Registration Dossier

Administrative data

Description of key information

The only repeat dose study available for 1-methyl piperazine is an OECD 422 repeated dose toxicity oral study, via the drinking water.  The NOAEL from this study can also be used to calculate DNELs for the dermal and inhalation routes.   There are also studies for the read across source substance2-piperazin-1-ylethylamine(aminoethyl piperazine). These include a 90-day inhalation, a 14-day dose ranging inhalation study to identify short term/local effects.

 

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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 study does address all the end points required for a repeat dose toxicity assessment at this tonnage level but unlike the 28 day study the females are pregnant which may have some influence on bodyweight etc.
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 315-362 g 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 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.


Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on oral 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.
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
Remarks:
Doses / Concentrations:
0 ppm
Basis:
nominal in water
Remarks:
Doses / Concentrations:
500 ppm
Basis:
other: Nominal in water (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:
other: 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:
other: Nominal in water (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.
Positive control:
Not applicable
Observations and examinations performed and frequency:
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
Sacrifice and pathology:
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 at AnaPath GmbH, Oberbuchsiten, Switzerland.
Other examinations:
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.
Statistics:
Due to the nature and quantity of this data please see section "any other information on material and methods including tables"
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "details on results" section
Histopathological findings: neoplastic:
no effects observed
Details on results:
Adult Responses
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
A summary of adult performance is given in Table 1. Group values for fertility are given in Table 13. Individual data are given in Appendix 11.
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.


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.


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

Offspring
Neither the type, incidence nor distribution of necropsy findings for decedent offspring or for offspring killed at Day 5 of age indicated any underlying adverse effect of treatment at 500, 2500 or 10000 ppm.

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.
Key result
Dose descriptor:
NOAEL
Effect level:
2 500 ppm
Based on:
dissolved
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical 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 (10000 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 conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
190 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
We have a Klimisch 1 OECD study carried out under GLP with a high 99.9% purity sample of N-methyl piperazine (1-methyl piperazine). The dose levels showed some adverse effects at the highest dose level and allowed a NOAEL to be established; therefore this is sufficient information to establish interim DNELs.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/DuCrl
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Kingston, New York)
- Age at study initiation: Animals were ~5 weeks at arrival and ~9 weeks of age at initiation of treatment
- Housing: After assignment to the study, animals were housed two or three per cage in stainless steel cages. Cages had solid floors with corncob bedding and paper nesting material for enrichment. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
- Diet: ad libitumexcept during acclimation to the nose-only exposure tubes and during nose-only exposure
- Water: ad libitumexcept during acclimation to the nose-only exposure tubes and during nose-only exposure
- Acclimation period: Rats were acclimated to nose-only exposure tubes prior to starting exposures to test material. Rats were loaded into exposure tubes and remained in the tubes for increasing amounts of time. They remained in the exposure tubes for one hour on the first day of acclimation. The amount of time in the tubes was increased by one hour/day until the rats remained in the exposure tubes for six consecutive hours.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

IN-LIFE DATES: From: To:
Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: The average mass median aerodynamic diameter (MMAD) of aerosol present in the exposure chamber test atmospheres was 0.85 ± 1.52, 1.96 ± 1.58, or 1.04 ± 2.39 microns (MMAD ± geometric standard deviation; GSD) for the 0.2, 5.1, or 53.5 mg AEP/m3 exposure chambers, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Forty-two liter, Dow-modified ADG flow-past, nose-only chambers [30 centimeters (cm) in diameter by 60 cm high]
- Source and rate of air: Compressed air supplied to the chamber was at ambient temperature. Airflow through the chamber was determined with a manometer which measured the pressure drop across a calibrated orifice plate and was maintained at approximately 30-35 liters per minute, which was sufficient to provide the normal concentration of oxygen to the animals and approximately 43-50 air changes per hour.
- Method of conditioning air: The 53.5 mg/m3 AEP exposure atmosphere was generated by metering the liquid test material with a syringe pump (KD Scientific, Inc., Holliston, Massachusetts) into an aerosol spray nozzle (TSI Inc., Shoreview, Minnesota). The test material was mixed with air in the spray nozzle, and passed through an aerosol mixing/conditioning chamber (TSE Systems, Inc., Chesterfield, Missouri) wrapped with heat tape before being passed into the exposure chamber. The carrier air and heat tape were heated to the minimum extent necessary to vaporize the test material aerosol. The air and test material vapors were mixed with the appropriate amount of humidified dilution air. The 0.2 and 5.1 mg/m3 AEP exposure atmospheres were generated using a glass J-tube method (Miller et al., 1980). Liquid test material was metered into a glass J-tube assembly with a syringe pump (KD Scientific, Inc., Holliston, Massachusetts) and vaporized by passing air (approximately 30 liters per minute) through the bead bed in the glass J-tube. The air was heated with a flameless heat torch to the minimum extent necessary to vaporize the test material. The mid (5.1 mg/m3) and low (0.2 mg/m3) exposure chamber atmospheres were generated using separate J-tube/syringe pump generation systems. The generation systems were electrically grounded and the J-tubes were changed as needed. The air and test material vapors were mixed with the appropriate amount of humidified dilution air to achieve the target chamber concentration. The test material was not recycled.
On the first day of exposure (Test Day 1), all chambers were monitored to ensure sufficient O2 levels via oxygen sensors with high and low alarms set at 24% and 19% O2, respectively. CO2 levels were also monitored.

- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: Exposure room temperature and chamber temperature, humidity, and airflow were recorded approximately every hour during the exposure periods from each exposure chamber.
- Air flow rate: Based on the approximately 30-35 liter per minute flow rate for the exposure chambers, the theoretical equilibrium time to 99% (T99) of the target concentration was ~5.5-6.5 minutes. The animals were placed on the chamber after the T99 had elapsed and were removed after ~360 minutes of exposure.
- Method of particle size determination: Due to the formation of AEP-carbamate, some aerosol was present in each of the exposure chambers. The mass median aerodynamic diameter (MMAD) was determined at least once per week for each exposure chamber either by drawing samples from within the animal breathing zone, at a set rate using a constant flow air sampling pump through a multi-stage cascade impactor (Sierra Instruments, Inc., Monterey, California), or by using a time-of-flight aerodynamic particle size spectrometer (APS 3321; TSI Incorporated, Shoreview, Minnesota). The MMAD and geometric standard deviation (σg) was determined for each sample as well as the average of the samples.
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of AEP present in each chamber was determined analytically, for each exposure group, 1-3 times during each six-hour exposure period. Each sample was taken by drawing chamber atmosphere from within the animal breathing zone at a set rate using a constant flow air sampling pump and collecting the test material using NITC treated XAD-2 sorbent tubes. Analyses of the chamber atmosphere samples were conducted using liquid chromatography with ultraviolet detection (LC/UV). This method measured the total concentration of AEP, both vapor and AEP-carbamate, present in the exposure atmosphere. See Appendix B for details of the analytical method used.
The time-weighted average (TWA) exposure concentration was calculated from the analytical measurements for each chamber.
Prior to exposure of animals to the test material, the distribution of the test material in the breathing zone of each chamber was determined.
Nominal concentrations for the exposure system which supplied test material to the exposure chambers were calculated from the amount of test material used in the generation apparatus each day and the total airflow through the exposure system for each exposure period.



VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of AEP present in each chamber was determined analytically, for each exposure group, 1-3 times during each six-hour exposure period. Each sample was taken by drawing chamber atmosphere from within the animal breathing zone at a set rate using a constant flow air sampling pump and collecting the test material using NITC treated XAD-2 sorbent tubes. Analyses of the chamber atmosphere samples were conducted using liquid chromatography with ultraviolet detection (LC/UV). This method measured the total concentration of AEP, both vapor and AEP-carbamate, present in the exposure atmosphere. The time-weighted average (TWA) exposure concentration was calculated from the analytical measurements for each chamber.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week for 13 weeks (65 days of exposure)
Remarks:
Doses / Concentrations:
0 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.2 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
5 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The target exposure levels selected, 0, 0.2, 5, or 50 mg/m3, were based on the results of a two-week nose-only inhalation study with AEP (Hotchkiss et al., 2015). The high-exposure concentration (53.5 mg/m3) was expected to result in exposure-related point-of-contact effects in the respiratory tract with no fatalities. The mid- and low-exposure concentrations were fractions of the high-exposure concentration and were expected to provide exposure-response data for any treatment-related effects observed in high-exposure group. A filtered air control group was included. A recovery group of males (most sensitive sex in the two-week study) was included to evaluate the reversibility of any treatment-induced effects.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once a day, at approximately the same time each day (usually in the morning).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pre-exposure and at least weekly throughout the exposure period

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period, on test day 1 prior to the first exposure, at least twice per week during the first 4 weeks of exposure, and at least weekly thereafter.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure and prior to the scheduled necropsy (test day 86) using indirect ophthalmoscopy
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained from the orbital sinus following anesthesia with isoflurane/O2 at the scheduled necropsy. Blood was not obtained from animals that were euthanized prior to their scheduled necropsy.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were obtained from all animals the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this procedure.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Means and standard deviations were calculated for all continuous data. Feed consumption, body weights, terminal body weight, organ weight (absolute and relative), urine volume, urine specific gravity, hematologic parameters (excluding RBC indices and differential WBC), coagulation and clinical chemistry parameters (excluding globulin and albumin/globulin ratio) were evaluated by Bartlett's test for homogeneity of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric ANOVA (Steel and Torrie, 1960) or nonparametric ANOVA (Hollander and Wolfe, 1973). If significant at alpha = 0.05, the ANOVA was followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973) with a Bonferroni correction for multiple comparisons to the control. The experiment-wise alpha level of 0.05 was reported for these two tests. Statistical outliers were identified by a sequential test (Grubbs, 1969).
DCO incidence data (scored observations only) were statistically analyzed by a z-test of proportions comparing each treated group to the control group at alpha = 0.05 (Bruning and Kintz, 1987). Data collected at different time points were analyzed separately.
Descriptive statistics only (means and standard deviations) were reported for body weight gains, globulin and albumin/globulin ratio, RBC indices, differential WBC counts, chamber concentration, temperature, relative humidity and airflow and exposure room temperature.
Data collected in the recovery phase was analyzed by the same methods as indicated during the exposure phase.
Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final toxicologic interpretation of the data considers other factors, such as dose-response relationships, biological plausibility and consistency, and historical control values.
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical ObservationsExaminations performed on all animals pre-exposure and at least weekly throughout the study noted thin appearance in two (of 20) male rats exposed to 0.2 mg/m3 on test day 47 (caused by an abnormally working lixit valve with restricted water flow in the animal housing cage), a broken incisor in one (of 20) male 53.5 mg/m3 group rat on test day 81, and a kinked tail tip due to a mechanical injury in 1 (of 10) female 53.5 mg/m3 group rat on test day 88. Mortality: Animal 15A0444, a female in the 5.1 mg/m3 group, was found in a cage with two male rats on test day 27; this animal was subsequently removed from the study and taken to necropsy on test day 29 due to a sperm positive slide. There were no instances of mortality related to test material exposure throughout this study.
Mortality:
no mortality observed
Description (incidence):
Clinical ObservationsExaminations performed on all animals pre-exposure and at least weekly throughout the study noted thin appearance in two (of 20) male rats exposed to 0.2 mg/m3 on test day 47 (caused by an abnormally working lixit valve with restricted water flow in the animal housing cage), a broken incisor in one (of 20) male 53.5 mg/m3 group rat on test day 81, and a kinked tail tip due to a mechanical injury in 1 (of 10) female 53.5 mg/m3 group rat on test day 88. Mortality: Animal 15A0444, a female in the 5.1 mg/m3 group, was found in a cage with two male rats on test day 27; this animal was subsequently removed from the study and taken to necropsy on test day 29 due to a sperm positive slide. There were no instances of mortality related to test material exposure throughout this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically identified differences in the body weights of any treated groups when compared to their respective controls. Body weight gains were also unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Statistically identified lower mean feed consumption values were observed from test days 1-5 in male recovery group rats exposed to 0.2, 5.1, or 53.5 mg/m3 and female rats exposed to 5.1 or 53.5 mg/m3 when compared to their respective controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Examinations performed on all animals pre-exposure and at termination revealed no treatment-related findings.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in any of the hematology parameters of males or females at any exposure level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in clinical chemistry parameters of males or females at any exposure level.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically significant changes in urinalysis parameters of males or females at any exposure level.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
. Final body weights and all other organ weights in both sexes were similar to controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross pathologic observations in males or females at any exposure level. All gross pathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to AEP.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes in the nasal tissues in mid and high dose groups and nasal tissue larynx, trachea, and lungs of high dose group
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY:
Animal 15A0444, a female in the 5.1 mg/m3 group, was found in a cage with two male rats on test day 27; this animal was subsequently removed from the study and taken to necropsy on test day 29 due to a sperm positive slide. There were no instances of mortality related to test material exposure throughout this study.
No treatment-related clinical observations were observed during the study. Examinations performed on all animals pre-exposure and at least weekly throughout the study noted thin appearance in two (of 20) male rats exposed to 0.2 mg/m3 on test day 47 (caused by an abnormally working lixit valve with restricted water flow in the animal housing cage), a broken incisor in one (of 20) male 53.5 mg/m3 group rat on test day 81, and a kinked tail tip due to a mechanical injury in 1 (of 10) female 53.5 mg/m3 group rat on test day 88.

BODY WEIGHT AND WEIGHT GAIN: There were no statistically identified differences in the body weights of any treated groups when compared to their respective controls. Body weight gains were also unaffected by treatment.

FOOD CONSUMPTION: There were no treatment-related effects in the amount of feed consumed by any treated group when compared to their respective controls. Statistically identified lower mean feed consumption values were observed from test days 1-5 in male recovery group rats exposed to 0.2, 5.1, or 53.5 mg/m3 and female rats exposed to 5.1 or 53.5 mg/m3 when compared to their respective controls. In addition, male 90-day exposure groups rats exposed to 5.1 mg/m3 were noted in one instance (test days 75-81) to have statistically identified higher mean feed consumption values when compared to their respective controls. The increases and/or decreases in feed consumption noted throughout the study were considered unrelated to treatment related due to their random occurrence.

OPHTHALMOSCOPIC EXAMINATION: Examinations performed on all animals pre-exposure and at termination revealed no treatment-related findings.

HAEMATOLOGY: There were no treatment-related alterations in any of the hematology parameters of males or females at any exposure level. Females exposed to 0.2 mg/m3 had statistically significant decreases in mean red blood cell count and mean hemoglobin concentration, relative to controls, which were interpreted to be unrelated to treatment because of the lack of a dose response for these parameters, and the values were within historical control ranges of studies recently conducted at this laboratory.

CLINICAL CHEMISTRY: There were no treatment-related alterations in clinical chemistry parameters of males or females at any exposure level. Males exposed to 53.5 mg/m3 had a statistically significant lower mean cholesterol concentration which was interpreted to be unrelated to treatment because of the lack of a clear dose-related response in the low- and mid-exposure levels, the minimal difference of the high-exposure level cholesterol concentration as compared to the historical control group range, and as compared to the concurrent control group.

URINALYSIS: There were no treatment-related or statistically significant changes in urinalysis parameters of males or females at any exposure level.

ORGAN WEIGHTS: Males and females exposed to 53.5 mg/m3 had higher absolute and relative mean lung weights as compared to controls. The higher relative lung weights of males and females exposed to 53.5 mg/m3 were statistically significant, relative to controls. The higher absolute and relative lung weight of females exposed to 53.5 mg/m3 were interpreted to be treatment-related because the values were clearly higher than the historical control ranges from studies recently conducted at the laboratory. The higher absolute and relative lung weights of males exposed to 53.5 mg/m3 were within or near the historical control range, were minimally different from the concurrent control values, and were therefore interpreted to be unrelated to treatment. Final body weights and all other organ weights in both sexes were similar to controls.

GROSS PATHOLOGY: There were no treatment-related gross pathologic observations in males or females at any exposure level. All gross pathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to AEP.

