Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

There is an OECD422 reproductive screening test available for 1-methyl piperazine which is of high quality with a Klimisch 1 rating. This study showed no effects on fertility or any other reproductive effects on the parental animals.

Link to relevant study records
Reference
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.
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
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.
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.
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
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
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 2 500 ppm (analytical)
Based on:
dissolved
Sex:
male/female
Basis for effect level:
mortality
Reproductive effects observed:
not specified
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
466 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
There is a high quality OECD422 study via the oral (drinking water) route. The database is therefore considered to be adequate.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
We do not have an inhalation test for reproduction toxicity, but there is a high quality OECD422 study via the oral (drinking water) route. ECHA guidance allows the NOAEL from this study to be used to calculate the systemic inhalation DNEL, with an appropriate addition assessment factor of 2. The database is therefore considered to be adequate.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
We do not have a dermal test for reproduction toxicity, but there is a high quality OECD422 study via the oral (drinking water) route. ECHA guidance allows the NOAEL from this study to be used to calculate the systemic dermal DNEL, as dermal absorption is assumed to be the same as oral ingestion, which is probably conservative for 1-methyl piperazine. The database is therefore considered to be adequate
Additional information

There is an OECD422 reproductive screening test available for 1-methyl piperazine which is of high quality with a Klimisch 1 rating. This study showed no effects on fertility or any other reproductive effects on the parental animals. The highest dose which was 10’000ppm in the drinking water, resulted in a ca. 50% reduction in water consumption so this was the maximum achievable dose level, as higher concentration would have resulted in even greater reduction in water consumption which would have been likely to compromise the rats survival. This concentration was equivalent to 466mg/kg bodyweight/day for the males and 574-1055 mg/kg bodyweight/day for the females, the highest dose which is in excess of the 1000mg/kg upper limit dose, was due to increased water consumption during lactation, but even then, actual consumption was some 33% compared to the controls due to the unpalatability of the test substance. The male dose of 466 mg/kg bodyweight/day will be used as the NOAEL for effects on fertility.

 

Effects on developmental toxicity

Description of key information

There is an OECD422 reproductive screening test available for 1-methyl piperazine which is of high quality with a Klimisch 1 rating.  This study is a screening study, which is capable of detecting adverse effects on the pre and post implantation losses etc and survival and development of the offspring up to day 4 after parturition. 

There are also modern Klimisch 1 OECD414 pre-natal development studies in rats and rabbits on the read across substance 2-piperazin-1-ylethylamine (Amino ethyl piperazine).

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
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:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test”
Version / remarks:
The OECD422 does not include skeletal examinations as included in the OECD414 guideline test for pre-natal development.
Deviations:
no
Principles of method if other than guideline:
DEVIATIONS FROM STUDY PLAN
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
Species:
rat
Strain:
Wistar
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 Deviations from Study Plan), 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. Certificates of analysis of the batches of diet used are given in Addendum 1. 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.
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 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.
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.
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.
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
Duration of test:
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.
Remarks:
Doses / Concentrations:
0 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
500 ppm
Basis:
analytical conc.
Mean dosages: Males - 44 mg/kg bw/day, Females - pre-pairing - 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:
analytical conc.
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
Basis:
analytical conc.
Mean dosages: 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.


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 trans
ferred 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.
Maternal 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


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 by the Study Pathologist (at AnaPath GmbH, Oberbuchsiten, Switzerland).
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 (Salewski 1964).
Fetal examinations:
Litter Data
On completion of parturition (Day 0 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)
Statistics:
Due to the nature and quantity of this data please see Section "any other information on materials and methods incl. tables"
Indices:
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 mated/number if animals paired) x 100

Pregnancy Index (%) = (number of pregnant females / number of animals mated) 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 females delivering live offspring / number of pregnant females) x 100
Historical control data:
Not applicable.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Please refer to "details on maternal toxic effects"

Details on maternal toxic effects:
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.


Laboratory Investigations

Haematology
For males at 2500 and 10000 ppm, lower group mean eosinophil count at the end of treatment attained statistical significance when compared to control; however group mean values at these dosages were close to that of the historical control data. Additionally, values for treated animals were all within the historical control range, while one control value exceeded it, and there was no corresponding statistically significant difference in overall total leucocyte count for males at these dosages, compared to control. In view of this, and in the absence of any histopathological correlates, this finding was considered incidental and of no toxicological significance.

