Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In-life phase: 12th April 2012 to 7th June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
The OECD422 guideline used only observes the offspring until day 4 rather than until weaning / sexual maturity as in a full reproduction toxicity study.
Deviations:
no
Principles of method if other than guideline:
The body weight range of male animals at the start of treatment on this study was 315-362g which slightly exceeded the predicted range in the Study Plan (190 to 350g). All animals were of the corrected age specification and were considered acceptable for use on the study. This deviation from Study Plan was considered to have had no impact on the scientific integrity of the study.

The formulations of the Test Item used for pre-study chemistry were prepared in distilled water and not water obtained by reverse osmosis. For the purposes of formulations these was considered to be no practical difference between distilled water and water obtained by reverse osmosis, therefore this deviation from Study Plan was considered to have had no impact on the scientific integrity of the study.

Achieved concentration was scheduled to be measured on three occasions during the study. On the third occasion achieved concentration was lower than anticipated at the low dosage, although it was anticipated that this represented a sampling error (the sample being taken before the formulation had been completely mixed) rather than a problem with the formulation procedure. In view of this an addition sampling occasion was instigated to confirm the accuracy of the formulation procedure. It is considered that this deviation from Study Plan had no adverse impact on the scientific integrity of the study.

For animals 12, 13 and 14 the water residue was not recorded on Day 4 of gestation in error. Water residue was recorded on Day 5 and therefore it was possible to calculate the amount consumed by these animals during Day 3 and 4. As the water consumption data was presented over the period Day 0 to Day 7 of gestation there was no overall loss of data and there was considered to be no impact on the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Sponsor's identification :N-methylpiperazine
Description : Clear colourless liquid
Chemical name :N-methylpiperazine
Purity : 99.9%
Batch number : 101124
Label : N-METHYLPIPERAZINE 101124 NMP F-5711
CAS number : 109-01-3
UN number : 2734
Date received : 19 August 2011
Storage conditions :Ambient temperature, in the dark, over silica gel
Expiry date : Not supplied

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for eight days during which time their health status was assessed. A total of ninety animals (fifty males and forty females) were accepted into the study. At the start of treatment the males weighed 315 to 362g (see Principles of Method if other than Guideline), the females weighed 191 to 225g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the non-recovery dose group 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. Recovery group animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage on the day of arrival and during the treatment-free recovery period. Reverse osmosis water was supplied from seven days prior to the start of treatment and throughout the treatment period (either untreated or containing the required concentration of Test Item). The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK, Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system and print-outs of hourly temperatures and humidities are included in the study records. Study Plan target ranges for temperature and relative humidity 22 ± 3°C and 50 ± 20% respectively and there were no deviations from these target ranges.

The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on exposure:
For the purpose of this study, the test item was prepared at the appropriate concentrations as a solution in water obtained by reverse osmosis. Prior to treatment in the preliminary study (Harlan Laboratories Ltd., Project Number 41102856) the pH of formulations containing concentrations of the test item between 1 and 15 mg/ml were investigated and found to be alkaline. After discussions with the sponsor it was decided that test item formulations used on the preliminary study and this main study would be adjusted to an approximate pH of 9. No adjustment of pH was made for the reverse osmosis water supplied to the control group or for tap water supplied to recovery animals during the treatment-free recovery period.

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in water obtained by reverse osmosis. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services (see Deviations from Study Plan). Results show the formulations were homogeneous and that formulations were to be stable for at least ten days at 4°C and at room temperature (during storage in the drinking bottle used to deliver water to the animals). Formulations were generally prepared on a weekly basis (or more frequently depending on the number of cages being used at certain points of the study) and stored at ambient temperature in the animal room in the dark.

Samples of test item formulations were taken on four occasions during the study and analysed for concentration of N-methylpiperazine at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 26. The results indicate that the prepared formulations were within 84 to 101% of nominal concentration indicating that the formulation procedure was sufficiently accurate for the purpose of this study.

Details on mating procedure:
Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary
The concentration of N-methyl piperazine in the test item formulations was determined by gas chromatography (GC) using an external standard technique.


Samples
The test item formulations were diluted with methanol to give a final, theoretical test item concentration of approximately 0.01 mg/ml.


Standards
Standard solutions of test item were prepared in methanol at a nominal concentration of 0.01 mg/ml.


