N,N-bis(2,3-epoxypropyl)aniline

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2,3-epoxypropyl)aniline

EC Number:

218-259-5

EC Name:

N,N-bis(2,3-epoxypropyl)aniline

Cas Number:

2095-06-9

Molecular formula:

C12H15NO2

IUPAC Name:

N,N-bis[(oxiran-2-yl)methyl]aniline

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK.

Sex:

male/female

Details on test animals or test system and environmental conditions:

On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 273 to 346g and were approximately eleven weeks old. The females weighed 193 to 236g and were approximately fourteen weeks old.

Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 6. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, 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 computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.

Vehicle:

polyethylene glycol

Details on oral exposure:

Animals were allocated to treatment groups as follows:

Treatment Group Dose Level Treatment Concentration Animal Numbers
(mg/kg bw/day) Volume (mL/kg) (mg/mL) Male Female
Control 0 4 0 12 (1-12) 12 (13-24)
Low 10 4 2.5 12 (25-36) 12 (37-48)
Intermediate 30 4 7.5 12 (49-60) 12 (61-72)
High 100 4 25 12 (73-84) 12 (85-96)

The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared every two weeks and stored at approximately 4 ºC in the dark.

Samples of three test item formulations were taken and analyzed for concentration of Formaldehyde, oligomeric reaction products with 2-Oxiranemethanamine, N-(2-oxiranylmethyl)-N-phenyl- (EP493) at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Annex 2. The results indicate that the prepared formulations were within 9% of the nominal concentration.

Duration of treatment / exposure:

28 day

Frequency of treatment:

once per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Chronological Sequence of Study

i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.

ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). During the pre-pairing period, vaginal smears were performed for females. The first day of dosing was designated as Day 1 of the study.

iii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.

iv. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

v. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

vi. On completion of the pairing phase, five selected males per dose group were evaluated for functional/sensory responses to various stimuli during Week 6.

vii. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance and visible nipple counts (male offspring) and clinical signs were also recorded during this period.

viii. On Day 4 post partum, where possible, blood sampling was performed on two randomly allocated offspring from each litter in order to obtain serum samples.

ix. At Day 12 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

x. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 43. The male dose groups were killed and examined macroscopically on Day 44 and 45.

xi. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 13 post partum. All females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. Any female which did not show positive evidence of mating or produce a pregnancy was also sacrificed and examined macroscopically around the same time as littering females.

xii. Where possible, blood samples to produce serum were taken from two randomly allocated offspring on Day 4 post partum for assessment of thyroid hormones. On Day 13 post partum, where possible, blood sampling (to produce serum) was performed on two randomly allocated offspring (one male and one female) per litter for assessment of thyroid hormones. Where possible, a further two randomly allocated offspring (one male and one female) per litter were sampled (to produce plasma). On Day 13 post partum all surviving offspring were sacrificed and examined externally; an internal examination was performed if abnormalities were detected externally. In addition, blood samples were taken from all adult males and females at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Examinations

Observations and examinations performed and frequency:

Serial Observations
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

Normal range data for body weight changes in pregnant and lactating females are shown in Annex 8.

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 for the periods covering post partum Days 1-4, 4-7, 7-14.

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

Normal range data for pregnant and lactating females are presented in Annex 8.

Water Consumption
Water intake was measured daily during the pre-pairing phase of the study.

Estrous Cycle Assessment
Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior 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 Behavioral 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 on each occasion (at least two hours after dosing), 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
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Reproductive Performance
Normal range data for reproductive parameters and offspring are presented in Annex 8 and Annex 9.

Mating
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 estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible 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

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4, 7 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 43 for males and Day 13 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

The methods used for hematological and blood chemical investigations are presented in Annex 7 and normal ranges are shown in Annex 10.

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)

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 Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)

Thyroid Hormone Analysis
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:

Where possible from each litter, serum samples from two randomly allocated offspring on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or less offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.

