Bis(2,3-epoxypropyl) phthalate

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 (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2,3-epoxypropyl) phthalate

EC Number:

230-566-6

EC Name:

Bis(2,3-epoxypropyl) phthalate

Cas Number:

7195-45-1

Molecular formula:

C14H14O6

IUPAC Name:

bis(oxiran-2-ylmethyl) phthalate

Test material form:

liquid

Details on test material:

As defined in Section 1.2 and 1.4.

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 283 to 340g and were approximately eleven weeks old. The females weighed 196 to 243g 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 5. 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 50 4 12.5 12 (25-36) 12 (37-48)
Intermediate 125 4 31.25 12 (49-60) 12 (61-72)
High 250 4 62.5 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 4,4’-isopropylidenediphenol and diethylenetriamine (EK110) 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.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

250 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 and 7 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 Research Centre, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: J Schofield). The tissues from five selected control and 250 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 250 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 250 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.


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.

There were no clinical signs specifically related to systemic toxicity elicited by the test item for any animal which survived until the end of the treatment period.

Animals of either sex treated with 250 mg/kg bw/day showed incidences of increased salivation from Day 6 and noisy respiration from Day 1. One female animal from this treatment group exhibited signs of decreased respiratory rate, hunched posture and pilo-erection but these signs were isolated to one or two instances only. Increased salivation was apparent (but to a lesser extent) in male animals treated with 125 mg/kg bw/day from Day 13, noisy respiration was also noted but this occurred in one animal for one day only. An isolated instance of increased salivation was noted in one female treated with 125 mg/kg bw/day. Noisy respiration was apparent in one female treated with 50 mg/kg bw/day on two separate occasions only. No clinical signs were apparent in any male animal treated with 50 mg/kg bw/day. One control animal of either sex exhibited noisy respiration for one day only.

Increased salivation is commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and is generally considered to be of no toxicological importance. Noisy respiration was considered to reflect occasional difficulties with dosing these animals (possibly causing reflux of the test item) rather than any underlying toxicological effect of the test item.

No significant clinical signs of toxicity were apparent prior to the animals being found dead during the study. The two male animals treated with 250 mg/kg bw/day that were killed in extremis on Days 42 or 43 exhibited increased salivation, labored respiration, noisy respiration and hunched posture. One of these animals also exhibited decreased respiratory rate and diarrhea, the other male animal also exhibited gasping respiration and lethargy. The female animal treated with 250 mg/kg bw/day that was killed in extremis on Day 51 did not exhibit any clinical signs on Day 51 but had shown substantial body weight loss and had exhibited noisy respiration and decreased respiratory rate on Day 50.

Description (incidence):

One control female and one male animal treated with 125 mg/kg bw/day were found dead on Day 9. One female treated with 250 mg/kg bw/day was found dead on Day 15. Two male animals treated with 250 mg/kg bw/day were killed in extremis on Day 42 or 43, one female from this treatment group was killed in extremis on Day 51. There were no further
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 250 mg/kg bw/day showed a statistically significant (p<0.01) reduction in body weight gain during the first week of treatment. Recovery was evident during the second week, however, fluctuations were noted throughout the remainder of the treatment period such that overall body weight gain was 30% lower than control by the end of the treatment period.

Males treated with 125 mg/kg bw/day showed a slight reduction in body weight gain during the first week of treatment. Recovery was generally evident thereafter, however, slight fluctuations were apparent during the remainder of the treatment period, these were not as marked as for males treated with 250 mg/kg bw/day but still resulted in an overall body weight gain which was 11% lower than control.

No such effects were detected in male animals treated with 50 mg/kg bw/day.

No adverse effects were detected in treated females during maturation. During the second week of maturation, females treated with 250 and 125 mg/kg bw/day exhibited statistically significant increases (p<0.05) when compared to control. Body weight gain during the first two weeks of gestation for females treated with 50 mg/kg bw/day was comparable to controls, however, a reduction in body weight gain was evident in these females during the final week of gestation but without attaining statistical significance, this was considered to reflect the lower litter size at this dosage. During lactation, body weight gains were reduced in animals treated with 50 mg/kg bw/day, statistical significance was achieved from Days 1 to 4 (p<0.05). Whilst slight signs of recovery were evident during the remainder of the lactation phase, body weight gains were still lower than control resulting in reductions in cumulative body weight gains which achieved statistical significance (p<0.01 - 0.05) from Days 1 to 7 and 1 to 14 respectively. This resulted in an overall reduction in body weight gain of approximately 31% when compared to control.

