being made specifically refer to the period where the animals were only receiving the higher dosage of 150 mg/kg bw/day).

ADULT RESPONSES
MORTALITY
There were no unscheduled deaths in adult animals on the study.

CLINICAL INVESTIGATIONS
Animals on the study did not show any abnormal clinical signs throughout the study at any of the dosages investigated.

FUNCTIONAL OBSERVATIONS
Behavioural Assessments:
Behavioural assessment of the animals in a standard arena did not reveal any obvious effects of treatment at any of the dosages investigated.

Functional Performance Tests:
Assessment of functional performance using grip strength and measurement of motor activity did not indicate any obvious effects of treatment at any of the dosages investigated.

Statistically significant intergroup differences from control for overall mobile activity were observed for all treated females, however there was no dosage relationship and all values were within the historical normal range. These differences were therefore considered to be incidental and unrelated to treatment.

Sensory Reactivity Assessments:
Sensory reactivity to different stimuli (auditory, visual and proprioceptive) appeared unaffected by treatment at any of the dosages investigated.

BODYWEIGHT:
At 150 mg/kg bw/day treatment for both sexes was associated with mean body weight loss during the first week of dosing. In view of the extent of body weight loss observed and the length of the treatment period for this study design, a decision was taken to reduce the dosage level for this dosage group to 100 mg/kg bw/day from Day 9. Following this reduction in dosage, recovery of body weight gain was observed in both sexes and overall gains for males at the end of the treatment period and for females at the end of the pre-pairing phase were similar to control. There were also no subsequent effects on body weight gain for females during gestation or early lactation.

At 75 mg/kg bw/day treatment of females was associated with a marginal body weight loss during the first week of dosing and males at this dosage also showed marginally lower body weight gain, compared with control, during this period. For both sexes these differences in body weight gain from control during the first week of dosing failed to attain statistical significance and thereafter body gains for the remainder of the study, including for females during the gestation and lactation phases were considered to be unaffected by treatment.

At 30 mg/kg bw/day there were no obvious adverse effects on body weight gains for either sex, including for females during the periods of gestation and lactation.

FOOD CONSUMPTION:
At 150 mg/kg bw/day treatment was associated with lower food consumption for both sexes during the first week of dosing. Following the reduction in dosage level to 100 mg/kg/day food intake was similar to control to termination, including for females during the periods of gestation and lactation.

At 30 and 75 mg/kg bw/day there was no clear adverse effect of treatment on food intake in either sex, including for females during the periods of gestation and lactation.

For females at 30 mg/kg bw/day mean food consumption appeared higher than control during early lactation. However, these differences were principally attributable to two females who showed atypically high food intake and the differences in mean values from control were considered to reflect normal biological variation rather than any effect of treatment.

FOOD EFFICIENCY
The value of analysing food conversion efficiency during periods where an overall body weight loss has been observed is equivocal.
For both sexes at 150 mg/kg bw/day and females at 75 mg/kg bw/day food conversion efficiency during the first week of treatment, was notably inferior to control reflecting the overall mean body weight loss observed for these animals at that time. Thereafter food utilisation for these animals was similar or slightly superior to control during the remaining pre-pairing period and, for males, the post-pairing period.
There was no adverse effect on food utilisation for both sexes at 30 mg/kg bw/day or males at 75 mg/kg bw/day.

WATER CONSUMPTION
Measurement of water consumption during the pre-pairing period did not reveal any consistent pattern in water intake that indicated any adverse effect of treatment for either sex.

LABORATORY INVESTIGATIONS
HAEMATOLOGY
There were no statistically significant differences in haematology parameters that were considered to be of toxicological significance.

For females at 75 and 150/100 mg/kg bw/day mean white blood cell counts were lower than control principally due to lower numbers of neutrophils and lymphocytes; differences attained statistical significance but mean values were within the historical control range. A decrease in these parameters is considered unlikely to occur in response to treatment without any associated histopathological changes. As no histopathological findings were observed for these animals the decreased blood counts were considered incidental and unrelated to treatment.

BLOOD CHEMISTRY
There were no statistically significant differences in blood chemistry parameters that were considered to be of toxicological significance.

For males at 150/100 mg/kg bw/day, mean bile acid levels were statistically significantly higher than control, and also exceeded the historical control range. This isolated finding, in the absence of any histopathological changes in the liver was considered to be incidental and of no toxicological significance.

For females at all dosages creatinine levels were lower than control with values attaining statistical significance; however all values were within the historical control range and in the absence of any dosage relationship differences were considered to be incidental and unrelated to treatment.
For females at 75 and 150/100 mg/kg bw/day, levels of aspartate aminotransferase (ASAT) were lower than control and alanine aminotransferase (ALAT) was also lower than control at 150/100 mg/kg bw/day; all values attained statistical significant but were within the historical control range. A decrease in the levels of these blood parameters is unlikely to result from any toxic insult and therefore these findings were considered to be incidental and unrelated to treatment.