Procedure
The standard and sample solutions were analysed by GC using the following conditions:

GC system : Agilent Technologies 5890, incorporating autosampler and workstation
Column : DB-5 (30 m x 0.53 mm id x 5 µm film)
Oven temperature program :initial 50 ºC for 1 mins
rate 10 ºC/min
final 260 ºC for 0 mins
Injection temperature :250 ºC
Flame ionisation detector temperature 250 ºC
Injection volume: : 1 µl
Retention time : ~ 4.8 mins


Homogeneity Determinations
The test item formulations were assessed visually.


Stability Determinations
The test item formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark and room temperature (ambient) for ten days. This storage was conducted in the same type of water bottles used to deliver the drinking water to the animals.


Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed within two days of preparation.


Conclusion
Formulations analysed after 10 days at room temperature were within ±5% of initial concentration confirming stability of the test item in the aqueous matrix over this period.

Samples of formulations prepared on four occasions during the study were within 84-101% of nominal concentration. On one occasion at 0.5 mg/ml achieved concentration was only 84% of nominal however on the remaining occasions it was 93% or greater. It is suspected that the lower concentration observed on this occasion was due to sampling before the test item has fully dispersed with the vehicle matrix. It is considered that the formulation method and the formulations produced were sufficiently accurate for the purposes of the study.

Duration of treatment / exposure:
Up to fifty-three consecutive days (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 500, 2500 and 10000 ppm. A control group of ten males and ten females received untreated drinking water (obtained by reverse osmosis) over the same treatment period. Two recovery groups, each of five males received via the drinking water the high dose (1000 ppm) or untreated water alone for forty-two consecutive days and then were maintained without treatment (tap water) for a further fourteen days.
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study

Non-Recovery Dose Groups
i) Groups of ten male and ten female animals received the appropriate concentration of test item via the drinking water throughout the study with the first day of administration being 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 the pairing phase (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. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded 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, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.


Recovery Dose Groups
i) Groups of five male rats were dosed according to dose group (control and high dose only) continuously up to the point of sacrifice of non-recovery males at which time administration of the test item was discontinued.

ii) The males were maintained without treatment for a further fourteen days.

iii) Blood samples were taken for haematological and blood chemical assessment on Day 56.

iv) After fourteen days of recovery, males were killed and examined macroscopically.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 ppm
Basis:
nominal in water
Remarks:
Doses / Concentrations:
500 ppm
Basis:
nominal in water
Mean dosages: Males - 44 mg/kg bw/day, Females - prepairing - 49 mg/kg bw/day, Females - gestation - 61 mg/kg bw/day, Females - lactation - 87 mg/kg bw/day
Remarks:
Doses / Concentrations:
2500 ppm
Basis:
nominal in water
Mean dosages: Males - 190 mg/kg bw/day, Females - pre-pairing - 231 mg/kg bw/day, Females - gestation - 268 mg/kg bw/day, Females - lactation - 416 mg/kg bw/day.
Remarks:
Doses / Concentrations:
10000 ppm
Basis:
nominal in water
Mean dosage: Males - 466 mg/kg bw/day, Females - pre-pairing - 574 mg/kg bw/day, Females - gestation - 653 mg/kg bw/day, Females - lactation - 1055 mg/kg bw/day.
No. of animals per sex per dose:
10 animals per sex per dose (including control).
Control animals:
yes, concurrent no treatment
Details on study design:
The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.

Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change on a daily basis. During the treatment-free period, recovery animals were also observed daily. All observations were recorded.


Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioural toxicity.

Functional performance tests were also performed on five selected males and females from each non-recovery 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 non-recovery animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin colour
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.


Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

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).

The following parameters were observed:
Grasp response
Touch escape
Vocalisation
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach


Body Weight
Individual body weights were recorded on Day 1 and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.


Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed for each cage of recovery group animals throughout the study period.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for non-recovery males (except during the mating phase) and recovery group animals throughout the study period and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females, during gestation and lactation.


Water Consumption
Water intake was measured daily throughout the study (with the exception of the pairing phase), including the first week prior to Test Item administration.


Reproduction Screening

Mating
Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.