No statistically significant differences from control for haematology parameters were apparent for males at 500 ppm at the end of treatment or for males at 10000 ppm at the end of the two week treatment-free recovery period.

For females at 10000 ppm, total leucocyte count was lower than control, principally due to lower numbers of neutrophils and lymphocytes; differences for all these parameters attained statistical significance. Values for lymphocytes were lower than the historical control range for two animals at 10000 ppm but only one of these animals showed a total leucocyte count lower than the historical control range. For the control group, neutrophil and total leucocyte count for one female exceeded the historical control. Overall, in the absence of any histopathological correlates, the decrease in these haematology parameters was considered unlikely to be of any toxicological significance.

No statistically significant differences from control for haematology parameters were apparent for females at 500 or 2500 ppm.


Blood Chemistry
For males at 2500 and 10000 ppm, lower mean total cholesterol levels attained statistical significance when compared to control. Group mean values for the treated animals were close to the historical control mean, while the mean control value exceeded the historical control range; the differences observed were, therefore, considered to be incidental and reflect atypically high control values rather than any effect of treatment.

At 10000 ppm, higher blood chloride levels for males attained statistical significance when compared to control; all individual values at 10000 ppm were within the historical control range and the mean value at 10000 ppm was close to the historical control mean. In isolation, and in the absence of any histopathological correlates, this finding was considered to be of no toxicological significance.

For males at all dosages, inorganic phosphorus levels were lower than control but, although differences attained statistical significance, there was no dosage relationship. Values for all treated animals were within the historical control range and the group mean values were close to the historical control mean. In the absence of any supporting histopathological findings, this finding was considered to be unrelated to treatment and to be of no toxicological significance.

No statistically significant differences from control for blood chemistry parameters were apparent for recovery males at 10000 ppm.
For females at all dosages, mean billirubin levels were lower than control but, although differences attained statistical significance, there was no dosage relationship. The mean values for all groups were higher than the historical control mean but the control mean also exceeded the historical control range. A decrease in billirubin levels, in the absence of any effects on erythrocyte parameters, is unlikely to indicate an adverse effect of treatment. This finding was, therefore, considered to be of no toxicological significance and to reflect atypically high control values.


Pathology

Necropsy


Adults
Necropsy of adult animals did not indicate any obvious adverse effect of treatment at 500, 2500 or 10000 ppm.

One male and one female at 500 ppm, one male and two females at 2500 ppm and five females at 10000 ppm showed reddened lungs at necropsy following the end of treatment. No similar necropsy findings were apparent for males at 10000 ppm at the end of treatment, although a similar finding was apparent for one male at this dosage following the two week recovery period. While the aetiology of this finding is uncertain, there was no evidence of any histopathological change in the tissues for these animals and, therefore, this finding was considered to be of no toxicological significance.

The incidence of other findings observed for other animals was unremarkable and considered to be of no toxicological significance.


Organ Weights
At 10000 ppm, lower absolute and body weight relative liver weights for both sexes attained statistical significance when compared with control. No similar statistically significant decrease was apparent for males at this dosage following the two week recovery period and, in the absence of any evidence of histopathological change, this finding was considered to be of no toxicological significance. Additionally for females at 10000 ppm, higher absolute and body weight relative kidney weights attained statistical significance compared with control; again there was no accompanying histopathological change observed and this finding was therefore considered to be of no toxicological significance.

For males at 10000 ppm and females at all dosages, lower spleen weights attained statistical significance compared with control. There was no statistically significant decrease apparent for recovery males at 10000 ppm at the end of the treatment-free recovery period and, in the absence of any evidence of histopathological change, this finding was considered to be of no toxicological significance.

For males at all dosages, lower absolute and body weight relative prostate and seminal vesicle weights attained statistical significance compared with control. No statistically significant decrease was apparent for recovery males at 10000 ppm at the end of the treatment-free recovery period. In the absence of any evidence of histopathological change and no adverse effect of fertility indicating any functional deficit, this finding was considered to be of no toxicological significance.

For males at 2500 ppm, lower absolute and body weight relative epididymal weights attained statistical significance compared with control, but no similar decrease was apparent males at 10000 ppm, and this finding was considered incidental and unrelated to treatment.