Serum samples from two randomly allocated offspring (one male and one female) on Day 13 post partum. Where possible from each litter, plasma samples were also taken from two randomly allocated offspring (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.

Serum and plasma samples were taken from all adult males and females at termination.

All serum samples were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator (I Komjarova). A complete Thyroid Hormone Analysis report is presented in Annex 3.

Sacrifice and pathology:

Necropsy
Surviving adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Surviving adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to mate or achieve pregnancy were killed around the same time as littering females.

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).

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 Examination of offspring was restricted to an macroscopic external examination except where abnormalities were observed, then an additional internal examination was performed.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in bold were weighed from all remaining animals:

Adrenals Prostate
Brain Seminal Vesicles (with Coagulating Gland)
Epididymides Spleen
Heart Testes
Kidneys Thymus
Liver Thyroid (weighed post-fixation with Parathyroid)
Ovaries Uterus (weighed with Cervix and oviducts)
Pituitary (weighed post-fixation)

Normal ranges for organ weights are given in Annex 11.

On Day 13 of age, where possible, for one male and one female offspring per litter, the whole or samples of thyroid/parathyroid were retained in 10% Buffered Formalin.

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:

Adrenals Mammary gland
Aorta (thoracic) Muscle (skeletal)
Bone & bone marrow (femur including stifle joint) Ovaries
Bone & bone marrow (sternum) Pancreas
Brain (including cerebrum, cerebellum and pons) Pituitary
Cecum Prostate
Colon Rectum
Cowpers glands Salivary glands (submaxillary)
Duodenum Sciatic nerve
Epididymides ♦ Seminal vesicles (with coagulating gland)
Esophagus Skin
Eyes * Spinal cord (cervical, mid thoracic and lumbar)
Glans penis Spleen
Gross lesions Stomach
Heart Testes ♦
Ileum (including peyer’s patches) Thyroid/Parathyroid
Jejunum Trachea
Kidneys Thymus
Liver Urinary bladder
LABC (levator ani-bulbocavernous) muscle Uterus & Cervix (with oviducts)
Lungs (with bronchi)# Vagina
Lymph nodes (mandibular and mesenteric)

* Eyes fixed in Davidson’s fluid
Preserved in Modified Davidsons fluid
# lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

Tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The tissues from five selected control and 500/400 mg/kg bw/day dose group animals and any animals dying during the study, were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 500/400 mg/kg bw/day animals and animals which did not mate or achieve a pregnancy were also processed. In addition, sections of testes from all control and 500/400 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the kidneys (males only) and the stomach and intestines (both sexes) from five selected animals from each sex in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS. A complete histopathology phase report is presented in Annex 1 and represents the consensus view of both pathologists.

Other examinations:

Data Evaluation
Data were processed to give summary incidence or group mean values and standard deviations where appropriate. All data were summarized in tabular form.

Treatment of Data
Data shown in the appendices are frequently rounded values for presentation purposes. Group mean values are generally calculated using non-rounded values therefore is it not always possible to calculate the exact group values from the individual values presented in the appendices.

For body weights and food consumptions during gestation, group mean values were calculated using data from females which were observed to give birth to offspring.

For body weights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 13 of lactation.
Reproductive Indices
Mating Performance and Fertility

The following parameters were calculated from the individual data during the mating period of the parental generation:

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

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

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

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

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

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

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

Parturition Index (%) = Number of females delivering live offspring x 100/Number of pregnant females

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).

i. Implantation Losses (%)
Group mean percentile post-implantation loss were calculated for each female/litter as follows:

Post–implantation loss (%) = (Number of implantation sites - Total number of offspring born) x 100/Number of implantation sites

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live Birth Index (%) = Number of offspring alive on Day 1 x 100/Number of offspring born

Viability Index (%) = Number of offspring alive on Day 4 x 100/Number of offspring alive on Day 1

Viability Index 2(%) = Number of offspring alive on Day 13 x 100/Number of offspring alive on Day 4

Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum.