HISTOPATHOLOGY: NON-NEOPLASTIC
There was no histopathological evidence of any primary systemic toxicity in any AEP exposure group (systemic NOEC = 53.5 mg/m3). The nasal tissue was the most sensitive target tissue in rats exposed to AEP. Compared to air-exposed (0 mg/m3) control rats of the same sex, male and female rats exposed to 5.1 or 53.5 mg/m3 had exposure-related histopathological changes in the nasal tissues. The larynx, trachea, and lungs of male and female rats exposed to 53.5 mg/m3 also had exposure-related histopathological effects. All of the exposure-related changes were consistent with localized irritant effects of the test material at the point of contact with the airway epithelium.
The majority of the nasal tissue effects were present in the anterior and dorsal aspects of the nasal passages. The most frequently affected site in the nasal tissue was the transitional epithelium lining the nasoturbinates, maxilloturbinates, and lateral walls of the nasal passages. All males exposed to 5.1 or 53.5 mg/m3, all females exposed to 53.5 mg/m3, and the majority of females exposed to 5.1 mg/m3 had multifocal atrophy (very slight, slight, or moderate), hyperplasia (very slight or slight), chronic-active inflammation (very slight or slight), and squamous metaplasia (very slight or slight) of the transitional epithelium. Focal or multifocal, very slight ulcers of the transitional epithelium were also present in a few males and females exposed to 5.1 or 53.5 mg/m3. Focal or multifocal atrophy (very slight, slight, or moderate) of the anterior respiratory epithelium occurred in the majority of males exposed to 53.5 mg/m3, and in lesser numbers of females exposed to 53.5 mg/m3 and males and females exposed to 5.1 mg/m3. Multifocal hyperplasia (very slight or slight) of the anterior respiratory epithelium occurred in the majority of males exposed to 53.5 mg/m3 and in lesser numbers of females exposed to 53.5 mg/m3 and males exposed to 5.1 mg/m3. Additional treatment-related effects of the anterior respiratory epithelium consisted of: increased incidence of multifocal, very slight or slight hyperplasia and hypertrophy of mucous cells lining the septum and lateral walls of the ventral meatus in males and females exposed to 5.1 or 53.5 mg/m3, as compared to the incidence in same sex control group animals; multifocal, very slight or slight chronic active inflammation in some males exposed to 5.1 or 53.5 mg/m3; multifocal, slight squamous metaplasia in 3/10 males exposed to 53.5 mg/m3; focal, slight mineralization in 2/10 males exposed to 5.1 mg/m3; and focal slight necrosis in 1/10 males exposed to 5.1 mg/m3. Focal or multifocal atrophy (very slight, slight or moderate) of the anterior olfactory epithelium occurred in the majority of females exposed to 5.1 or 53.5 mg/m3, and in lesser numbers of males exposed to 5.1 or 53.5 mg/m3. Additional treatment-related effects of the anterior olfactory epithelium consisted of: multifocal, very slight or slight chronic active inflammation in 1/10 males exposed to 5.1 mg/m3 and 1/10 females exposed to 53.5 mg/m3; focal or multifocal, very slight or slight respiratory epithelial metaplasia in a few males and females exposed to 5.1 or 53.5 mg/m3, and a focal, very slight ulcer with accompanying slight olfactory epithelial mineralization in 1/10 males exposed to 5.1 mg/m3. A very slight or slight suppurative exudate was present in the lumen of the nasal passages in some males exposed to 5.1 or 53.5 mg/m3, and in 2/10 females exposed to 53.5 mg/m3.
Some treatment-related histopathological effects were present in the posterior and ventral aspects of the nasal passages. The majority of males and females exposed to 53.5 mg/m3 had very slight or slight hyaline droplet formation in the olfactory epithelium. Very slight or slight hyaline droplet formation was also present in the respiratory epithelium in 5/10 males and 5/10 females exposed to 53.5 mg/m3. The hyaline droplet formation in the olfactory and respiratory epithelium was most commonly present on the ventral ethmoid turbinates and the ventral septum in the posterior aspect of the nasal passages. An increase in the incidence of multifocal, very slight or slight eosinophilic inflammation was present in males exposed to 53.5 mg/m3 and females exposed to 5.1 or 53.5 mg/m3, as compared to the incidence of same sex control group animals. The inflammation, which consisted of mainly eosinophils, was present in the lamina propria of the ventral aspect of the ethmoid turbinates, subjacent to the dorsal aspect of the pharyngeal duct, and/or in the lamina propria of the ventral and posterior aspects of the nasal septum. Very slight mucous cell hypertrophy of cells lining the pharyngeal duct was present in 6/10 males and 5/10 females exposed to 53.5 mg/m3. One male exposed to 53.5 mg/m3 had slight hyperplasia and hypertrophy of mucous cells lining the pharyngeal duct. One male exposed to 5.1 mg/m3 had a focal, very slight ulcer of the epithelium of the pharyngeal duct, which was accompanied by slight granulomatous inflammation in the underlying lamina propria.
The most common treatment-related effects of the larynx, present in all males and females exposed to 53.5 mg/m3, consisted of: multifocal, slight squamous metaplasia of the respiratory epithelium; multifocal, slight or moderate subacute to chronic inflammation of the lamina propria; and multifocal, slight or moderate fibrosis in the lamina propria. The chronic inflammation was composed of a mixture of neutrophils, lymphocytes and plasma cells, with nodular clusters of lymphocytes present in the lamina propria at the base of the epiglottis. Treatment-related multifocal, slight or moderate hyperplasia of the respiratory epithelium was present in the larynx of all males and 9/10 females exposed to 53.5 mg/m3. All of these laryngeal effects were most prominent in the anterior larynx, near the base of the epiglottis at the level of the seromucinous gland, and some of these effects were present with lesser frequency on the medial aspect of the arytenoid cartilage. Additional treatment-related effects of the larynx in rats exposed to 53.5 mg/m3 consisted of: multifocal, very slight erosions of the respiratory epithelium at the base of the epiglottis in 1/10 males; slight suppurative exudate in lumen of the larynx in 1/10 males and 4/10 females; multifocal, very slight necrosis of individual cells in the respiratory epithelium of ventral aspect of the larynx at the level of the ventral pouch in 1/10 males; and focal or multifocal, very slight, slight, or moderate ulceration of the respiratory epithelium in 4/10 males and 5/10 females. The locations of the laryngeal ulcers varied from animal to animal, and were noted in the respiratory epithelium at the base of the epiglottis, dorsal to the ventral pouch, and/or on the medial aspect of the arytenoid cartilage.
The most common treatment-related effect of the lungs was multifocal, very slight epithelial alteration of the bronchi, observed in all males and females exposed to 53.5 mg/m3. The epithelial alteration was characterized by loss of cilia, flattening, and minimal stratification of the bronchial epithelium. The epithelial alteration was noted in the epithelium that lines the portion of the bronchial walls which project into the airways at some of the branching points of the bronchi. One male exposed to 53.5 mg/m3 had focal, slight bronchiolo-alveolar hyperplasia of the lungs, which was interpreted to be treatment related. Alveolar macrophages and perivascular eosinophils were present in the focal area of bronchiolo-alveolar hyperplasia.
Treatment-related focal or multifocal, very slight epithelial alteration of the respiratory epithelium of the trachea was present in all males and 7/10 females exposed to 53.5 mg/m3. The epithelial alteration was characterized by loss of cilia, flattening, and minimal stratification of respiratory epithelial cells at the level of bifurcation of the trachea.
There were no treatment-related histopathological effects in the upper or lower respiratory tract of males or females exposed to 0.2 mg/m3.
All males from the control and high-dose (53.5 mg/m3) groups had degeneration of testicular seminiferous tubules and associated degenerative spermatic elements of the epididymides of variable severities and distributions. These alterations were caused by physical compression of the testes when the rats were present in the nose-only exposure chambers. All other histopathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to AEP.
Dose descriptor:
NOEC
Effect level:
0.2 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: point of contact irritant effects
Dose descriptor:
NOEC
Effect level:
53.5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Critical effects observed:
not specified

Female Red Blood Cell Counts and Hemoglobin Concentrations

 Sex  Females            
 Exposure Level (mg/m3)  Historical Control@  0  0.2  5.1  53.5
 Red Blood Cell Count (106/µ L)  8.35 -9.41  8.92  8.73*  8.85  9.03
 Hemoglobin Concentration (g/dL)  14.5 -16.8  15.4  15.0*  15.4  15.6

@Historical control from data obtained from two nose-only 90-day inhalation toxicity studies and eleven data sets with dietary 90-day toxicity endpoints in F344 rats reported from 2011-2015 (seven 90-day studies and four 2-year studies using 90-day time points).