For recovery males at 10000 ppm following at the end of the two week treatment-free period, 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 for these organs were apparent for males at the end of treatment. The lower thymus and thyroid weights for recovery males were therefore 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.

Dose descriptor:
NOAEL
Effect level:
2 500 ppm (analytical)
Based on:
dissolved
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
10 000 ppm (analytical)
Based on:
dissolved
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: Please refer to "details on embryotoxic/teratogenic effects"

Details on embryotoxic / teratogenic effects:
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.


Dose descriptor:
NOAEL
Effect level:
>= 2 500 ppm (analytical)
Based on:
dissolved
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

The OECD422 does not include skeletal examinations as included in the OECD414 guideline test for pre-natal development.

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 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.
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.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, Margate
- Age at study initiation: ca 9-10 weeks old
- Weight at study initiation: 222-298 g
- Housing: Animals were housed 2 per cage in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings which were provided with a certificate of analysis for significant contaminants. An analytical certificate for each batch of bedding used has been retained at Charles River, Edinburgh.
- Diet: SDS VRF-1 breeder diet was provided ad libitum throughout the study, except during designated procedures.The feed is analysed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are retained at Charles
River, Edinburgh. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): The animals had access to water ad libitum from the public supply from polycarbonate water bottles which were changed as necessary throughout the course of the study. The water used by Charles River Edinburgh is analysed at regular intervals for dissolved materials, heavy metals, pesticide residues, pH, nitrates and nitrites. Microbiological screening is also conducted. An analytical certificate for each analysis is retained at Charles
River, Edinburgh. The water used is considered not to contain any additional substances, in sufficient concentration, to have any influence on the outcome of the study.
- Acclimation period: Animals were allowed to acclimatise to the Charles River rodent toxicology accommodation from arrival until dosing on Day 6 of gestation (3-5 days).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 44% to 77%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. All formulations were adjusted to pH 9 with HCl. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulations were also stirred continuously during dosing. Any residual volumes were discarded.

VEHICLE
The control item, Milli-Q water, was prepared weekly, stored in a refrigerator set to maintain 4°C and dispensed daily for administration to Group 1 control animals. The control item was removed from the refrigerator and stirred for at least 30 minutes before dosing. The control item was also stirred continuously during dosing. Any residual volumes were discarded.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All samples to be analysed were transferred to the analytical laboratory at the Test Facility and analysed within the established stability period. Any residual/retained analytical samples have been discarded. Analyses were performed by Gas Chromatography with Flame Ionisation using a validated analytical procedure.

Duplicate top, middle and bottom sets of samples (duplicate middle only for control) at volumes of 0.5 mL (by weight) for Group 1 and 0.1 mL (by weight) for Groups 2 – 4 and triplicate top, middle and bottom (triplicate middle samples only for control) sets of back up samples (of the same volumes) were taken from formulations prepared for use on Day 1 and Week 2. The analytical samples were transferred to the analytical laboratory for analysis, stored in a refrigerator set to maintain 4°C and were analysed within the established stability period. Concentration results were accepted if mean sample concentration results were
within or equal to ± 10%. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of ≤ 10% for each group.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
The test and control items were administered to the appropriate animals by once daily oral gavage from Days 6-19 of gestation
Frequency of treatment:
Daily
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral gavage route of administration was selected for this study as this was defined as an appropriate method to define prenatal development toxicity and may be a possible route of human exposure.
The dose levels were selected in agreement with the Sponsor following review of the data from a preliminary prenatal developmental toxicity study using the same test item (Charles River Study No. 497008) and the recommended limit dose of 1000 mg/kg/day from the regulatory guidelines (OECD Guideline 414).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once in the morning and once as late as possible during the working day, throughout the study, animals were observed for general health/mortality and moribundity. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, from the start of dosing animals were removed from their cage for examination. Prior to dosing and regularly throughout the day, all the animals were examined for reaction to treatment with particular attention being paid to the animals during and for the first hour after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed once during pretrial on Day 5 of gestation and daily during the dosing period from Day 6 – 20 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was quantitatively measured for all animals daily from Day 6 of gestation (first measured quantity given on Day 5 of gestation).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored by visual inspection of the water bottles on a regular basis
throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All adult animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: Fetuses were examined for external abnormalities. Late embryonic deaths were examined for external abnormalities to the extent possible. Each implant was classified as being live, or a dead fetus (dead full term fetus that showed no sign of maceration), or a late embryonic death (macerated tissue identifiable as an embryo fetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may be of varying size).