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4 and 13 post partum, using the following formula:

Number of male offspring x 100/Total number of offspring

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Post-Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights and Thyroid Hormone (Thyroxine).

Results and discussion

Results of examinations

Description (incidence and severity):

A summary incidence of daily clinical observations is given in Table 2. Individual data are presented in Appendix 1.

Noisy respiration and increased salivation were evident in the majority of animals of either sex treated with 100 mg/kg bw/day from Day 1 onwards. These observations were also evident in animals of either sex treated with 30 mg/kg bw/day albeit to a lesser extent. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and reflects the distaste of the formulation or potential difficulty in dosing particular animals on occasions rather than true systemic toxicity.

No such effects were evident in animals of either sex treated with 10 mg/kg bw/day.

One male and one female treated with 10 mg/kg bw/day had generalised fur loss towards the end of the treatment period. An observation of this nature is commonly observed in group housed males and in females during lactation and is considered to be incidental.

Description (incidence):

There were no unscheduled deaths.

Description (incidence and severity):

Group mean weekly body weights and standard deviations are given in Table 7 and are presented graphically in Figure 1 and Figure 2. Group mean weekly body weight gains and standard deviations are given in Table 8 (statistically significant differences are indicated). Individual data are given in Appendix 7 and Appendix 8.

Males treated with 100 mg/kg bw/day showed an 8% reduction in overall body weight gain when compared to controls.

No such effects were evident in males treated with 30 or 10 mg/kg bw/day.

No effect in body weight development was evident in treated females during maturation, gestation or lactation. Statistical analysis of the data did not reveal any significant intergroup differences.

Males treated with 10 mg/kg bw/day showed a statistically significant increase in body weight gain during Week 4 of treatment. An increase in body weight gain is considered not to represent an adverse effect of treatment.

Description (incidence and severity):

Group mean food consumptions are given in Table 9 and are presented graphically in Figure 3 and Figure 4. Individual values for females during gestation and lactation are presented in Appendix 9. Food efficiency for males and for females during the pre-mating phase is given in Table 10.

There was no effect of treatment on food consumption or food conversion efficiency for males throughout the pre-pairing and post-pairing phases of the study at 10, 30 or 100 mg/kg bw/day.

There was no effect of treatment on food consumption for females during the pre-pairing, gestation or lactation phases of the study at 10, 30 or 100 mg/kg bw/day.

Description (incidence and severity):

There was no effect of treatment on water consumption at 10, 30 or 100 mg/kg bw/day. Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Description (incidence and severity):

Group mean values and standard deviations for test and control group animals are given in Table 20 (statistically significant differences are indicated). Individual data are given in Appendices 19 to 21.

There were no toxicologically significant effects detected in the hematological parameters measured.

Females treated with 100 mg/kg bw/day showed statistically significant reductions (p<0.05-0.01) in mean corpuscular hemoglobin concentration, total leukocyte count, lymphocytes and activated partial thromboplastin time. Males from all treatment groups showed a statistically significant increase in reticulocyte count (p<0.05-0.01). Males treated with 100 mg/kg bw/day also showed a statistically significant increase (p<0.05) in prothrombin time. All of the individual values were within historical control ranges and no associated histopathological correlates were evident. The intergroup differences were therefore considered not to be of toxicological significance.

Description (incidence and severity):

Group mean values and standard deviations for test and control group animals are given in Table 21 (statistically significant differences are indicated). Individual data are given in Appendices 22 and 23.

There were no toxicologically significant effects detected in the blood chemical parameters measured.

Males from all treatment groups showed a statistically significant increase in bilirubin (p<0.05). Males treated with 100 mg/kg bw/day also showed a statistically significant reduction (p<0.01) in chloride concentration. All of the individual values were within historical control ranges, no true dose related response was evident in bilirubin and no associated histopathological correlates were evident. The intergroup differences were therefore considered not to be of toxicological significance. Males treated with 100 mg/kg bw/day also showed a statistically significant increase (p<0.05) in bile acids. Although the majority of the individual values were above the historical control range, no associated histopathological correlates were evident, therefore, the intergroup difference was considered not to be of toxicological significance.