Pregnancy and Parturition
Each pregnant female was observed at approximately 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 non-recovery test and control group prior to termination (Day 42 for males and Day 4 post partum for females). In addition haematological and blood chemical investigations were performed on all recovery group animals after the fourteen day treatment-free period at termination (Day 56). 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
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded.
Sperm parameters (parental animals):
Testes weight
Testes histopathology
Litter observations:
Litter Data
On completion of parturition (Day 0 of post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)


Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Pathology
Adult non-recovery males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult non-recovery 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 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
Recovery group animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 57.
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)
Pituitary (post fixation)


Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides•
Skin (hind limb)
Eyes*
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes•
Lungs (with bronchi) #
Thymus
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cervix
Muscle (skeletal)
Vagina

All tissues were despatched to the histology processing Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing The tissues from five selected non-recovery control and 10000 ppm 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. The tissues shown in bold from the remaining control and 10000 ppm and any animals which did not achieve a pregnancy, were also processed. In addition, sections of testes and epididymides from all control and 10000 ppm males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Since there were no indications of treatment-related changes, examination was not extended to include similarly prepared sections of animals from the low, intermediate and recovery groups.

Microscopic examination was conducted (at AnaPath GmbH, Oberbuchsiten, Switzerland).
Postmortem examinations (offspring):
All offspring, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Due to the nature and quantity of this data please see section "any other information on material and methods including tables".
Reproductive indices:
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation.

i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:

Mating Index (%) = (Number of animals paired ÷ Number of animals mated) x 100

Pregnancy Index (%) = (Number of animals mated ÷ Number of pregnant females) x 100


Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:

Parturition Index (%) = (Number of pregnant females ÷ Number of females delivering live offspring) x 100
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

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 1 (%) = (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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Please refer to "details on results" section
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Please refer to "details on results" section
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 10000 ppm, water intake for both sexes was noticeably lower than control (and also from previous consumption prior to treatment) throughout the treatment period, and including for females the gestation and lactation phases of the study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Mortality
There were no unscheduled deaths on the study.


Clinical Observations
No clinical signs considered to be of any toxicological significance were apparent for adult animals during the study.

Yellow staining of the cage bedding was observed at 10000 ppm from Day 20 and 500 and 2500 ppm from Day 21. This was considered to reflect staining due to the test item and was considered to be of no toxicological significance.


Functional Observations

Behavioural Assessments
Assessment of the animals in an open arena did not reveal any adverse effects of treatment at 500, 2500 or 10000 ppm.


Functional Performance Tests
Functional performance, as assessed by measurement of grip strength and motor activity did not indicate any adverse effect of treatment at 500, 2500 or 10000 ppm.

At 10000 ppm, higher male fore limb grip strength during test 1 attained statistical significance compared with control. No further statistically significance differences were observed during the remainder of the test and, in isolation this finding was considered incidental and of no toxicological significance.


Sensory Reactivity Assessments
Sensory reactivity to different stimuli (auditory, visual and proprioceptive) did not indicate any adverse effect of treatment at 500, 2500 or 10000 ppm.


Body Weight
At 10000 ppm, body weight gain of males was generally slightly lower than control throughout the treatment period, with differences occasionally attaining statistical significance. At the end of treatment period, overall body weight gain was approximately 80% of the control. Recovery of body weight gain was apparent during the treatment-free recovery period with overall gain being similar to control at the end of the study.

Body Weight Gain during Days
1-43 43-57 1-57
Control 90.1 12.0 97.6
500 ppm 93.4 (104) - -
2500 ppm 82.5 (92)
10000 ppm 72.6** (81) 26.2* (218) 103.8 (106)
( ) = % Control

For females at 10000 ppm, body weight gains during the two week pre-pairing phase were slightly lower than control but, these differences from control failed to attain statistical significance and, at the level observed, may represent normal biological variation. However, subsequent body weight gains during gestation and lactation were clearly lower than control with both body weight and body weight gain frequently attaining statistical significance. The differences from control for body weight gain during gestation could not be attributed to differences in litter size for the pregnant females and appeared to represent an underlying effect on maternal body weight gain. Supporting this mean body weight on Day 1 of lactation was statistically significantly lower than control.

There was no adverse effect of treatment on body weight performance of either sex at 500 or 2500 ppm, including for females the gestation and lactation phases of the study.


Food Consumption and Food Efficiency
There were no adverse effects of treatment on food consumption for males observed during the study at 500, 2500 or 10000 ppm.