- Visceral examinations: Yes: Half of the viable fetuses from each uterus were fixed in methylated ethyl alcohol. Following initial fixation fetuses were examined internally for sex and eviscerated; the viscera were not examined from fetuses prior to disposal. The remaining half of viable fetuses from each uterus were fixed in Bouin's fluid. The fetuses fixed in Bouin's fluid were examined for soft tissue abnormalities and sex using a freehand sectioning technique derived from that of Wilson.

- Skeletal examinations: Yes:The eviscerated carcasses were macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification.

Statistics:
Means and standard deviations were calculated for body weight, food consumption, pregnancy data and fetal weights and group incidence data was calculated for fetal abnormalities and variations.
Where required to assist interpretations, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. All statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group. Body weight and food consumption, data were analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appear homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F-test is significant. If the variances are heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remain heterogeneous, then a Kruskal-Wallis nonparametric ANOVA were used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical Observations: All animals dosed at 300 and 1000 mg/kg/day had evidence of yellow staining of the cage shavings for the majority of the study. There were no other clinical observations that were considered to be related to test item administration.

Body Weights and Body Weight Changes: At 1000 mg/kg/day approximately 50% of animals had a slight body weight loss after the first day of dosing. This was recovered by Day 2 of dosing (Day 7 of gestation); however, the overall body weight gains for the dosing period were reduced compared to controls. At dose levels up to 300 mg/kg/day the group mean body weight gains were similar to the control group for the duration of the study.

Feed Consumption: At 1000 mg/kg/day, when compared with Controls, there was a slight reduction in group mean food consumption for the majority of the dosing period with a maximum daily reduction of -28%. This was most notable during the start and end of the dosing period. At dose levels up to 300 mg/kg/day the group mean food consumption performance was similar to the control group for the duration of the study.

Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The type and distribution of all fetal abnormalities and variations, including those indicating the extent of skeletal ossification were similar in all groups and did not indicate any association with test item administration.
There were no treatment related differences in fetal weights, group mean gravid uterine weight or any of the pregnancy performance parameters evaluated. Any slight intergroup differences are considered to be due to natural variation and are considered to be unrelated to test item administration.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Administration of Aminoethylpiperazine at 1000 mg/kg/day was associated with slight reductions in food consumption and body weight gain along with yellow staining of the cage shavings at 300 and 1000 mg/kg /day. At dose levels up to 1000 mg/kg/day there were no test item-related effects on gross
pathology findings, pregnancy performance parameters or fetal weights, fetal abnormalities and variants.
In conclusion, under the conditions of this study, the maternal no observed effect level (NOEL) was considered to be 100 mg/kg/day based on yellow staining of the cage shavings at 300 mg/kg/day, and the maternal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day. There was no effect on embryofetal development or survival detected at any dose level; therefore, the embryofetal no observed effect level (NOEL) was considered to be 1000 mg/kg/day.
Executive summary:

The objective of this study was to determine the potential maternal and developmental toxicity of Aminoethylpiperazine when given orally during the period of organogenesis to pregnant rats. Four groups of 24 female Sprague-Dawley rats were dosed orally by gavage on Days 6-19 of gestation (where Day 0 was detection of mating).

The following parameters and end points were evaluated in this study: clinical observations, body weights and body weight changes, food consumption, gross necropsy findings, gravid uterine weight, examination of pregnancies and fetal evaluations (external abnormalities, fetal weights, visceral and skeletal evaluations). Animals were killed on Day 20 of gestation.

There were 2 premature decedents in this study (Animal 37 on Day 15 of gestation at 100 mg/kg/day and Animal 91 on Day 18 of gestation at 1000 mg/kg/day). The cause of these deaths is unknown; however, there was no evidence to indicate any association with the dosing procedure or the test item and, therefore, both deaths are considered to be incidental.

Administration of Aminoethylpiperazine at 1000 mg/kg/day was associated with slight reductions in food consumption and body weight gain along with yellow staining of the cage shavings at 300 and 1000 mg/kg /day. At dose levels up to 1000 mg/kg/day there were no test item-related effects on gross pathology findings, pregnancy performance parameters or fetal weights, fetal abnormalities and variants.