Description (incidence and severity):

Functional Observations
Summary incidence of behavioral assessment observations are given in Table 3 and group mean behavioral assessment scores are given in Table 4. Group mean functional performance test values and standard deviations are given in Table 5. Individual values are given in Appendices 2 to 5. Group mean sensory reactivity assessment scores are given in Table 6. Individual responses are given in Appendix 6.

Behavioral Assessments
There were no toxicologically significant changes in the behavioral parameters at 10, 30 or 100 mg/kg bw/day.

One male and one female treated with 10 mg/kg bw/day showed an isolated incident of noisy respiration during week 2 assessment or during assessment on Day 11 of gestation. No such effects were evident in these animals during the routine daily clinical observations, therefore, this was considered to be the result of difficulty in dosing these particular animals on these occasions rather than evidence of true systemic toxicity.

Functional Performance Tests
There were no treatment related changes in functional performance considered to be related to treatment at 10, 30 or 100 mg/kg bw/day.

Sensory Reactivity Assessments
There were no intergroup differences in sensory reactivity scores that were considered to be related to treatment at 10, 30 or 100 mg/kg bw/day.

Description (incidence and severity):

Group mean absolute and body weight-relative organ weights and standard deviations for test and control group animals are presented in Table 24 (statistically significant differences are indicated). Individual data are given in Appendices 26 and 27.

Males treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.01) in seminal vesicles weights both absolute and relative to terminal body weights. Although the majority of individual values were within historical control ranges, in view of the microscopic changes evident in the seminal vesicles in these males, a relationship to treatment cannot be excluded.

No toxicologically significant effects were evident in treated females or in males treated with 30 or 10 mg/kg bw/day.

Males treated with 100 and 30 mg/kg bw/day showed a statistically significant reduction (p<0.05-0.01) in pituitary weight both absolute and relative to terminal body weight. The majority of individual values were within historical control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance. Males treated with 100 mg/kg bw/day also showed statistically significant increases in kidney and liver weights both absolute and relative to terminal body weight. Although the majority of individual relative kidney weights and all individual relative liver weights were above historical control ranges, all but one absolute individual kidney and liver weights were within historical control ranges. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance. Females treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.01) in thymus weight both absolute and relative to terminal body weight. All of the individual values were within historical control ranges and no associated histopathological correlates were evident, therefore, the intergroup differences were considered not to be of toxicological significance.

Description (incidence and severity):

A summary incidence of necropsy findings for offspring and adults is given in Tables 23 and 24. Individual data are given in Appendices 24 and 25.

Offspring
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any obvious effect of maternal treatment at 10, 30 or 100 mg/kg bw/day.

Adults
Macroscopic necropsy findings did not indicate any effect of treatment for either sex at dosages of 10, 30 or 100 mg/kg bw/day.

The following macroscopic findings observed were not associated with any microscopic abnormalities or true dose related responses and were considered to be incidental and unrelated to treatment. Increased pelvic space in the left kidney in one male treated with 30 mg/kg bw/day, pale areas on the right kidney in one female treated with 100 mg/kg bw/day, pale areas on the liver in one female treated with 10 mg/kg bw/day, a dark spleen in one control female and two masses in one female treated with 10 mg/kg bw/day.

Description (incidence and severity):

A complete histopathology phase report is presented in Annex 1.

The following treatment-related microscopic abnormality was detected:
Seminal Vesicles: Decreased secretion in one horn was evident in two control males, three males treated with 10 mg/kg bw/day, two males treated with 30 mg/kg bw/day and five males treated with 100 mg/kg bw/day. Decreased secretion in both horns was evident in two males treated with 30 mg/kg bw/day and four males treated with 100 mg/kg bw/day.