Although females treated with 250 and 125 mg/kg bw/day were all non-pregnant, body weight gains during the first week post coitum were comparable to control females. Subsequent body weight gains for these females was reduced; with the majority of females showing actual body weight losses.

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.

There were no adverse effects in male food consumption throughout the treatment period. Males treated with 250 mg/kg bw/day showed marginal reductions when compared to control throughout the treatment period. No such effects were apparent for male animals treated with 125 or 50 mg/kg bw/day.

No effects were detected in food consumption for treated females during maturation. Food consumption during gestation for females treated with 50 mg/kg bw/day was comparable to controls throughout gestation, however, food consumption was reduced in this treatment group during lactation which resulted in statistical significance being achieved (p<0.01 - 0.05) from Days 4 to 7 and 7 to 14 respectively. Although females treated with 250 and 125 mg/kg bw/day were all non-pregnant, food consumption during the first week post coitum was comparable to control females. Food consumption then progressively reduced in these animals during the next two weeks of treatment.

Description (incidence and severity):

Food efficiency for males and for females during the pre-mating phase is given in Table 10.

Reductions in food conversion efficiency was evident in animals of either sex treated at 250 mg/kg bw/day during the first week of treatment, this was noted in the males from this group during the final two weeks of treatment also, however, these generally followed the fluctuations in body weight gain seen in these animals.

No such effects were apparent in animals of either sex treated with 50 or 125 mg/kg bw/day.

Description (incidence and severity):

Group mean daily water consumptions during the pre-pairing phase are given in Table 11.

No effects were detected in water consumption for any treated male animal. Females treated with 250 mg/kg bw/day exhibited an increase in water consumption throughout maturation. No such effects were noted in female animals treated with 125 or 50 mg/kg bw/day.

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 19 to 21.

Male animals treated with 500 mg/kg bw/day and 300 mg/kg bw/day exhibited statistically significant increases in neutrophils (p<0.05). One male animal treated with 500 mg/kg bw/day exhibited a neutrophil value that was extremely high when compared to the normal control data range. Due to the effects noted in the stomach at histopathological examination an association with treatment cannot be discounted.

There were no further effects which could be associated with treatment on the hematological parameters measured. Male animals treated with 500 mg/kg bw/day and 300 mg/kg bw/day exhibited statistically significant increases in reticulocytes (p<0.01). All individual reticulocyte values were within the historical control data range and there were no further supporting findings in other erythrocyte parameters measured. As there were also no histopathological correlates these intergroup differences were considered not to be of any toxicological significance. Male animals treated with 500 mg/kg bw/day exhibited a statistically significant reduction (p<0.05) in activated partial thromboplastin time. All individual values were within the normal historical control data range and in the absence of any histopathological correlates these intergroup differences were considered not to be of any toxicological significance. No such effects were noted in male animals treated with 100 mg/kg bw/day. Female animals from all treatment groups exhibited statistically significant increases in reticulocytes (p<0.05-0.01). All reticulocyte values were within the historical control data range and as there were no histopathological correlates these intergroup differences were considered not to be of any toxicological significance. A statistically significant reduction in erythrocytes was noted in females treated with 500 mg/kg bw/day, however, this was not dose related and all individual values were within the normal historical control data range for the treated female animals, one control female exhibited a value that was increased. As there were no other supporting findings in other erythrocyte parameters measured and in the absence of any histopathological correlates these intergroup differences were considered not to be of any toxicological significance.

Platelet counts were statistically significantly elevated (p<0.05) in females treated with 500 and 300 mg/kg bw/day. However, as all control female platelet counts were lower than the normal control data range, this finding is considered to have been over emphasised and as such it is considered not to be of toxicological significance. It must also be noted that two females treated with 500 mg/kg bw/day and one female treated with 300 mg/kg bw/day also exhibiting values that were lower.

Lymphocytes were statistically significantly reduced (p<0.05) in females treated with 500 mg/kg bw/day. However, all values were found to be within the historical control data range. This finding is considered to have been overemphasized by one control value which was higher than the historical control data range. As such this finding is considered not to be of any 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 19 to 21.

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

Statistically significant reductions in hemoglobin (p<0.01), hematocrit (p<0.05) and mean corpuscular hemoglobin concentration (p<0.05) were noted in males treated with 250 mg/kg bw/day. Statistically significant reductions in erythrocytes (p<0.01) was noted across all male treatment groups with reticulocytes being statistically significantly increased (p<0.01) across all male treatment groups. However, the vast majority of these findings were within the normal historical control data range and in the absence of any histopathological correlates these findings were considered not to be of any toxicological significance.