In conclusion, under the conditions of this study, the maternal no observed effect level (NOEL) was considered to be 100 mg/kg/day based on yellow staining of the cage shavings at 300 mg/kg/day, and the maternal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day. There was no effect on embryofetal development or survival detected at any dose level; therefore, the embryofetal no observed effect level (NOEL) was considered to be 1000 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
According to OECD 414 guidelines. Contingent on the outcome of the study in the rat.
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK
- Age at study initiation: 4-5 months old
- Weight at study initiation: 2.5 and 4 kg.
- Fasting period before study:
- Housing: Females were housed individually in appropriately sized stainless steel cages with a ‘Noryl’ dual level interior and perforated floor. Beneath each cage was a suspended tray containing absorbent paper. Bedding material was provided with a certificate of analysis for significant contaminants. An analytical certificate for each batch of bedding used was retained at Charles River Laboratories, Edinburgh. Animals were allowed a period of exercise in a separate floor pen.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 to 5 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17°C to 18°C
- Humidity (%): 35% to 61%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures.
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Test item dosing formulations were prepared based on a method established at the Test Facility under Study No. 432775 at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 4C, and dispensed daily. All formulations were adjusted to pH9 with HCl. The dosing formulations were removed from the refrigerator and were stirred for at least 30 minutes before dosing. The dosing formulation was also stirred continuously during dosing. Details of the preparation and dispensing of the test item have been retained in the Study Records.
- Storage temperature of food: The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 4C, and dispensed daily

VEHICLE
The control item, Milli-Q water, was prepared weekly, stored in a refrigerator set to maintain 4C, and dispensed daily. The prepared control item was removed from the refrigerator and stirred for at least 30 minutes before dosing. The control item was also stirred continuously during dosing. Details of the preparation and dispensing of the control item have been retained in the Study Records.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by Gas Chromatography with Flame Ionisation using a validated analytical procedure .
Concentration and Homogeneity Analysis:
Samples for Analysis: Duplicate top, middle, and bottom samples (duplicate middle only for Groups 1 and 3); sent for analysis as noted in Section 8.6.3 (see Appendix 1 for deviations and other events).
Backup Samples: Triplicate top, middle, and bottom samples (triplicate middle only for Groups 1 and 3); maintained at the Test Facility (see Appendix 1 for deviations and other events).
Sampling Containers: Appropriate sized glass containers.
Sample Volume: Group 1: 0.5 mL (by weight) for analysis and backup samples. Groups 2 to 4: 0.1 mL (by weight) for analysis and backup samples.
Storage Conditions:2-8ºC
Acceptance Criteria: For concentration, the criteria for acceptability were the mean sample concentration results within or equal to ± 10% of theoretical concentration. For homogeneity, the criteria for acceptability will be a relative standard deviation (RSD) of concentrations of £ 10% for each group

Stability Analysis: Stability analyses performed previously in conjunction with Charles River Study No.432775 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study