*Statistically different from control mean by Dunnett’s test, alpha=0.05. Male Cholesterol Concentrations
 Sex  Males            
 Dose (mg/m3)  Historical Control@  0  0.2  5.1  53.5
 Cholesterol Concentration (mg/dL)  52 -70  54  49  50  48*

@Historical control from data obtained from two nose-only 90-day inhalation toxicity studies and eleven data sets with dietary 90-day toxicity endpoints in F344 rats reported from 2011-2015 (seven 90-day studies and four 2-year studies using 90-day time points).

*Statistically different from control mean by Dunnett’s test, alpha=0.05.
Conclusions:
There were no treatment-related changes in in-life observations, ophthalmological exams, body weight or body weight gains, feed consumption, clinical chemistry, coagulation, hematology, urinalysis, or gross pathologic observations in male or female rats at any exposure level.
Females exposed to 53.5 mg/m3 had higher absolute and relative mean lung weights (statistically significant for relative weight) as compared to controls, which were interpreted to be treatment-related.
In the 13-week exposure group, there was no histopathological evidence of any primary systemic toxicity in any AEP exposure group (systemic NOEC = 53.5 mg/m3). The nasal tissue was the most sensitive target tissue in rats exposed to AEP. Compared to air-exposed (0 mg/m3) control rats of the same sex, male and female rats exposed to 5.1 or 53.5 mg/m3 had exposure-related histopathological changes in the nasal tissues. The larynx, trachea, and lungs of male and female rats exposed to 53.5 mg/m3 also had exposure-related histopathological effects. All of the exposure-related changes were consistent with localized irritant effects of the test material at the point of contact with the airway epithelium.
Treatment-related histopathologic effects persisted following 13-weeks of recovery in the nasal tissues of males exposed to 5.1 or 53.5 mg/m3, and in the larynx and lungs of males exposed to 53.5 mg/m3.
Based on treatment-related effects in the nasal tissues of males and females exposed to 5.1 or 53.5 mg/m3, the no-observed-effect concentration (NOEC) for point of contact irritant effects for male and female F344/DuCrl rats repeatedly exposed to AEP for 13 weeks (65 exposures) was 0.2 mg/m3. The no-observed-effect concentration (NOEC) for systemic toxicity was 53.5 mg/m3.
Executive summary:

This study was designed to evaluate the potential for local (portal-of-entry) and systemic toxicity from inhalation of aminoethylpiperazine (AEP). Groups of ten male and ten female F344/DuCrl rats were exposedvianose-only inhalation to AEP six hours/day, five consecutive days/weekfor 13 weeks (a total of 65 exposures). Additional groups of 10 male rats per exposure concentration were contemporaneously exposed and held (unexposed) for an additional 13 weeks following the last exposure day to serve as post-exposure recovery groups to determine the persistence or reversibility of any AEP-dependent effects identified at the end of exposure. The exposure atmosphere contained both AEP vapor and an AEP-carbamate aerosol that formed spontaneously in the humidified chamber atmosphere. The rats were exposed to analytically-determined concentrations of0, 0.2 ± 0.1, 5.1 ± 1.1, or 53.5 ± 6.0 mg AEP/m3(study mean±standard deviation). The analytical method measured total AEP present in the exposure chambers (AEP vapor + AEP-carbamate aerosol). The average mass median aerodynamic diameter (MMAD) of the aerosol fraction in each of the exposure chambers was0.85±1.52,1.96 ± 1.58, or 1.04 ± 2.39microns (MMAD ± geometric standard deviation; GSD) for the 0.2, 5.1, or 53.5 mg AEP/m3exposure chambers, respectively. In-life observations(including ophthalmology), body weights/body weight gains, feed consumption,urinalysis,hematology, coagulation, clinical chemistry, and organ weights were evaluated. A gross necropsy was conducted and a detailed histopathological examination of the entire respiratory tract was performed to assess treatment-dependent portal-of-entry effects. In addition, a detailed histopathologic examination of other specified tissues/organs was performed on the control- and high-exposure group rats to identify treatment-related systemic toxicity.

There were no treatment-related changes in in-life observations, ophthalmologic examinations, body weight/body weight gains, feed consumption, clinical chemistry, coagulation, hematology, urinalysis, or gross pathologic observations in male or female rats at any exposure level.

Females exposed to 53.5 mg/m3had higher absolute and relative mean lung weights (statistically significant for relative weight) as compared to controls, which were interpreted to be treatment-related.

In the 13-week exposure group, there was no histopathological evidence of any primary systemic toxicity in any AEP exposure group (systemic NOEC ≥ 53.5 mg/m3). The nasal mucosa was the most sensitive target tissue in rats exposed to AEP. Compared to air-exposed (0 mg/m3) control rats of the same sex, male and female rats exposed to 5.1 or 53.5 mg/m3had exposure-related histopathological changes in the nasal tissues. The larynx, trachea, and lungs of male and female rats exposed to 53.5 mg/m3also had exposure-related histopathological effects. All of the exposure-related changes were consistent with localized irritant effects of the test material at the point of contact with the airway epithelium.

There were no treatment-related histopathologic effects in males or females exposed to 0.2 mg/m3. In the 13-week recovery group, treatment-related histopathological effects persisted in the nasal tissues of males exposed to 5.1 or 53.5 mg/m3, and in the larynx and lungs of males exposed to 53.5 mg/m3.

Based on treatment-related effects in the nasal tissues of males and females exposed to 5.1 or 53.5 mg/m3, the no-observed-effect concentration (NOEC) for point of contact irritant effects for male and female F344/DuCrl rats repeatedly exposed toAEPfor 13 weeks (65 exposures) was 0.2 mg/m3. The no-observed-effect concentration (NOEC) for systemic toxicity was 53.5 mg/m3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
53.5 mg/m³
Study duration:
subchronic
Experimental exposure time per week (hours/week):
30
Species:
rat
Quality of whole database:
There is a good quality recent fully GLP compliant Klimisch 1 90-day inhalation study for the read across source substance 2-piperazin-1-ylethylamine (aminoethyl piperazine). Due to the corrosive nature of this substance which is classified as category 1B as is 1-methyl piperazine, adverse effects were mainly due to local irritation/damage in the respiratory tract, the top concentration can be used as a NOAEC for systemic toxicity.
System:
respiratory system: upper respiratory tract
Organ:
larynx
nasal cavity
lungs

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/DuCrl
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Kingston, New York)
- Age at study initiation: Animals were ~5 weeks at arrival and ~9 weeks of age at initiation of treatment
- Housing: After assignment to the study, animals were housed two or three per cage in stainless steel cages. Cages had solid floors with corncob bedding and paper nesting material for enrichment. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
- Diet: ad libitumexcept during acclimation to the nose-only exposure tubes and during nose-only exposure
- Water: ad libitumexcept during acclimation to the nose-only exposure tubes and during nose-only exposure
- Acclimation period: Rats were acclimated to nose-only exposure tubes prior to starting exposures to test material. Rats were loaded into exposure tubes and remained in the tubes for increasing amounts of time. They remained in the exposure tubes for one hour on the first day of acclimation. The amount of time in the tubes was increased by one hour/day until the rats remained in the exposure tubes for six consecutive hours.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