Assessment of hematology parameters for females at 250 and 125 mg/kg bw/day has to be treated with caution as no females at these dosages were maintaining a litter at the time of blood sampling and all of these females were non-pregnant. The female animals at these dosages were therefore in a different physiological state in comparison to the other females on the study and also those females contributing to the historical control data.

Females treated with 250 mg/kg bw/day showed a statistically significant decrease in clotting time (p<0.05). Females treated with 250 and 125 mg/kg bw/day exhibited statistically significant increases in erythrocytes (p<0.05) and reductions in mean corpuscular volume (p<0.05). Reticulocytes were statistically significantly increased (p<0.01) across all treatment groups. The vast majority of these findings were within the normal historical control data range and in the absence of any histopathological correlates these findings were considered not to be of any 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 (statistically significant differences are indicated). 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 treatment-related changes in the behavioral parameters at 50, 125 or 250 mg/kg bw/day. One female animal treated with 50 mg/kg bw/day exhibited diarrhea during the second week of treatment, however, this was an isolated incident and considered unlikely to be specifically related to treatment as none of the animals from the other two (higher) dose groups had shown similar signs at this observation time point.

Functional Performance Tests
There were no treatment related changes in functional performance considered to be related to treatment at 50, 125 or 250 mg/kg bw/day.

All treated female animals exhibited a statistically significantly higher (p<0.01) hind limb grip strength, but this was only apparent during the first run. Female animals treated with 250 mg/kg bw/day then exhibited a statistically significantly reduced (p<0.05) hind limb grip strength during the second run. However, as these increases or decreases were only noted in one out of the three runs completed and there were no clinical signs observed to signify a neurotoxic effect of the test item these findings were considered to be incidental and of no toxicological relevance.

Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 50, 125 or 250 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 28 (statistically significant differences are indicated). Individual data are given in Appendix 29 and Appendix 30.

No toxicologically significant effects were detected in the organ weights measured in animals of either sex treated with 50, 125 and 250 mg/kg bw/day.

Male animals treated with 250 mg/kg bw/day showed statistically significant increases in kidney weights both absolute and relative to terminal body weight. Four absolute values were outside of the normal historical control data range with all relative values outside of these ranges also. However, in the absence of any histopathological correlates these findings were considered to be of no toxicological significance.

Although females treated with 250 mg/kg bw/day were in a different physical state to controls, these females also showed an increase in absolute and relative kidney weights (p<0.05). Females treated with 250 and 125 mg/kg bw/day also showed absolute and relative uterus/cervix weights which were statistically significantly increased (p<0.01) when compared to control. All kidney absolute and relative values were outside of the normal historical control data ranges. Two absolute and one relative uterus weights for females treated with 250 mg/kg bw/day were outside of the normal historical control data ranges. Six relative and six absolute uterus weights in animals treated with 125 mg/kg bw/day were also outside of the normal historical control data ranges. Four control females also showed values that were lower than the historical control which would have accentuated the apparent effect on uterus weights. However, in the absence of any histopathological correlates these findings were considered not to be of any 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 Appendix 24 and Appendix 25.

Offspring
The necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment at 50 mg/kg bw/day.

Adults
One control female that was found dead on Day 9 exhibited gaseous distension of cecum, dark area on the liver, reddened lungs, torn esophagus, dark spleen, sloughing of the stomach and a dark area in the non-glandular region of the stomach. The male animal treated with 125 mg/kg bw/day which was also found dead Day 9 exhibited gaseous distension throughout the gastro-intestinal tract, yellow colored contents in the colon and jejunum, dark liver, reddened lungs, sloughing of the stomach and a raised limiting ridge in the stomach. One female treated with 250 mg/kg bw/day which was found dead on Day 15 exhibited gaseous distension throughout the gastro-intestinal tract (excluding the duodenum), a blue duodenum, a dark area on the liver, the stomach showed sloughing and a raised limiting ridge and a thin non-glandular region was also apparent. The two male animals treated with 250 mg/kg

bw/day that were killed in extremis on Day 42 or 43 generally exhibited gaseous distension throughout the gastro-intestinal tract and a raised limiting ridge in the stomach. The male that was killed in extremis on Day 42 also exhibited a reddened cecum, yellow colored contents in the colon, small epididymides, small seminal vesicles, small levator ani-bulbocavernous muscle, small testes, small liver and sloughing of the non-glandular region of the stomach. The male that was killed in extremis on Day 43 also exhibited an enlarged cecum and a thin non-glandular region of the stomach. The female from this treatment group which was killed in extremis on Day 51 exhibited gaseous distension throughout the gastro-intestinal tract, sloughing of the stomach and the non-glandular region of the stomach was thin and the limiting ridge was raised.