Duration of treatment / exposure:
Day 6 to Day 28 of gestation
Frequency of treatment:
once daily
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were decided after evaluation of existing toxicity data including a Preliminary Developmental Toxicity Study of Aminoethylpiperazine in the rabbit (Charles River Study Number 497029) where administration at 150 mg/kg/day resulted in slight reductions in food consumption and fetal weight
- Other: At study assignment, each animal was identified using a subcutaneously implanted electronic cylindrical, ‘glass-sealed’ TROVAN microchip.
For each mated female, records were supplied of its parentage (mother and father) and the identity of the inseminating male was also supplied
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once at the start and once towards the end of the working day throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Day 4 of gestation, then daily from Day 6 to 29 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 4 of gestation, then daily from Day 6 to 29 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was quantitatively measured. In the event that food consumption for an animal was low, hay consumption was monitored to assess the welfare of the animals. In addition, when food consumption was low animals were given some time in an “exercise area”, at the technicians discretion. The full details were recorded and retained within the study data.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #29
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Fetal examinations:
- External examinations: Yes: Fetuses were examined for external abnormalities including macroscopic examination of the eyes, cranial bones, brain, nasal passage and tongue following removal of the skin. Late embryonic deaths and dead fetuses were examined for external abnormalities to the extent possible
- Soft tissue examinations: Yes: Prior to fixation, the fetuses were sexed and examined by open dissection for abnormalities of the thoracic and abdominal viscera. For half of the fetuses there was macroscopic examination of the eyes and cranial bones, following removal of the skin from these areas; the cranium was sectioned once through the coronal suture to allow inspection of the brain in that region. For the remaining fetuses, the head was removed from the spine (as close to the head as possible) and placed in Bouins fluid for subsequent serial sectioning and evaluation (see Appendix 1 for deviations and other events).
The internal structure of the heart and kidneys of all fetuses was examined.
The thoracic and abdominal viscera were then discarded and the fetuses were fixed in methylated ethyl alcohol
- Skeletal examinations: Yes: All of the eviscerated carcasses were then macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. All the preparations were then examined for the presence of skeletal abnormalities and for the extent of ossification.
- Head examinations: Yes:The heads fixed in Bouins fluid were examined for soft tissue abnormalities using a free hand sectioning technique derived from that of Wilson et al. 1965.
Statistics:
Means and standard deviations were calculated for body weight, food consumption, pregnancy data and fetal weights and group incidence data was calculated for fetal abnormalities and variations.
All statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Body weight and food consumption, data were analysed for homogeneity of variance using the ‘F Max' test. If the group variances appear homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F test is significant. If the variances are heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remain heterogeneous, then a Kruskal-Wallis non-parametric ANOVA were used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical Observations: Administration at 150 mg/kg/day was associated with intermittent abnormal faecal output in 5/24 animals including small, decreased, abnormal colour and liquid faeces. Periods of decreased or small faeces in individual animals in the 150 mg/kg/day group were associated with extended periods of greatly reduced food consumption). Also at this dose level one animal was found to have red staining of the cage tray paper over Days 26-27 of gestation.
At dose levels up to and including 75 mg/kg/day there were no clinical observations that were considered to be test item-related. Minor observations such as sparse hair, fur staining, scab(s) etc were found at a similar incidence throughout the dose groups including controls, therefore, were considered to be incidental and not related to test item administration.

Body Weights Body Weight Changes: Administration at 150 mg/kg/day was associated with a slight reduction in group mean body weight gain throughout the dosing period (-17%), when compared to controls. When the body weight gain over Days 6-29 of gestation is adjusted for gravid uterine weight all groups show a body weight loss; however, it is noted that the group mean body weight loss at 150 mg/kg/day is slightly higher than controls.
The group mean body weight gain at dose levels up to and including 75 mg/kg/day was similar to control throughout the dosing period.

Food Consumption: Administration at 150 mg/kg/day was associated with a reduction in food consumption throughout the majority of the dosing period, when compared to controls. This was most notable over Days 7-21 of gestation where the daily group mean food consumption was found to be reduced by up to -38%.
The group mean food consumption at dose levels up to and including 75 mg/kg/day was similar to control throughout the dosing period.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal Abnormalities and Variants: When compared to controls, administration at 150 mg/kg/day was associated with an increase in the number of fetuses with unossified/incompletely ossified bones including skull bone(s), hyoid, odontoid process, cervical centrum, pubis(es), epiphyses, metacarpals and phalanges. The incidence of all other skeletal abnormalities and variations were similar to controls in all dose groups, including the number of ribs.
At dose levels up to and including 150 mg/kg/day there were no effects on the number or type of fetal abnormalities or variations, with the exception of at 150 mg/kg/day where there was a slight increase in fetuses noted as being small.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Pregnancy Performance, Gravid Uterine and Fetal Weights:

Administration at 150 mg/kg/day was associated with an increase in dead implants (early deaths, late deaths and dead fetuses) with a corresponding reduction in live implants, when compared to total implants and controls. The percentage of dead implants increased from 8% in controls to 20% at 150 mg/kg/day, resulting in an increase in post-implantation loss at this dose level. A slight decrease in fetal weight and gravid uterine weight was also noted at this dose level, with a 7% and 11% reduction in the mean litter mean fetal weight and mean gravid uterine weight, respectively, compared to controls. 

There were no similar test item-related effects on implant survival, fetal or gravid uterine weights at dose levels up to and including 75 mg/kg/day.