For females at 150/100 mg/kg bw/day, mean levels of total protein and inorganic phosphorus were higher than control, with differences attaining statistical significance. However, mean values were within the historical control range and the increase in total protein occurred without any corresponding statistically significant differences from control being observed for albumin or albumin/globulin ratio. In isolation these changes were considered to be incidental and unrelated to treatment.

PATHOLOGY
ORGAN WEIGHTS
For males at 150/100 mg/kg bw/day, absolute and body weight relative prostate weight was higher than control; differences from control attaining statistical significance. However, mean organ weights were within the historical control range and in the absence of any evidence of histopathological change this finding was considered to be incidental and unrelated to treatment.

There were no other differences on male organ weights that attained statistical significance at this dosage or at 30 and 75 mg/kg bw/day.

At all dosages, absolute and body weight relative kidney weights were higher than control; differences from control attaining statistical significance. However, mean values were within the historical control range and in the absence of any consistent dosage relationship or evidence of histopathological change, these differences were considered to be incidental and unrelated to treatment.

For females at 150/100 mg/kg bw/day, absolute and body weight relative spleen and thymus weights were higher than control with differences attaining statistical significance. Mean values were within the historical control range and, in the absence of any evidence of histopathological change these differences were considered not to be of any toxicological significance.

NECROPSY:
Offspring:
Neither the type, incidence nor distribution of macroscopic findings observed for decedent offspring or offspring killed at termination indicated any obvious adverse effect of treatment at any of the dosages investigated.

Adults:
Macroscopic examination of animals at terminal necropsy did not indicate any adverse effect of treatment at any of the dosages investigated

One male (number 49) at 75 mg/kg bw/day showed small and flaccid testes; the female partner to this animal failed to achieve pregnancy. In isolation, and in the absence of any similar findings at 150/100 mg/kg bw/day, this finding was considered to be incidental and unrelated to treatment.

One female at 150/100 mg/kg bw/day was observed to have an enlarged right kidney which had a pale granular internal appearance. The left kidney was small. It is not unusual when a kidney is not fully functioning for the opposite kidney to increase in size in order to cope with the increased physiological demand placed on it. In isolation these kidney findings were considered incidental and unrelated to treatment.

HISTOPATHOLOGY
The test item Carbohydrazide at a dosage of 150/100 mg/kg bw/day produced no histological evidence of toxicological properties in the organs and tissues examined.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

adult toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See discussion on results

2

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See discussion on results

3

Dose descriptor:

NOEL

Remarks:

offspring growth and development

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See discussion on results

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Reproductive Performance:

Mating. There was no adverse effect of treatment on mating performance.

Fertility. Fertility, as assessed by the number of pregnant females, was unaffected by treatment.

Gestation Lengths. There was a tendency towards a longer gestation length at 75 and 150/100 mg/kg bw/day in comparison to control females and those receiving 30 mg/kg bw/day.

Litter Responses:

Offspring Litter Size and Viability. At 150 mg/kg bw/day litter size at birth/Day 1 was lower than control due to higher post-implantation loss and, despite this lower litter size, survival to Day 4 was slightly inferior to control. One female showed total litter loss post partum and, although a similar incidence was observed for control and 30 mg/kg bw/day, an association with treatment at this higher dosage could not be discounted.

Offspring Growth and Development. There was no adverse effect of treatment on offspring body weight on Day 1 or body weight gain to Day 4 of age, although at 150/100 mg/kg/day litter weight was lower than control as a consequence of the lower litter size at this dosage.

Clinical signs and assessment of surface righting did not indicate any obvious adverse effect of treatment

Applicant's summary and conclusion

Conclusions:

Initial effects on body weights and food intake during the first week of treatment precluded classifying either 75 or 150/100 mg/kg bw/day as a No Observed Effect Level (NOEL) for adult toxicity. However, as these effects did not persist following the lowering of the high dose level, a dosage of 100 mg/kg bw/day is classified as a No Observed Adverse Effect Level (NOAEL) for adult toxicity.

At 75 mg/kg bw/day, a tendency towards longer gestation length was observed but, in the absence of any adverse effect on post-implantation loss or litter size at birth/Day 1, this dosage is classified as a NOAEL for reproduction.

No effects were apparent on offspring body weights and survival at 75 mg/kg bw/day and this dosage is classified as a NOEL for offspring growth and development.