One female treated with 50 mg/kg bw/day, one female treated with 125 mg/kg bw/day and five males and four females treated with 250 mg/kg bw/day exhibited a thin non-glandular region of the stomach and a raised limiting ridge.

One male treated with 125 mg/kg bw/day and two males treated with 250 mg/kg bw/day exhibited a raised limiting ridge in the stomach. One male and one female treated with 250 mg/kg bw/day exhibited raised white patches on the non-glandular region of the stomach. One female treated with 250 mg/kg bw/day exhibited gaseous distension of the cecum and ileum.

There were no further findings that were considered to be directly related to treatment with the test item.

Three males treated with 250 mg/kg bw/day exhibited enlarged kidneys (both), one control male and one male treated with 50 mg/kg bw/day exhibited an increased pelvic space in the left kidney. One control female exhibited a pale area on the liver. One male treated with 125 mg/kg bw/day exhibited large kidneys and one female treated with 125 mg/kg bw/day exhibited enlarged adrenals. Due to the isolated nature of these findings they were considered to be incidental and unrelated to treatment with the test item.

Description (incidence and severity):

A complete histopathology phase report is presented in Annex 1.

Premature Decedents
In Animal 13F (Control) many of the tissues were autolyzed and no histopathological changes to account for death were noted, but necropsy confirmed a dosing error.

Animal 52 M (Group 3) and 75M (Group 4) showed minor changes in the thymus indicative of stress. Animals 88 and 92 F (Group 4) both showed minimal or mild hyperplasia in the non-glandular stomach and stress related changes were present in animal 92. Animal 77M showed notable stress related change, mild hyperplasia in the non-glandular stomach and changes in the reproductive tract including tubular atrophy in the testes. None of these animals had any indication of a cause of death and it is likely after taking into account the clinical and necropsy findings that reflux may be occurring and histological examination of the nasopharynx, likely to be definitive, was not possible.

Terminal Kill
Stomach
Changes in the non-glandular region of the stomach, namely diffuse or focal (limiting ridge) hyperplasia, were present in 4/7 males and 2/5 females in Group 4 at a minimal or mild severity. In Group 3 minimal hyperplasia, focal (limiting ridge), was present in 2/5 males and one female in Group 3. No changes were apparent in Group 2 animals.

Trachea
Metaplasia, minimal or mild was present in the trachea of one male and three females in Group 4 only. This is indicative of a reaction to irritation possibly caused by reflux or inadvertent deposition of an irritant from the end of the gavage tube.

No other changes which could be related to the administration of the test item were noted and all are considered to be incidental. There was no histological change to account for the increase in weight of the kidneys and this may be related to functional change.

Non-Productive Matings
No pregnancies occurred in Group 3 and 4 animals and 2/12 matings in Group 2. With the exception of animal 77M (Group 4 premature decedent), which showed moderate degeneration in the testes there were no changes in males or females which could account for the lack of pregnancy. Females showed an expected range of cyclical stages.

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
Group mean values and standard deviations for test and control group animals are given in Table 29. A complete Thyroid Hormone Analysis phase report is presented in Annex 3.

Levels of thyroxine (T4) in adult males did not indicate any obvious effect of treatment or indication of endocrine disruption at 50, 125 or 250 mg/kg bw/day. Levels of thyroxine (T4) in offspring at Day 13 of age did not indicate any obvious effect of maternal treatment or indication of endocrine disruption at 50 mg/kg bw/day.

Reproductive Performance
Estrous Cycles
A summary incidence of estrous cycle assessment is presented in Table 12. Individual data are given in Appendix 10.

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with all females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy. The majority of surviving female animals exhibited di-estrus whereas three animals exhibited early estrus (two treated with 250 mg/kg bw/day and one treated with 125 mg/kg bw/day).

Mating
A summary of adult performance is presented in Table 1. A summary incidence for mating performance is presented in Table 13. Individual data are given in Appendix 11.

No treatment-related effects were detected in mating performance. Statistically significant reductions (p<0.01 - 0.05) were apparent in the mean pre-coital intervals for all treated animals. All but one treated pairings showed evidence of mating at the first estrus opportunity. This difference was considered to be due to three control animals exhibiting pre-coital intervals of between 12 and 13 days and as such the effects in treated animals are considered not to be of any toxicological significance.

Fertility
A summary of adult performance is presented in Table 1. Group values for fertility, litter data and implantation losses are given in Tables 13, 14 and 16. Individual data are given in Appendices 11, 12 and 14.