At dose levels up to and including 150 mg/kg/day there were no test item-related effects on pregnancy frequency, number of corpora lutea and implants or the pre-implantation loss. 

Conclusions:
The reduced body weight gain observed in animals administered Aminoethylpiperazine at 150 mg/kg/day corresponded with a reduction in food consumption throughout the majority of the dosing period along with an associated delay in fetal development, as observed by the reduced fetal weights and decreased bone ossification at this dose level.
Also at 150 mg/kg/day, the decrease in live implants correlated with an increase in dead implants on Day 29 of gestation and of the surviving fetuses, several fetuses were noted as being small at necropsy. With the exception of the decreased embryofetal survival, delayed ossification and reduced fetal weight, there were no other fetal abnormalities and variations that could be positively associated with treatment at this dose level.
Executive summary:

The objective of this study was to detect effects ofAminoethylpiperazineon embryofetal development in the rabbit following oral administration of the test item from Day 6 to 28 of gestation. The study also provided information on any maternal toxicity.

The study design was as follows:

Text Table 1: Experimental design

 Group No.  Test Item  Dose Level (mg/kg/day)  Dose Volume (mL/kg)  Dose Concentration (mg/mL)  No. of Animals (Females
 1  Control  0  5  0  24
 2  AEP  25  5  5  24
 3  AEP  75  5  15  24
 4  AEP  150  5 30   24

Vehicle/control was Milli-Q water.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, gross necropsy findings, fetal weights and examinations and gravid uterus weights.

Administration ofAminoethylpiperazine at 150 mg/kg/day was associated with abnormal faecal output (small, decreased, abnormal colour and liquid faeces), reductions in food consumption and body weight gain along with a decrease in embryofetal survival, decrease in fetal and gravid uterine weight and a decrease in fetal bone ossification. 

At dose levels up to 75 mg/kg/day there were no treatment related effects noted in any of the parameters examined including clinical observations, body weight gain, food consumption, gross pathology findings, pregnancy performance parameters, fetal and gravid uterine weights, fetal abnormalities and variants.

In conclusion, under the conditions of this study, the maternal and fetal no observed adverse effect level (NOAEL) and no observed effect level (NOEL) were considered to be 75 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The OECD 442 oral (drinking water) combined repeat dose toxicity study with reproduction/developmental screening test in the rat is a high quality modern study which has a Klimisch 1 rating. The test substance was clearly defined and of high purity a certificate of analysis is included in the report. There are also modern OECD414 studies in both rats and rabbits for the read across substance, 2-piperazin-1-ylethylamine (Amino ethyl piperazine). The database is therefore considered to be adequate.The OECD 442 oral (drinking water) combined repeat dose toxicity study with reproduction/developmental screening test in the rat is a high quality modern study which has a Klimisch 1 rating. The test substance was clearly defined and of high purity a certificate of analysis is included in the report. There are also modern OECD414 studies in both rats and rabbits for the read across substance, 2-piperazin-1-ylethylamine (Amino ethyl piperazine). The Rabbit study showed adverse effects and so provides the lowest NOAEL. The database is therefore considered to be adequate.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
There is no inhalation study available for 1-methyl piperazine or the read across source substance 2-piperazin-1-ylethylamine that provides information on possible developmental toxicity. There is a high-quality oral (drinking water) OECD 422 study which can provide information on developmental toxicity via that exposure route. There are also oral OECD414 studies in rats and rabbits for the read across source substance, 2-piperazin-1-ylethylamine. The Rabbit study showed adverse effects and so provides the lowest NOAEL. ECHA guidance allows the NOAEL from this study to be used to calculate the systemic inhalation DNEL, with an appropriate addition assessment factor of 2. The database is therefore considered to be adequate
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
We do not have a dermal test for developmental toxicity, but there is a high quality OECD422 study via the drinking water (oral) route. There are also oral OECD414 studies in rats and rabbits for the read across source substance, 2-piperazin-1-ylethylamine. The Rabbit study showed adverse effects and so provides the lowest NOAEL. ECHA guidance allows the NOAEL from this study to be used to calculate the systemic dermal DNEL, as dermal absorption is assumed to be the same as oral ingestion, which is probably conservative for 1-methyl piperazine. The database is therefore considered to be adequate
Additional information