There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

Description (incidence and severity):

Thyroid Hormone Analysis
A complete Thyroid Hormone Analysis phase report is presented in Annex 3. Mean values for serum T4 concentrations are presented in Table 25.

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any adverse effect of treatment or indication of endocrine disruption at 10, 30 or 100 mg/kg bw/day.

Males treated with 100 and 30 mg/kg bw/day showed a statistically significant increase in Thyroxine (T4) when compared to controls. In the absence of any changes in thyroid weights or histopathological correlates in the thyroids, the intergroup differences were considered to be of no toxicological significance.

Reproductive Performance
Estrous Cycles
A summary incidence of estrous cycle assessments is presented in Table 11. Individual data are given in Appendix 10.

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 10, 30 or 100 mg/kg bw/day, with all females showing regular cycling.

Mating
A summary of adult performance is presented in Table 1. A summary incidence for mating performance is presented in Table 12. Individual data are given in Appendix 11.

Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at dosages of 10, 30 or 100 mg/kg bw/day.

Fertility
A summary of adult performance is presented in Table 1. Group values for fertility, litter data and implantation losses are given in Tables 12, 13 and 15. Individual data are given in Appendices 11, 12 and 14.

There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy, at dosages of 10, 30 or 100 mg/kg bw/day.

Two female treated with 10 mg/kg bw/day and two females treated with 30 mg/kg bw/day showed positive evidence of mating but were non-pregnant. No changes were noted at histopathology in either the females or the males to account for these lack of pregnancies. In the absence of any non-pregnant females at 100 mg/kg bw/day, these were considered incidental and unrelated to treatment.

Gestation Length
A summary of gestation lengths is presented in Table 12. Individual lengths are given in Appendix 11.

The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 10, 30 or 100 mg/kg bw/day.

Litter Responses
In total twelve females from the control group and ten females from the 10, 30 and 100 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 13 of age. One female treated with 100 mg/kg bw/day showed evidence of implantation sites in the uterus however no offspring were observed to be born. A further female from this dosage exhibited a total litter loss. The following assessment of litter response is based on all litters reared to termination on Day 13 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
Group mean implantation counts, litter size, implantation losses, survival indices and sex ratio are given in Tables 13, 15 and 16. Individual data are given in Appendices 12, 14 and 15.

There was no effect of maternal treatment on the number of implantations, post-implantation loss and live birth index, and subsequent offspring survival to Day 13 of age at dosages of 10, 30 or 100 mg/kg bw/day. Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.

Offspring Growth and Development
Group mean values for total litter weights, offspring body weights and body weight changes, a summary incidence of clinical signs, ano-genital distance and visible nipple counts (male offspring) are given in Tables 14, 17, 18 and 19. Individual values and observations are given in Appendices 13, 16, 17 and 18.

There was no effect of treatment with the test item indicated by offspring body weight or body weight gain, ano-genital distance on Day 1 post partum or visible nipple count in male offspring on Day 13 post partum at 10, 30 or 100 mg/kg bw/day.

Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment.

Female offspring from females treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.05) in body weight gain between Days 4 (after cull) and 7 of lactation. No such effect was evident in male offspring from these litters and the body weight gain for the female offspring during this period was within historical control range. Body weight development throughout the remainder of lactation was similar to controls, therefore this intergroup difference was considered not to be of toxicological importance.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral administration of 2-Oxiranemethanamine, N-(2-oxiranylmethyl)-N-phenyl- (EP493) to rats by gavage, at dose levels of 10, 30 and 100 mg/kg bw/day, resulted in treatment related effects in males treated with 100 mg/kg bw/day. No treatment-related effects were evident in females. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 100 mg/kg bw/day for females and 30 mg/kg bw/day for males.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 100 mg/kg bw/day.

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), to evaluate some endocrine disruptor relevant endpoints and is designed to be 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 29 July 2016).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to nine weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 10, 30 and 100 mg/kg bw/day.  A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) over the same period.