IN-LIFE DATES: From: To:
Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: The average mass median aerodynamic diameter (MMAD) of aerosol present in the exposure chamber test atmospheres was 0.85 ± 1.52, 1.96 ± 1.58, or 1.04 ± 2.39 microns (MMAD ± geometric standard deviation; GSD) for the 0.2, 5.1, or 53.5 mg AEP/m3 exposure chambers, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Forty-two liter, Dow-modified ADG flow-past, nose-only chambers [30 centimeters (cm) in diameter by 60 cm high]
- Source and rate of air: Compressed air supplied to the chamber was at ambient temperature. Airflow through the chamber was determined with a manometer which measured the pressure drop across a calibrated orifice plate and was maintained at approximately 30-35 liters per minute, which was sufficient to provide the normal concentration of oxygen to the animals and approximately 43-50 air changes per hour.
- Method of conditioning air: The 53.5 mg/m3 AEP exposure atmosphere was generated by metering the liquid test material with a syringe pump (KD Scientific, Inc., Holliston, Massachusetts) into an aerosol spray nozzle (TSI Inc., Shoreview, Minnesota). The test material was mixed with air in the spray nozzle, and passed through an aerosol mixing/conditioning chamber (TSE Systems, Inc., Chesterfield, Missouri) wrapped with heat tape before being passed into the exposure chamber. The carrier air and heat tape were heated to the minimum extent necessary to vaporize the test material aerosol. The air and test material vapors were mixed with the appropriate amount of humidified dilution air. The 0.2 and 5.1 mg/m3 AEP exposure atmospheres were generated using a glass J-tube method (Miller et al., 1980). Liquid test material was metered into a glass J-tube assembly with a syringe pump (KD Scientific, Inc., Holliston, Massachusetts) and vaporized by passing air (approximately 30 liters per minute) through the bead bed in the glass J-tube. The air was heated with a flameless heat torch to the minimum extent necessary to vaporize the test material. The mid (5.1 mg/m3) and low (0.2 mg/m3) exposure chamber atmospheres were generated using separate J-tube/syringe pump generation systems. The generation systems were electrically grounded and the J-tubes were changed as needed. The air and test material vapors were mixed with the appropriate amount of humidified dilution air to achieve the target chamber concentration. The test material was not recycled.
On the first day of exposure (Test Day 1), all chambers were monitored to ensure sufficient O2 levels via oxygen sensors with high and low alarms set at 24% and 19% O2, respectively. CO2 levels were also monitored.

- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: Exposure room temperature and chamber temperature, humidity, and airflow were recorded approximately every hour during the exposure periods from each exposure chamber.
- Air flow rate: Based on the approximately 30-35 liter per minute flow rate for the exposure chambers, the theoretical equilibrium time to 99% (T99) of the target concentration was ~5.5-6.5 minutes. The animals were placed on the chamber after the T99 had elapsed and were removed after ~360 minutes of exposure.
- Method of particle size determination: Due to the formation of AEP-carbamate, some aerosol was present in each of the exposure chambers. The mass median aerodynamic diameter (MMAD) was determined at least once per week for each exposure chamber either by drawing samples from within the animal breathing zone, at a set rate using a constant flow air sampling pump through a multi-stage cascade impactor (Sierra Instruments, Inc., Monterey, California), or by using a time-of-flight aerodynamic particle size spectrometer (APS 3321; TSI Incorporated, Shoreview, Minnesota). The MMAD and geometric standard deviation (σg) was determined for each sample as well as the average of the samples.
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of AEP present in each chamber was determined analytically, for each exposure group, 1-3 times during each six-hour exposure period. Each sample was taken by drawing chamber atmosphere from within the animal breathing zone at a set rate using a constant flow air sampling pump and collecting the test material using NITC treated XAD-2 sorbent tubes. Analyses of the chamber atmosphere samples were conducted using liquid chromatography with ultraviolet detection (LC/UV). This method measured the total concentration of AEP, both vapor and AEP-carbamate, present in the exposure atmosphere. See Appendix B for details of the analytical method used.
The time-weighted average (TWA) exposure concentration was calculated from the analytical measurements for each chamber.
Prior to exposure of animals to the test material, the distribution of the test material in the breathing zone of each chamber was determined.
Nominal concentrations for the exposure system which supplied test material to the exposure chambers were calculated from the amount of test material used in the generation apparatus each day and the total airflow through the exposure system for each exposure period.



VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of AEP present in each chamber was determined analytically, for each exposure group, 1-3 times during each six-hour exposure period. Each sample was taken by drawing chamber atmosphere from within the animal breathing zone at a set rate using a constant flow air sampling pump and collecting the test material using NITC treated XAD-2 sorbent tubes. Analyses of the chamber atmosphere samples were conducted using liquid chromatography with ultraviolet detection (LC/UV). This method measured the total concentration of AEP, both vapor and AEP-carbamate, present in the exposure atmosphere. The time-weighted average (TWA) exposure concentration was calculated from the analytical measurements for each chamber.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week for 13 weeks (65 days of exposure)
Remarks:
Doses / Concentrations:
0 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.2 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
5 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The target exposure levels selected, 0, 0.2, 5, or 50 mg/m3, were based on the results of a two-week nose-only inhalation study with AEP (Hotchkiss et al., 2015). The high-exposure concentration (53.5 mg/m3) was expected to result in exposure-related point-of-contact effects in the respiratory tract with no fatalities. The mid- and low-exposure concentrations were fractions of the high-exposure concentration and were expected to provide exposure-response data for any treatment-related effects observed in high-exposure group. A filtered air control group was included. A recovery group of males (most sensitive sex in the two-week study) was included to evaluate the reversibility of any treatment-induced effects.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once a day, at approximately the same time each day (usually in the morning).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pre-exposure and at least weekly throughout the exposure period

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period, on test day 1 prior to the first exposure, at least twice per week during the first 4 weeks of exposure, and at least weekly thereafter.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure and prior to the scheduled necropsy (test day 86) using indirect ophthalmoscopy
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained from the orbital sinus following anesthesia with isoflurane/O2 at the scheduled necropsy. Blood was not obtained from animals that were euthanized prior to their scheduled necropsy.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were obtained from all animals the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this procedure.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Means and standard deviations were calculated for all continuous data. Feed consumption, body weights, terminal body weight, organ weight (absolute and relative), urine volume, urine specific gravity, hematologic parameters (excluding RBC indices and differential WBC), coagulation and clinical chemistry parameters (excluding globulin and albumin/globulin ratio) were evaluated by Bartlett's test for homogeneity of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric ANOVA (Steel and Torrie, 1960) or nonparametric ANOVA (Hollander and Wolfe, 1973). If significant at alpha = 0.05, the ANOVA was followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973) with a Bonferroni correction for multiple comparisons to the control. The experiment-wise alpha level of 0.05 was reported for these two tests. Statistical outliers were identified by a sequential test (Grubbs, 1969).
DCO incidence data (scored observations only) were statistically analyzed by a z-test of proportions comparing each treated group to the control group at alpha = 0.05 (Bruning and Kintz, 1987). Data collected at different time points were analyzed separately.
Descriptive statistics only (means and standard deviations) were reported for body weight gains, globulin and albumin/globulin ratio, RBC indices, differential WBC counts, chamber concentration, temperature, relative humidity and airflow and exposure room temperature.
Data collected in the recovery phase was analyzed by the same methods as indicated during the exposure phase.
Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final toxicologic interpretation of the data considers other factors, such as dose-response relationships, biological plausibility and consistency, and historical control values.
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical ObservationsExaminations performed on all animals pre-exposure and at least weekly throughout the study noted thin appearance in two (of 20) male rats exposed to 0.2 mg/m3 on test day 47 (caused by an abnormally working lixit valve with restricted water flow in the animal housing cage), a broken incisor in one (of 20) male 53.5 mg/m3 group rat on test day 81, and a kinked tail tip due to a mechanical injury in 1 (of 10) female 53.5 mg/m3 group rat on test day 88. Mortality: Animal 15A0444, a female in the 5.1 mg/m3 group, was found in a cage with two male rats on test day 27; this animal was subsequently removed from the study and taken to necropsy on test day 29 due to a sperm positive slide. There were no instances of mortality related to test material exposure throughout this study.
Mortality:
no mortality observed
Description (incidence):
Clinical ObservationsExaminations performed on all animals pre-exposure and at least weekly throughout the study noted thin appearance in two (of 20) male rats exposed to 0.2 mg/m3 on test day 47 (caused by an abnormally working lixit valve with restricted water flow in the animal housing cage), a broken incisor in one (of 20) male 53.5 mg/m3 group rat on test day 81, and a kinked tail tip due to a mechanical injury in 1 (of 10) female 53.5 mg/m3 group rat on test day 88. Mortality: Animal 15A0444, a female in the 5.1 mg/m3 group, was found in a cage with two male rats on test day 27; this animal was subsequently removed from the study and taken to necropsy on test day 29 due to a sperm positive slide. There were no instances of mortality related to test material exposure throughout this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically identified differences in the body weights of any treated groups when compared to their respective controls. Body weight gains were also unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Statistically identified lower mean feed consumption values were observed from test days 1-5 in male recovery group rats exposed to 0.2, 5.1, or 53.5 mg/m3 and female rats exposed to 5.1 or 53.5 mg/m3 when compared to their respective controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Examinations performed on all animals pre-exposure and at termination revealed no treatment-related findings.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in any of the hematology parameters of males or females at any exposure level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in clinical chemistry parameters of males or females at any exposure level.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically significant changes in urinalysis parameters of males or females at any exposure level.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
. Final body weights and all other organ weights in both sexes were similar to controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross pathologic observations in males or females at any exposure level. All gross pathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to AEP.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes in the nasal tissues in mid and high dose groups and nasal tissue larynx, trachea, and lungs of high dose group
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY:
Animal 15A0444, a female in the 5.1 mg/m3 group, was found in a cage with two male rats on test day 27; this animal was subsequently removed from the study and taken to necropsy on test day 29 due to a sperm positive slide. There were no instances of mortality related to test material exposure throughout this study.
No treatment-related clinical observations were observed during the study. Examinations performed on all animals pre-exposure and at least weekly throughout the study noted thin appearance in two (of 20) male rats exposed to 0.2 mg/m3 on test day 47 (caused by an abnormally working lixit valve with restricted water flow in the animal housing cage), a broken incisor in one (of 20) male 53.5 mg/m3 group rat on test day 81, and a kinked tail tip due to a mechanical injury in 1 (of 10) female 53.5 mg/m3 group rat on test day 88.

