Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
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
Details on test animals 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 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.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
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.
Duration of test:
See duration of treat/exposure.
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:
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.

Examinations

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

REPRODUCTION SCREENING:
PREGNANCY AND PARTURITION:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition


LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

HAEMATOLOGY:
Parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant.

BLOOD CHEMISTRY:
Parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant.

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

All adult animals 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 tissuess were removed from all animals and preserved in buffered 10% formalin.































Ovaries and uterine content:
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.
Fetal examinations:
Not examined.
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
Indices:
Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of implantation sites) ÷ Number of corpora lutea] x 100
% post – implantation loss =[(Number of implantation sites - Total number of offspring born) ÷ Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 ÷ Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1 ) x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100
Historical control data:
Normal range data for the different parameters examined were used in comparison against the test data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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:
no effects observed
Details on 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.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, treatment-related
Dead fetuses:
effects observed, treatment-related
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, treatment-related
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
REPRODUCTIVE PERFORMANCE
MATING
There was no adverse effect of treatment on mating performance at any of the dosages investigated. The majority of animals mated within the first five days of pairing (i.e. at the first oestrous opportunity) and only one mating failed to result in a pregnancy.

FERTILITY
Fertility, as assessed by the number of pregnant females, was unaffected by treatment at any of the dosages investigated; all females were pregnant with the exception of one female at 75 mg/kg bw/day.

GESTATION LENGTH:
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.

For the Control and 30 mg/kg bw/day groups the majority of females showed gestation lengths of 23 days or less; the only two females that showed a longer gestation period (one control female, one female at 30 mg/kg bw/day) both showed total litter loss post partum. At 75 and 150/100 mg/kg bw/day the majority of females delivered on Day 23 ½ or later but despite the comparatively later delivery only one female at 150/100 mg/kg bw/day showed total litter loss post partum.

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
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:
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 (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
NOEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Changes in postnatal survival:
effects observed, treatment-related
External malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Effect levels (fetuses)

Dose descriptor:
other: NOEL/ 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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

LITTER RESPONSE:

In total nine, eight, nine and nine females from control, 30, 75 and 150/100 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is generally based on all litters reared to termination on Day 5 of lactation/age.

OFFSPRING LITTER SIZE, VIABILITY AND SEX RATIO:

At 150/100 mg/kg bw/day, while corpora lutea count, pre-implantation loss and implantation count were similar to control, subsequent post partum litter size was lower due to higher post-implantation loss. Despite this lower litter size, survival to Day 4 appeared slightly inferior to control, although differences for the viability index did not attain statistical significance.

At 30 and 75 mg/kg bw/day, there was no obvious effect on corpora lutea and implantation counts, pre and post-implantation losses, post partum litter size or neonatal survival to Day 5 of age.

One control female, one female at 30 mg/kg bw/day and another female at 150/100 mg/kg bw/day showed total litter loss post partum. The control and 30 mg/kg bw/day both females showed a tendency to extended gestation length. In the absence of any effects on offspring survival at 30 mg/kg bw/day, the litter loss at this dosage was considered to be coincidental and unrelated to treatment. At the higher dosage of 150/100 mg/kg bw/day, lower offspring survival was observed for the other litters reared to Day 5 and an association with the litter loss and treatment at this dosage could not be discounted.

At 30 mg/kg bw/day, the one female that was not observed to give birth was found to have one implantation site at necropsy and was suspected as having total litter loss in utero. It is not unusual for females with such a low litter size to fail to retain their litter to parturition and this occurrence was considered to be incidental and unrelated to treatment.

There was no obvious adverse effect of treatment on sex ratio as assessed by the percentage male at birth, Day 1 or Day 4 at all dose levels.

At 150/100 mg/kg bw/day sex ratio was noted to be higher than control (indicating a greater number of males compared to females siblings), however differences were not statistically significant and were considered insufficient to indicate that any selective effect on survival for either sex had occurred.

OFFSPRING GROWTH AND DEVELOPMENT

There was no obvious adverse effect on body weight of the offspring on Day 1 and Day 4 or on body weight gain to Day 4 of age at any of the dosages investigated.

At 150/100 mg/kg/day litter weight was lower than control on Day 1 and Day 4 as a consequence of the lower litter size at this dosage; differences in litter weight attaining statistical significance at Day 1 of age. Litter weights on Day 1 and 4 were similar to control at 30 and 75 mg/kg bw/day.

The type and incidence of clinical signs and the performance of the offspring during assessment of surface righting at Day 1 of age did not indicate any obvious adverse effect of treatment at any of the dosages investigated.

At 150/100 mg/kg/day the group incidence of clinical signs appeared slightly higher than the control and other dosages, however the majority of offspring showing clinical signs belonged to the same female, which subsequently showed total litter loss before Day 4. This increased incidence was therefore considered likely to be incidental rather than treatment related.

PATHOLOGY

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.

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 partumHaematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Day 43, followed by the termination of 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.