At 10000 ppm there was a suggestion of slightly inferior food conversion efficiency during the first week of the study but subsequent food utilisation was similar to control for the remainder of the study. There were no obvious effects of treatment on food conversion efficiency for males observed during the study at 500 or 2500 ppm.

There was no adverse effect of treatment on food consumption or food conversion efficiency of females during the pre-pairing phase of the study at 500, 2500 or 10000 ppm.

At 10000 ppm, food consumption of females was lower than control during gestation and lactation; differences from control were most marked during lactation, a period of high physiological demand on the female due to the demands of the litter.

There was no obvious effect of treatment on food intake of females at 500 or 2500 ppm during gestation and lactation.


Water Consumption
At 10000 ppm, water intake for both sexes was noticeably lower than control (and also from previous consumption prior to treatment) throughout the treatment period, and including for females the gestation and lactation phases of the study.


Water consumption (g/rat/day) during Weeks
-1 1 2 5 6
Males
Control 31.6 34.1 35.2 37.0 34.7
500 ppm 29.7 (94) 32.0 (94) 31.8 (91) 36.4 (98) 35.4 (102)
2500 ppm 31.4 (99) 28.3 (83) 26.0 (74) 31.0 (84) 30.1 (87)
10000 ppm 28.5 (90) 18.5 (54) 17.1 (49) 17.5 (47) 16.3 (47)

Females
Control 21.1 20.5 20.4
500 ppm 20.0 (95) 21.1 (103) 20.9 (103)
2500 ppm 24.3 (115) 20.7 (101) 18.7 (92)
10000 ppm 21.1 (100) 12.9 (63) 11.4 (56)
( ) = % Control

At 500 and 2500 ppm there was no clear effect of treatment on water intake for either sex.


Reproductive Performance

Mating
There were no effects of treatment on mating performance at 500, 2500 and 10000 ppm; the majority of animals mated within the first five days of pairing (this probably representing the first oestrus opportunity).

Fertility
There were no adverse effects of treatment on fertility, with the majority of matings leading to successful pregnancy at 500, 2500 and 10000 ppm.

Gestation Length
There was no adverse effects of treatment on gestation length at 500, 2500 and 10000 ppm.

At 500 ppm one female showed an extended gestation length of 25 days and subsequently showed total litter loss post partum. This female only had three implantations and a low litter size of this nature can lead to an extended gestation length, although a length of 25 days is unusual. The offspring continue to grow throughout gestation and by Day 25 post coitum may be a very large size for the female to give birth to. It is common for either the female or offspring to be compromised under these circumstances and the subsequent litter loss post partum was not unexpected. There was no indication that treatment was associated with any extension of gestation length at any of the dosages investigated and therefore this occurrence on the study was considered incidental and of no toxicological significance.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Adult toxicity
Effect level:
2 500 ppm (analytical)
Based on:
dissolved
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOEL
Remarks:
Fertility and reproduction
Effect level:
10 000 ppm (analytical)
Based on:
dissolved
Sex:
male/female
Basis for effect level:
reproductive performance

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Please refer to "details on results" section
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results" section
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Litter Responses
Two females at 500 ppm and one female at 10000 ppm were non-pregnant; additionally, as previously discussed one female at 500 ppm showed post partum litter loss. The following assessment is based on the 10, 7, 10 and 9 litters successfully reared to Day 5 of age for the control, 500, 2500 and 10000 ppm dosage groups respectively.


Offspring Litter Size, Sex Ratio and Viability
There was no adverse effect of treatment on corpora lutea and implantation counts or on subsequent initial post-natal litter size at 500, 2500 and 10000 ppm.

At 10000 ppm offspring survival to Day 4 was marginally lower than control, however this was mainly due to only two litters and the lower litter size on Day 4 did not attain statistical significance when compared with control.

Offspring survival from birth to Day 4 of age at 500 and 2500 ppm was unaffected by treatment. One female at 500 ppm showed total litter loss post partum but this was considered to be incidental and related to extended gestation length rather than any treatment related effect on offspring survival.

Sex ratio of offspring on Day 1 and Day 4 of age was unaffected by treatment at 500, 2500 and 10000 ppm indicating that there was no selective effect on survival for either sex.