BODY WEIGHT AND WEIGHT GAIN: There were no statistically identified differences in the body weights of any treated groups when compared to their respective controls. Body weight gains were also unaffected by treatment.

FOOD CONSUMPTION: There were no treatment-related effects in the amount of feed consumed by any treated group when compared to their respective controls. Statistically identified lower mean feed consumption values were observed from test days 1-5 in male recovery group rats exposed to 0.2, 5.1, or 53.5 mg/m3 and female rats exposed to 5.1 or 53.5 mg/m3 when compared to their respective controls. In addition, male 90-day exposure groups rats exposed to 5.1 mg/m3 were noted in one instance (test days 75-81) to have statistically identified higher mean feed consumption values when compared to their respective controls. The increases and/or decreases in feed consumption noted throughout the study were considered unrelated to treatment related due to their random occurrence.

OPHTHALMOSCOPIC EXAMINATION: Examinations performed on all animals pre-exposure and at termination revealed no treatment-related findings.

HAEMATOLOGY: There were no treatment-related alterations in any of the hematology parameters of males or females at any exposure level. Females exposed to 0.2 mg/m3 had statistically significant decreases in mean red blood cell count and mean hemoglobin concentration, relative to controls, which were interpreted to be unrelated to treatment because of the lack of a dose response for these parameters, and the values were within historical control ranges of studies recently conducted at this laboratory.

CLINICAL CHEMISTRY: There were no treatment-related alterations in clinical chemistry parameters of males or females at any exposure level. Males exposed to 53.5 mg/m3 had a statistically significant lower mean cholesterol concentration which was interpreted to be unrelated to treatment because of the lack of a clear dose-related response in the low- and mid-exposure levels, the minimal difference of the high-exposure level cholesterol concentration as compared to the historical control group range, and as compared to the concurrent control group.

URINALYSIS: There were no treatment-related or statistically significant changes in urinalysis parameters of males or females at any exposure level.

ORGAN WEIGHTS: Males and females exposed to 53.5 mg/m3 had higher absolute and relative mean lung weights as compared to controls. The higher relative lung weights of males and females exposed to 53.5 mg/m3 were statistically significant, relative to controls. The higher absolute and relative lung weight of females exposed to 53.5 mg/m3 were interpreted to be treatment-related because the values were clearly higher than the historical control ranges from studies recently conducted at the laboratory. The higher absolute and relative lung weights of males exposed to 53.5 mg/m3 were within or near the historical control range, were minimally different from the concurrent control values, and were therefore interpreted to be unrelated to treatment. Final body weights and all other organ weights in both sexes were similar to controls.

GROSS PATHOLOGY: There were no treatment-related gross pathologic observations in males or females at any exposure level. All gross pathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to AEP.