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

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and ano-genital distance and visible nipple count (male offspring only).

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 12 post partum.  Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.  Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating.

Vaginal smears were also performed in the morning on the day of termination for all treated females.  Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively.  Any female which did not produce a pregnancy was terminated around the same time as littering females.  All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.  All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Results

Adult Responses

Mortality

There were no unscheduled deaths.

Clinical Observations

Incidences of noisy respiration and increased salivation were evident in either sex treated with 100 mg/kg bw/day throughout the treatment period and to a lesser extent in animals of either sex treated with 30 mg/kg bw/day.  No such effects were evident in animals of either sex treated with 10 mg/kg bw/day.

Behavioral Assessment

There were no toxicologically significant changes in the behavioral parameters at 10, 30 or 100 mg/kg bw/day.

Functional Performance Tests

There were no treatment related changes in functional performance considered to be related to treatment at 10, 30 or 100 mg/kg bw/day.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 10, 30 or 100 mg/kg bw/day.

Body Weight

Males treated with 100 mg/kg bw/day showed a reduction in overall body weight gain when compared to controls.  No such effects were evident in males treated with 30 or 10 mg/kg bw/day.

No effect in body weight development was evident in treated females during maturation, gestation or lactation.

Food Consumption

No effect on food consumption was evident in treated males.

No effect on food consumption was evident in treated females during maturation, gestation or lactation.

Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Reproductive Performance

Estrous Cycle

Assessment of estrous cycles during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 10, 30 or 100 mg/kg bw/day.

Mating

There was no effect of treatment on mating at dosages of 10, 30 or 100 mg/kg bw/day.

Fertility

There was no effect of treatment on fertility at dosages of 10, 30 or 100 mg/kg bw/day.

Gestation Lengths

There was no effect of treatment on gestation length at dosages of 10, 30 or 100 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of maternal treatment on the number of implantations, post-implantation loss, sex ratio, live birth index, and subsequent offspring survival to Day 13 of age at dosages of 10, 30 or 100 mg/kg bw/day.

Offspring Growth and Development

There was no effect of treatment with the test item indicated by offspring body weight or body weight gain, ano-genital distance on Day 1 post partum, or visible nipple count in male offspring on Day 13 post partum at 10, 30 or 100 mg/kg bw/day.  

Laboratory Investigations

Hematology

There were no toxicologically significant effects detected in the hematological parameters measured.

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters measured.

Pathology

Necropsy

Offspring

Macroscopic necropsy findings for offspring on the study did not indicate any effect of maternal treatment at 10, 30 or 100 mg/kg bw/day.

Adults  

Macroscopic necropsy findings did not indicate any effect of treatment for either sex at dosages of 10, 30 or 100 mg/kg bw/day.  

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 10, 30 or 100 mg/kg bw/day.

Organ Weights

Males treated with 100 mg/kg bw/day showed a reduction in seminal vesicle weights both absolute and relative to terminal body weight.  No such effects were detected in males treated with 30 or 10 mg/kg bw/day.  

No treatment-related effects were evident in treated females.

Histopathology

The following treatment-related microscopic abnormality was detected:

Seminal Vesicles: Decreased secretion in one horn was evident in two control males, three males treated with 10 mg/kg bw/day, two males treated with 30 mg/kg bw/day and five males treated with 100 mg/kg bw/day.  Decreased secretion in both horns was evident in two males treated with 30 mg/kg bw/day and four males treated with 100 mg/kg bw/day.

Conclusion

The oral administration of 2-Oxiranemethanamine, N-(2-oxiranylmethyl)-N-phenyl- (EP493) to rats by gavage, at dose levels of 10, 30 and 100 mg/kg bw/day, resulted in treatment related effects in males treated with 100 mg/kg bw/day.  No treatment-related effects were

evident in females.  The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 100 mg/kg bw/day for females and 30 mg/kg bw/day for males.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 100 mg/kg bw/day.