Offspring Growth and Development
At 10000 ppm, there was no clear adverse effect of treatment on offspring body weight on Day 1 or subsequent body weight gain to Day 4 of age. Although mean body weights and body weight gain were marginally lower than control, they were adversely influenced by the same two litters that showed low post-natal survival and mean values did not attain statistical significance when compared to control. Mean values for offspring body weights and body weight gains at 10000 ppm were similar to the other treated groups and no adverse effect on offspring growth at 10000 ppm was considered proven. Litter weight at Day 1 and on Day 4, where statistical significance was reached, was more influenced by the marginally lower litter size at this dosage rather than offspring body weight.

At 500 and 2500 ppm, there were no statistically significant differences from control for offspring body weight or litter weight at Day 1 or Day 4 or body weight gain between Day 1 and Day 4.

The type, incidence and distribution of clinical signs observed on the study were typical for the age observed and did not indicate any underlying adverse effect on offspring development. At 10000 ppm, there was a higher incidence of offspring observed to be small, weak and have no milk in stomach, although many of these findings were attributable to one litter, which showed high offspring losses.

At 2500 and 10000 ppm, performance of offspring at Day 1 of age in the assessment of surface righting ability was slightly inferior to control, with differences attaining statistical significance at the high dosage. However, only one litter value at 10000 ppm was outside the historical range and group mean litter values compared well with that of the historical mean (87.5%). These differences probably represent particularly good performance for control litters rather than any treatment-related effect on offspring performance.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 2 500 ppm (analytical)
Based on:
dissolved
Sex:
male/female
Basis for effect level:
mortality

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Most findings observed at 10000 ppm were considered to be of little toxicological significance and were probably influenced, at least in part, by the marked reduction in water consumption due to palatability. However, clear effects on bodyweight gain and food consumption during lactation and gestation preclude this dosage from being a no observed adverse effect level (NOAEL) for the adult animal. The NOAEL for adult toxicity was therefore considered to be 2500 ppm.
The no observed effect level (NOEL) for fertility and reproduction was considered to be 10000 ppm. At 10000 ppm, lower offspring survival and growth from birth to Day 4 was observed and while an association with treatment was not considered proven, it was also difficult to discount. The NOAEL for offspring survival, growth and development was therefore considered to be at least 2500 ppm.
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 via the drinking water to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to fifty-three consecutive days (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 500, 2500 and 10000 ppm. A control group of ten males and ten females received untreated drinking water (obtained by reverse osmosis) over the same treatment period. Two recovery groups, each of five males received via the drinking water the high dose (1000 ppm) or untreated water alone for forty-two consecutive days and then were maintained without treatment (tap water) for a further fourteen days. Achieved dosages were as follows:

Study Phase

Mean dosage (mg/kg bw/day) at

500 ppm

2500 ppm

10000 ppm

Males

44

190

466

Females – pre-pairing

49

231

574

Females – gestation

61

268

653

Females – lactation

87

416

1055

Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study. 

Pairing of non-recovery 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 evaluations of litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partum. Five non-recovery males and females from each dose group were selected for haematology and blood chemistry assessments prior to termination.

Adult non-recovery males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Following forty-two days of treatment, recovery group animals were maintained without treatment for a further fourteen days. Haematological and blood chemical assessments were performed on all recovery group animals at the end of the treatment-free period. These animals were then subjected to a gross necropsy and histopathological examinations of selected tissues was performed.

Results.

Adult Responses:

Mortality.There was no unscheduled deaths on the study.

Clinical Observations.No clinical signs considered to be of any toxicological significance were apparent for adult animals.

Behavioural Assessment.Behavioural assessments did not indicate any adverse effects of treatment at 500, 2500 or 10000 ppm.

Functional Performance Tests.Grip strength and motor activity did not indicate any adverse effect of treatment at 500, 2500 or 10000 ppm. 

Sensory Reactivity Assessments.Sensory reactivity assessments did not indicate any adverse effect of treatment at 500, 2500 or 10000 ppm.

Body Weight.At 10000 ppm, male body weight gain tended to be slightly lower than control, with overall body weight gain being approximately 80% of control by the end of treatment; recovery of body weight gain was apparent at the end of the recovery period. For females at 10000 ppm, body weight gains during gestation and lactation were lower than control with differences for body weight and body weight gain frequently attaining statistical significance.   

Body weight gain of both sexes was unaffected by treatment at 500 or 2500 ppm.

Food Consumption.There were no adverse effects of treatment on food consumption for males at 10000 ppm, although slightly inferior food conversion efficiency was apparent during the first week of the study. For females at 10000 ppm, food consumption was lower than control during gestation and lactation with differences from control being most marked during lactation.