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 maturation phase, pairing, gestation and early lactation for females), at initial dose levels of 30, 75 and 150 mg/kg bw/day. Following adverse toxicity the high dosage was reduced from 150 mg/kg bw/day to 100 mg/kg bw/day from Day 9 of the study. A control group of ten males and ten females was dosed with vehicle alone (Distilled water).

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 mating phase, and for five selected parental females from each dose group on Day 4 post 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 females and offspring on Day 5 post partum. 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 in adult animals on the study.

Clinical Observations. No abnormal clinical signs were observed throughout the study.

Behavioural Assessment. Assessment of the animals in a standard arena did not reveal any obvious effects of treatment.

Functional Performance Tests. Functional performance assessments did not indicate any adverse effects of treatment.

Sensory Reactivity Assessments. Sensory reactivity assessments did not indicate any adverse effects of treatment.

Body Weight. At 150 mg/kg bw/day treatment for both sexes was associated with mean body weight loss during the first week of dosing. The dosage level was reduced to 100 mg/kg bw/day from Day 9 and no adverse effects on body weight or body weight gain were subsequently observed for either sex, including gestation and lactation for females, throughout the remainder of the study.

At 75 mg/kg bw/day slight body weight loss for females and marginally lower body weight gain for males was observed during the first week of treatment. Thereafter body weight gain during the remainder of the study, including gestation and lactation phases, was unaffected by treatment.

At 30 mg/kg bw/day, for both sexes, there were no adverse effects on body weight or body weight gains throughout the study.

Food Consumption. At 150 mg/kg bw/day treatment was associated with lower food consumption for both sexes during the first week of dosing. Following the reduction in dosage level to 100 mg/kg bw/day food intake for both sexes was unaffected by treatment throughout the remainder of the study and including the gestation and lactation phases for females.

At 30 and 75 mg/kg bw/day there was no clear adverse effect of treatment on food intake for either sex throughout the study.

Food Efficiency.

For both sexes at 150 mg/kg bw/day and females at 75 mg/kg bw/day food conversion efficiency during the first week of treatment was notably inferior to control (reflecting the overall mean body weight loss observed). 

There was no adverse effect on food utilisation for both sexes at 30 mg/kg bw/day or males at 75 mg/kg bw/day.

Water Consumption.

There was no obvious effect of treatment on water consumption throughout the study.

Reproductive Performance:

Mating. There was no adverse effect of treatment on mating performance.

Fertility. Fertility, as assessed by the number of pregnant females, was unaffected by treatment.

Gestation Lengths. There was a tendency towards a longer gestation length at 75 and 150/100 mg/kg bw/day in comparison to control females and those receiving 30 mg/kg bw/day.

Litter Responses:

Offspring Litter Size and Viability. At 150 mg/kg bw/day litter size at birth/Day 1 was lower than control due to higher post-implantation loss and, despite this lower litter size, survival to Day 4 was slightly inferior to control. One female showed total litter losspost partumand, although a similar incidence was observed for control and 30 mg/kg bw/day, an association with treatment at this higher dosage could not be discounted.

Offspring Growth and Development. There was no adverse effect of treatment on offspring body weight on Day 1 or body weight gain to Day 4 of age, although at 150/100 mg/kg/day litter weight was lower than control as a consequence of the lower litter size at this dosage.

Clinical signs and assessment of surface righting did not indicate any obvious adverse effect of treatment

Laboratory Investigations:

Haematology. There were no statistically significant differences in haematology parameters that were considered to be of toxicological significance.

Blood Chemistry. There were no statistically significant differences in blood chemistry parameters that were considered to be of toxicological significance.

Pathology:

Necropsy. Macroscopic findings for decedent offspring and both adults and offspring at scheduled termination did not indicate any adverse effect of treatment.

Organ Weights. There were no statistically significant differences in adult organ weights that, in the absence of any evidence of histopathological change, were considered to be of any toxicological significance.

Histopathology. Dosage of 150/100 mg/kg bw/day produced no histological evidence of toxicological properties in the organs and tissues examined.

Conclusion.

Initial effects on body weights and food intake during the first week of treatment precluded classifying either 75 or 150/100 mg/kg bw/day as a No Observed Effect Level (NOEL) for adult toxicity. However, as these effects did not persist following the lowering of the high dose level, a dosage of 100 mg/kg bw/day is classified as a No Observed Adverse Effect Level (NOAEL) for adult toxicity.

At 75 mg/kg bw/day, a tendency towards longer gestation length was observed but, in the absence of any adverse effect on post-implantation loss or litter size at birth/Day 1, this dosage is classified as a NOAEL for reproduction.

No effects were apparent on offspring body weights and survival at 75 mg/kg bw/day and this dosage is classified as a NOEL for offspring growth and development.