HISTOPATHOLOGY: NON-NEOPLASTIC
There was no histopathological evidence of any primary systemic toxicity in any AEP exposure group (systemic NOEC = 53.5 mg/m3). The nasal tissue was the most sensitive target tissue in rats exposed to AEP. Compared to air-exposed (0 mg/m3) control rats of the same sex, male and female rats exposed to 5.1 or 53.5 mg/m3 had exposure-related histopathological changes in the nasal tissues. The larynx, trachea, and lungs of male and female rats exposed to 53.5 mg/m3 also had exposure-related histopathological effects. All of the exposure-related changes were consistent with localized irritant effects of the test material at the point of contact with the airway epithelium.
The majority of the nasal tissue effects were present in the anterior and dorsal aspects of the nasal passages. The most frequently affected site in the nasal tissue was the transitional epithelium lining the nasoturbinates, maxilloturbinates, and lateral walls of the nasal passages. All males exposed to 5.1 or 53.5 mg/m3, all females exposed to 53.5 mg/m3, and the majority of females exposed to 5.1 mg/m3 had multifocal atrophy (very slight, slight, or moderate), hyperplasia (very slight or slight), chronic-active inflammation (very slight or slight), and squamous metaplasia (very slight or slight) of the transitional epithelium. Focal or multifocal, very slight ulcers of the transitional epithelium were also present in a few males and females exposed to 5.1 or 53.5 mg/m3. Focal or multifocal atrophy (very slight, slight, or moderate) of the anterior respiratory epithelium occurred in the majority of males exposed to 53.5 mg/m3, and in lesser numbers of females exposed to 53.5 mg/m3 and males and females exposed to 5.1 mg/m3. Multifocal hyperplasia (very slight or slight) of the anterior respiratory epithelium occurred in the majority of males exposed to 53.5 mg/m3 and in lesser numbers of females exposed to 53.5 mg/m3 and males exposed to 5.1 mg/m3. Additional treatment-related effects of the anterior respiratory epithelium consisted of: increased incidence of multifocal, very slight or slight hyperplasia and hypertrophy of mucous cells lining the septum and lateral walls of the ventral meatus in males and females exposed to 5.1 or 53.5 mg/m3, as compared to the incidence in same sex control group animals; multifocal, very slight or slight chronic active inflammation in some males exposed to 5.1 or 53.5 mg/m3; multifocal, slight squamous metaplasia in 3/10 males exposed to 53.5 mg/m3; focal, slight mineralization in 2/10 males exposed to 5.1 mg/m3; and focal slight necrosis in 1/10 males exposed to 5.1 mg/m3. Focal or multifocal atrophy (very slight, slight or moderate) of the anterior olfactory epithelium occurred in the majority of females exposed to 5.1 or 53.5 mg/m3, and in lesser numbers of males exposed to 5.1 or 53.5 mg/m3. Additional treatment-related effects of the anterior olfactory epithelium consisted of: multifocal, very slight or slight chronic active inflammation in 1/10 males exposed to 5.1 mg/m3 and 1/10 females exposed to 53.5 mg/m3; focal or multifocal, very slight or slight respiratory epithelial metaplasia in a few males and females exposed to 5.1 or 53.5 mg/m3, and a focal, very slight ulcer with accompanying slight olfactory epithelial mineralization in 1/10 males exposed to 5.1 mg/m3. A very slight or slight suppurative exudate was present in the lumen of the nasal passages in some males exposed to 5.1 or 53.5 mg/m3, and in 2/10 females exposed to 53.5 mg/m3.
Some treatment-related histopathological effects were present in the posterior and ventral aspects of the nasal passages. The majority of males and females exposed to 53.5 mg/m3 had very slight or slight hyaline droplet formation in the olfactory epithelium. Very slight or slight hyaline droplet formation was also present in the respiratory epithelium in 5/10 males and 5/10 females exposed to 53.5 mg/m3. The hyaline droplet formation in the olfactory and respiratory epithelium was most commonly present on the ventral ethmoid turbinates and the ventral septum in the posterior aspect of the nasal passages. An increase in the incidence of multifocal, very slight or slight eosinophilic inflammation was present in males exposed to 53.5 mg/m3 and females exposed to 5.1 or 53.5 mg/m3, as compared to the incidence of same sex control group animals. The inflammation, which consisted of mainly eosinophils, was present in the lamina propria of the ventral aspect of the ethmoid turbinates, subjacent to the dorsal aspect of the pharyngeal duct, and/or in the lamina propria of the ventral and posterior aspects of the nasal septum. Very slight mucous cell hypertrophy of cells lining the pharyngeal duct was present in 6/10 males and 5/10 females exposed to 53.5 mg/m3. One male exposed to 53.5 mg/m3 had slight hyperplasia and hypertrophy of mucous cells lining the pharyngeal duct. One male exposed to 5.1 mg/m3 had a focal, very slight ulcer of the epithelium of the pharyngeal duct, which was accompanied by slight granulomatous inflammation in the underlying lamina propria.
The most common treatment-related effects of the larynx, present in all males and females exposed to 53.5 mg/m3, consisted of: multifocal, slight squamous metaplasia of the respiratory epithelium; multifocal, slight or moderate subacute to chronic inflammation of the lamina propria; and multifocal, slight or moderate fibrosis in the lamina propria. The chronic inflammation was composed of a mixture of neutrophils, lymphocytes and plasma cells, with nodular clusters of lymphocytes present in the lamina propria at the base of the epiglottis. Treatment-related multifocal, slight or moderate hyperplasia of the respiratory epithelium was present in the larynx of all males and 9/10 females exposed to 53.5 mg/m3. All of these laryngeal effects were most prominent in the anterior larynx, near the base of the epiglottis at the level of the seromucinous gland, and some of these effects were present with lesser frequency on the medial aspect of the arytenoid cartilage. Additional treatment-related effects of the larynx in rats exposed to 53.5 mg/m3 consisted of: multifocal, very slight erosions of the respiratory epithelium at the base of the epiglottis in 1/10 males; slight suppurative exudate in lumen of the larynx in 1/10 males and 4/10 females; multifocal, very slight necrosis of individual cells in the respiratory epithelium of ventral aspect of the larynx at the level of the ventral pouch in 1/10 males; and focal or multifocal, very slight, slight, or moderate ulceration of the respiratory epithelium in 4/10 males and 5/10 females. The locations of the laryngeal ulcers varied from animal to animal, and were noted in the respiratory epithelium at the base of the epiglottis, dorsal to the ventral pouch, and/or on the medial aspect of the arytenoid cartilage.
The most common treatment-related effect of the lungs was multifocal, very slight epithelial alteration of the bronchi, observed in all males and females exposed to 53.5 mg/m3. The epithelial alteration was characterized by loss of cilia, flattening, and minimal stratification of the bronchial epithelium. The epithelial alteration was noted in the epithelium that lines the portion of the bronchial walls which project into the airways at some of the branching points of the bronchi. One male exposed to 53.5 mg/m3 had focal, slight bronchiolo-alveolar hyperplasia of the lungs, which was interpreted to be treatment related. Alveolar macrophages and perivascular eosinophils were present in the focal area of bronchiolo-alveolar hyperplasia.
Treatment-related focal or multifocal, very slight epithelial alteration of the respiratory epithelium of the trachea was present in all males and 7/10 females exposed to 53.5 mg/m3. The epithelial alteration was characterized by loss of cilia, flattening, and minimal stratification of respiratory epithelial cells at the level of bifurcation of the trachea.
There were no treatment-related histopathological effects in the upper or lower respiratory tract of males or females exposed to 0.2 mg/m3.
All males from the control and high-dose (53.5 mg/m3) groups had degeneration of testicular seminiferous tubules and associated degenerative spermatic elements of the epididymides of variable severities and distributions. These alterations were caused by physical compression of the testes when the rats were present in the nose-only exposure chambers. All other histopathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to AEP.
Dose descriptor:
NOEC
Effect level:
0.2 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: point of contact irritant effects
Dose descriptor:
NOEC
Effect level:
53.5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Critical effects observed:
not specified

Female Red Blood Cell Counts and Hemoglobin Concentrations

 Sex  Females            
 Exposure Level (mg/m3)  Historical Control@  0  0.2  5.1  53.5
 Red Blood Cell Count (106/µ L)  8.35 -9.41  8.92  8.73*  8.85  9.03
 Hemoglobin Concentration (g/dL)  14.5 -16.8  15.4  15.0*  15.4  15.6

@Historical control from data obtained from two nose-only 90-day inhalation toxicity studies and eleven data sets with dietary 90-day toxicity endpoints in F344 rats reported from 2011-2015 (seven 90-day studies and four 2-year studies using 90-day time points).

*Statistically different from control mean by Dunnett’s test, alpha=0.05. Male Cholesterol Concentrations
 Sex  Males            
 Dose (mg/m3)  Historical Control@  0  0.2  5.1  53.5
 Cholesterol Concentration (mg/dL)  52 -70  54  49  50  48*

@Historical control from data obtained from two nose-only 90-day inhalation toxicity studies and eleven data sets with dietary 90-day toxicity endpoints in F344 rats reported from 2011-2015 (seven 90-day studies and four 2-year studies using 90-day time points).

*Statistically different from control mean by Dunnett’s test, alpha=0.05.
Conclusions:
There were no treatment-related changes in in-life observations, ophthalmological exams, body weight or body weight gains, feed consumption, clinical chemistry, coagulation, hematology, urinalysis, or gross pathologic observations in male or female rats at any exposure level.
Females exposed to 53.5 mg/m3 had higher absolute and relative mean lung weights (statistically significant for relative weight) as compared to controls, which were interpreted to be treatment-related.
In the 13-week exposure group, there was no histopathological evidence of any primary systemic toxicity in any AEP exposure group (systemic NOEC = 53.5 mg/m3). The nasal tissue was the most sensitive target tissue in rats exposed to AEP. Compared to air-exposed (0 mg/m3) control rats of the same sex, male and female rats exposed to 5.1 or 53.5 mg/m3 had exposure-related histopathological changes in the nasal tissues. The larynx, trachea, and lungs of male and female rats exposed to 53.5 mg/m3 also had exposure-related histopathological effects. All of the exposure-related changes were consistent with localized irritant effects of the test material at the point of contact with the airway epithelium.
Treatment-related histopathologic effects persisted following 13-weeks of recovery in the nasal tissues of males exposed to 5.1 or 53.5 mg/m3, and in the larynx and lungs of males exposed to 53.5 mg/m3.
Based on treatment-related effects in the nasal tissues of males and females exposed to 5.1 or 53.5 mg/m3, the no-observed-effect concentration (NOEC) for point of contact irritant effects for male and female F344/DuCrl rats repeatedly exposed to AEP for 13 weeks (65 exposures) was 0.2 mg/m3. The no-observed-effect concentration (NOEC) for systemic toxicity was 53.5 mg/m3.
Executive summary:

This study was designed to evaluate the potential for local (portal-of-entry) and systemic toxicity from inhalation of aminoethylpiperazine (AEP). Groups of ten male and ten female F344/DuCrl rats were exposedvianose-only inhalation to AEP six hours/day, five consecutive days/weekfor 13 weeks (a total of 65 exposures). Additional groups of 10 male rats per exposure concentration were contemporaneously exposed and held (unexposed) for an additional 13 weeks following the last exposure day to serve as post-exposure recovery groups to determine the persistence or reversibility of any AEP-dependent effects identified at the end of exposure. The exposure atmosphere contained both AEP vapor and an AEP-carbamate aerosol that formed spontaneously in the humidified chamber atmosphere. The rats were exposed to analytically-determined concentrations of0, 0.2 ± 0.1, 5.1 ± 1.1, or 53.5 ± 6.0 mg AEP/m3(study mean±standard deviation). The analytical method measured total AEP present in the exposure chambers (AEP vapor + AEP-carbamate aerosol). The average mass median aerodynamic diameter (MMAD) of the aerosol fraction in each of the exposure chambers was0.85±1.52,1.96 ± 1.58, or 1.04 ± 2.39microns (MMAD ± geometric standard deviation; GSD) for the 0.2, 5.1, or 53.5 mg AEP/m3exposure chambers, respectively. In-life observations(including ophthalmology), body weights/body weight gains, feed consumption,urinalysis,hematology, coagulation, clinical chemistry, and organ weights were evaluated. A gross necropsy was conducted and a detailed histopathological examination of the entire respiratory tract was performed to assess treatment-dependent portal-of-entry effects. In addition, a detailed histopathologic examination of other specified tissues/organs was performed on the control- and high-exposure group rats to identify treatment-related systemic toxicity.