The available information concerning the potential for 1-methyl piperazine to cause reproductive toxicity is from the OECD422 screening study. This study is capable of detecting many aspects of developmental toxicity such as pre-and post implantation loss and effects on survival and development from birth to day 4 post-partum. This oral OECD422 (drinking water) study showed some indications of slightly lower litter size and survival in the 10’000ppm group, with offspring bodyweight on day 1 and bodyweight gain to day 4 were marginally lower than control. Lower litter weight at day 4 was statistically significant but this was more influenced by marginally lower litter size than offspring bodyweight. This was at a dose that was for the parental females during lactation 1055mg/kg bodyweight/day and in the presence of maternal toxicity and female water consumption still 33% lower than controls. It cannot be excluded that these effects on the offspring are related to treatment with 1-methyl piperazine, but the maternal low water consumption and marked lower food consumption during gestation and particularly lactation may have contributed and could have reduced the available milk for the offspring. Due to these marginal effects on the offspring at 10’000ppm, the next dose level of 2’500 ppm has been selected as the NOAEL for developmental toxicity but this is very conservative. As the effects seen are considered to have been mediated via the parental female this NOAEL equates to a dose level of 268 mg/kg bodyweight/day using the effective dose level for the females during the gestation period of the study, which is significantly lower than the lactation period.

 

We also have data in the OECD414 study in rats for the read across source substance 2-piperazin-1-ylethylamine, there was also no evidence of developmental toxicity, the NOAEL for embryo/fetal development was 1000 mg/kg. However, in the OECD414 study in rabbits for 2-piperazin-1-ylethylamine at the 150 mg/kg top dose there was decrease in embryo/fetal survival, decrease in fetal and gravid uterine weight and a decrease in fetal bone ossification. This was in the presence of some maternal toxicity at this dose level abnormal faecal output (small, decreased, abnormal colour and liquid faeces), reductions in food consumption and body weight gain. The NOAEL for development was therefore 75 mg/kg in the rabbit OECD414 study, which was the lowest NOAEL.

Mode of Action Analysis / Human Relevance Framework

There is no specific information on the mode of action behind the adverse effects seen in the foetuses in the rabbit pre-natal development study OECD414. The NOAEL of 75mg/kg was also the NOAEL for effects on the parental female. There were indications of maternal toxicity at the LOAEL of 150mg/kg, it was associated with abnormal faecal output (small, decreased, abnormal colour and liquid faeces), reductions in food consumption and body weight gain. The effects seen in the foetuses were mainly indicative of embryo/fetal toxicity not teratogenicity in the presence of moderate maternal toxicity, this may indicate an indirect mode of action via the mother. It is clear there were no adverse effects in rats, it is therefore not clear what the relevance of these relatively minor effects on development seen in rabbits to human health hazard. In the absence of evidence, they are not relevant, they have to be considered relevant.

Justification for classification or non-classification

The high quality OECD422 oral (drinking water) reproductive screening study in rats, showed no effect on fertility or other parental reproductive parameters, at dose levels of 466 mg/kg bodyweight/day for males and 574-1055mg/kg bodyweight/day in females. In this study there were some marginal effects for the offspring on survival, bodyweight at day 1 and bodyweight gain to day 4 post-partum. Due the maternal toxicity at the 10’000ppm level consisting of reduced bodyweight gain during lactation and particular gestation, together with reduced water consumption during lactation which was 33% lower than the controls, together with reduced food consumption these marginal effects are not considered to indicate any specific potential of 1-methyl piperazine for developmental toxicity or for effects on fertility or reproduction. There is also no evidence of adverse effects on development in the OECD414 pre-natal development study in rats, with the read across source substance,2-piperazin-1-ylethylamine. So, based on the data in rats there would be no requirement for classification for developmental toxicity. However, the effects seen in the rabbit OECD414 study forread across source substance,2-piperazin-1-ylethylaminewhile they may have been secondary to toxicity in mother they do indicate some developmental toxicity. Therefore, there is a requirement to consider 1-methyl piperazine for classification for effects on developmental toxicity according to the EU CLP (GHS) criteria. As the effects seen in rabbits were more indicative of a toxicity with no evidence of teratogenicity and in the presence of some maternal toxicity a classification of category 2 for developmental toxicity according to the EU CLP (GHS)criteria is proposed.