Food consumption and food conversion efficiency was unaffected by treatment at 500 and 2500 ppm.

Water Consumption.At 10000 ppm, water intake for both sexes was approximately 50% lower than control (and also lower than previous consumption prior to treatment) throughout the study.

At 500 and 2500 ppm there was no clear effect of treatment on water intake for either sex.

Reproductive Performance:

Mating.Mating performance was unaffected by treatment at 500, 2500 and 10000 ppm.

Fertility.Fertility was unaffected by treatment at 500, 2500 and 10000 ppm.

Gestation Lengths.Gestation length was unaffected by treatment at 500, 2500 and 10000 ppm.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.Corpora lutea, implantation counts, subsequent post-natal litter size and sex ratio were not adversely affected by treatment at 500, 2500 and 10000 ppm. 

At 10000 ppm, offspring survival from birth to Day 4 was marginally lower than control but an association with treatment was considered unproven. Offspring survival to Day 4 of age at 500 and 2500 ppm was unaffected by treatment.

Offspring Growth and Development.At 10000 pm, offspring body weight on Day 1 and subsequent body weight gain to Day 4 of age were marginally lower than control but an association with treatment was considered unproven. Lower litter weight at Day 4 attained statistical significance but was more influenced by marginally lower litter size than offspring body weight.

There was considered to be no adverse effect of treatment on offspring body weight and litter weight at Day 1 and Day 4 and body weight gain to Day 4 at 500 and 2500 ppm. 

Offspring clinical signs and assessment of surface righting ability were considered not to indicate any underlying effect on offspring development at 500, 2500 and 10000 ppm.

Laboratory Investigations:

Haematology.There was no adverse effects of treatment on haematology parameters at 500, 2500 and 10000 ppm.

Blood Chemistry.There was no adverse effects of treatment on blood chemistry parameters at 500, 2500 and 10000 ppm.

Pathology:

Necropsy.

Offspring

Necropsy findings for decedent offspring or offspring killed at Day 5 of age did not indicate any underlying adverse effect of treatment at 500, 2500 or 10000 ppm.

Adults

Necropsy findings for both sexes did not indicate any adverse effect of treatment at 500, 2500 or 10000 ppm.

Organ Weights.For both sexes at 10000 ppm, lower absolute and body weight relative liver and spleen weights attained statistical significance when compared with control. A statistically significant decrease in spleen weights was also apparent for females at 500 and 2500 ppm. For females at 10000 ppm, higher absolute and body weight relative kidney weights attained statistical significance compared with control. No similar effect was apparent for these organ weights for recovery males at 10000 ppm. In the absence of any corresponding histopathological change, these finding were considered to be of no toxicological significance.

For males at all dosages, absolute and body weight relative prostate and seminal vesicle weights were statistically significantly lower than control. There was no statistically significant decrease for recovery males at 10000 ppm, no evidence of histopathological change and no adverse effect of fertility; therefore this finding was considered to be of no toxicological significance. 

For males at 2500 ppm, absolute and body weight relative epididymal weights were statistically significantly lower than control but, with no similar decrease for males at 10000 ppm, this finding was considered incidental and unrelated to treatment.

For recovery males at 10000 ppm, absolute and body weight relative thymus and thyroid weights were statistically significantly lower than control. No significant decrease for these organ weights or evidence of histopathological change was apparent at the end of treatment and these findings were considered incidental and unrelated to treatment.

Histopathology.The test item N-methylpiperazine produced no histological evidence of toxicological properties in the organs and tissues examined.

Conclusion.Most findings observed at 10000 ppm were considered to be of little toxicological significance and were probably influenced, at least in part, by the marked reduction in water consumption due to palatability. However, clear effects on bodyweight gain and food consumption during lactation and gestation preclude this dosage from being a no observed adverse effect level (NOAEL) for the adult animal. The NOAEL for adult toxicity was therefore considered to be 2500 ppm.

The no observed effect level (NOEL) for fertility and reproduction was considered to be 10000 ppm. At 10000 ppm, lower offspring survival and growth from birth to Day 4 were observed and while an association with treatment was not considered proven, it was also difficult to discount. The NOAEL for offspring survival, growth and development was therefore considered to be at least 2500 ppm.