There were no treatment-related changes in in-life observations, ophthalmologic examinations, body weight/body weight gains, feed consumption, clinical chemistry, coagulation, hematology, urinalysis, or gross pathologic observations in male or female rats at any exposure level.

Females exposed to 53.5 mg/m3had higher absolute and relative mean lung weights (statistically significant for relative weight) as compared to controls, which were interpreted to be treatment-related.

In the 13-week exposure group, there was no histopathological evidence of any primary systemic toxicity in any AEP exposure group (systemic NOEC ≥ 53.5 mg/m3). The nasal mucosa was the most sensitive target tissue in rats exposed to AEP. Compared to air-exposed (0 mg/m3) control rats of the same sex, male and female rats exposed to 5.1 or 53.5 mg/m3had exposure-related histopathological changes in the nasal tissues. The larynx, trachea, and lungs of male and female rats exposed to 53.5 mg/m3also had exposure-related histopathological effects. All of the exposure-related changes were consistent with localized irritant effects of the test material at the point of contact with the airway epithelium.

There were no treatment-related histopathologic effects in males or females exposed to 0.2 mg/m3. In the 13-week recovery group, treatment-related histopathological effects persisted in the nasal tissues of males exposed to 5.1 or 53.5 mg/m3, and in the larynx and lungs of males exposed to 53.5 mg/m3.

Based on treatment-related effects in the nasal tissues of males and females exposed to 5.1 or 53.5 mg/m3, the no-observed-effect concentration (NOEC) for point of contact irritant effects for male and female F344/DuCrl rats repeatedly exposed toAEPfor 13 weeks (65 exposures) was 0.2 mg/m3. The no-observed-effect concentration (NOEC) for systemic toxicity was 53.5 mg/m3.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
0.2 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
There is a good quality recent fully GLP compliant Klimisch 1 90-day inhalation study for the read across source substance 2-piperazin-1-ylethylamine (aminoethyl piperazine).

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
No repeat dose dermal study is available but the use of the oral NOAEL will allow the calculation of systemic dermal DNELs. It is not considered scientifically justified to carry out test as 1-methyl piperazine is corrosive to skin. The data base is adequate for the calculation of the DNELs.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
There is not study available to determine a NOAEL for local dermal effects, as the corrosive and skin sensitizing properties of 1-methyl piperazine require the use of gloves and protective clothing, the potential for skin contact will be minimized. It is not considered scientifically justified to carry out a dermal test as 1-methyl piperazine is corrosive to skin. The available information is sufficient for the safe use of 1-methyl piperazine.

Mode of Action Analysis / Human Relevance Framework

I-methyl piperazine did not product any systemic toxic effects in the OECD422 study in rats. The read across source substance2-piperazin-1-ylethylamine(aminoethyl piperazine) also did not produce and systemic toxic effects in the 90-inhalation study. Both substances are classified as category 1B for being corrosive to skin. The effects seen in the 90-day inhalation study were related to local irritant/damaging effects in the respiratory tract, in particular in the nasal passages, larynx and at the highest concentration in the lungs. Such local effects can be expected to also be relevant to humans exposed to similar airborne concentrations as the mechanism is local irritant effects which is dependent on local concentration in the respiratory tract. The irritant effects would limit human exposure as individuals would remove themselves form irritant atmospheres.

Additional information

There is Klimisch 1 OECD422 repeat dose study via the drinking water, this study on N-methyl piperazine (1-methyl piperazine) established NOAELs levels for the parental animals after 43 days dosing in males and 57-days dosing in females via the drinking water to allow the calculation of DNELs. The highest dose level 10’000ppm in the drinking water resulted in a significant reduction in water consumption including during the gestation and lactation phases for the females, consumption was approximately 50-60 % of the controls and the pre-treatment period. This indicated that this was a maximum dose as increasing further would not have resulted in a higher ingested dose, as water consumption would have decreased further and could have resulted in mortality.  There were also effects on the males of reduced body weight gain at the highest dose level and on female bodyweight gain during gestation which was not due to a difference in litter size. This confirmed that 10’000ppm was a suitable maximum dose level, which produced adverse effects, this corresponded to a dose level of 466mg/kg bodyweight/day for the males and between 574 and 1055 mg/kg bodyweight/day at different stages of the study for the females. 2’500ppm the next dose level was a NOAEL, this corresponded to a dose level in the males 190 mg/kg bodyweight/day and for the females between 231 and 416 mg/kg bodyweight/day at different stages in the study. The females being forced to drink more during the lactation phase in particular. The male NOAEL being the lowest can be used as the basis for calculating the DNELs.

There were no histopathological changes seen in any of the organs even at the highest dose level, some effects on organ weights were seen but were not accompanied by any function effect of histopathological changes and therefore not considered to be adverse effects due to treatment.

There is a good quality recent fully GLP compliant Klimisch 1 90-day inhalation study for the read across source substance2-piperazin-1-ylethylamine(aminoethyl piperazine). Due to the corrosive nature of this substance which is classified as category 1B as is 1-methyl piperazine, adverse effects were mainly due to local irritation/damage in the respiratory tract, specifically in the nasal passage, larynx and lungs at the highest concentration. There were no systemic adverse effects seen in this study. Therefore, the top concentration 53.5 mg/m3 can be used as a NOAEC for systemic toxicity. This study also provides a NOAEC of 0.2mg/m3 for local effects in the respiratory tract in particular the nasal passages.

Justification for classification or non-classification

Classification for Specific Target Organ Toxicity (STOT), by the EU CLP(GHS) guideline requires serious adverse effects on organs at dose levels of less than 100mg/kg bodyweight/day in a 90-day study or <300mg/kg bodyweight/day in a 28-day study. In the OECD 422 study the duration was 43 days for males and 5-day post-partum for the females (more than 43 days). These values for STOT could therefore be between the 28 and 90-day i.e. less than 200 mg/kg bodyweight/day. The OECD 422 showed no adverse effects that could be classified as STOT at the highest dose, which was well in excess of 300mg/kg bodyweight/day. Therefore, there were no effects that would require classification for STOT for the oral or dermal routes of exposure.

However, In the 90-day study forthe read across source substance2-piperazin-1-ylethylamine(aminoethyl piperazine), done by the inhalation route, there were marked effects in the respiratory tract due to corrosive nature of the test substance. The 90-day point of contact NOEC was 0.2mg/m3. Exposure related lesions were seen in the larynx, trachea and lungs in rats exposed to 53.5mg/m3. Based on these findings2-piperazin-1-ylethylamineis classified as STOT RE Category 1 for Specific Target Organ Toxicity in the respiratory tract. There were no systemic toxic effects at these exposure levels. Both substances are classified as category 1B corrosive to skin and 1-methylpiperazine has a similar but higher vapour pressure thanthe read across source substance2-piperazin-1-ylethylamine (aminoethyl piperazine)so reading across the inhalation test results is appropriate.

 

As we are reading across to 2-piperazin-1-ylethylamine (aminoethyl piperazine) for the Annex IX tests for the 1-methyl piperazine, with this new study we need to read across this classification. Based on this the classification of 1-methylpiperazine, is also STOT RE Category 1, for inhalation due to local effects in the respiratory tract based on EU CLP(GHS) criteria.