All of the females treated with 250 and 125 mg/kg bw/day and two females treated with 50 mg/kg bw/day were non-pregnant following positive evidence of mating.

Gestation Length
A summary of gestation lengths is presented in Table 13. Individual lengths are given in Appendix 11.

Gestation lengths for controls and females treated with 50 mg/kg bw/day were generally between 22 and 23½ days. One female treated with 50 mg/kg bw/day exhibited a gestation length that was 24½ days. Overall, the distribution of gestation lengths for the females treated with 50 mg/kg bw/day was essentially similar to control.

Litter Responses
None of the animals from the 125 or 250 mg/kg bw/day treatment groups achieved pregnancy during the study. Two animals treated with 50 mg/kg bw/day failed to achieve pregnancy and one control female was found dead prior to the mating phase of the study. The following assessment is generally made using the 11 and 10 litters which were reared to Day 13 of age from the control and 50 mg/kg bw/day treatment groups respectively.

Offspring Litter Size, Sex Ratio and Viability
Group mean implantation counts, litter size, post-implantation losses, survival indices and sex ratio are given in Tables 14, 16 and 17. Individual data are given in Appendices 12, 14 and 15.

Females treated with 50 mg/kg bw/day showed a statistically significant reduction (p<0.01) in the number of implantation sites. Of the litters born, litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum were statistically significantly lower (p<0.01 - 0.05) for females treated with 50 mg/kg bw/day when compared to control litters. Live birth index and offspring viability in females treated with 50 mg/kg bw/day were comparable to controls. Sex ratio was also comparable to controls and did not indicate any selective effect of maternal treatment on survival for either sex.

Offspring Growth and Development
Group mean values for total litter weights, offspring body weights and body weight changes, a summary incidence of clinical signs and group mean ano-genital distance and visible nipple counts in male offspring are given in Tables 15, 18, 19 and 20. Individual values and observations are given in Appendices 13, 16, 17 and 18.

As a consequence of the reduced litter size at 50 mg/kg bw/day, litter weights on Days 1, 4, 7, and 13 post partum were statistically significantly reduced (p<0.01 - 0.05) when compared to controls. However, offspring body weights and body weight gains at birth and subsequently on Days 1, 4, 7, and 13 post partum actually exceeded control litters with female offspring showing statistically significantly higher (p<0.05) body weight on Day 4 after cull.

The clinical signs apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 50 mg/kg bw/day.

Ano-genital distance in offspring on Day 1 post partum and visible nipple count for male offspring on Day 13 post partum appeared unaffected by maternal treatment at 50 mg/kg bw/day.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

50 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 Isophthalic acid, oligomeric reaction products with 1-chloro-2,3-epoxypropane and phthalic anhydride (EA100) to Wistar Han™:RccHan™:WIST strain rats for approximately six weeks (males) and up to eight weeks (females) (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 50, 125 and 250 mg/kg bw/day resulted in the early deaths/terminations of two males and two female animals treated with 250 mg/kg bw/day and one male animal treated with 125 mg/kg bw/day. The deaths of these animals was considered to be potentially related to the administration of the test item. The clinical signs noted, including gasping or laboured respiration, indicate respiratory distress. However, in the absence of significant lung lesions, these signs, along with gaseous distension of the gastro-intestinal tract are indicative of reflux related responses to the gavage procedure, combined with the irritancy or consistency of the test item (Damsch et al 2011). Histological change in the trachea in animals surviving to termination is supportive of this. The changes in the non-glandular stomach of animals surviving to termination are likely to reflect a minor irritant effect of the test item on the non-glandular mucosa where there is prolonged exposure due to the unique structure of the rodent stomach. Whilst this is an adverse change in the animals affected it is not generally considered to be significant to man as the corresponding anatomical area is not present. The changes in the trachea of some animals treated with 250 mg/kg bw/day is also likely to reflect the irritant effect of the test item, due to reflux or accidental deposition during dosing.

A No Observed Adverse Effect Level (NOAEL) can be established at 50 mg/kg bw/day for animals of either sex because the findings in general (reductions in body weight gain and food consumption during lactation) do not reflect true systemic toxicity as the effects noted are considered to be potentially related to the irritant nature of the test item.

Analysis of sperm concentration, motility, morphology and homogenisation resistant spermatid counts revealed reductions in sperm motility, but without achieving statistical significance in animals treated with 250 and 125 mg/kg bw/day. A reduction in concentration was also noted in animals treated with 250 mg/kg bw/day but without achieving statistical significance. Due to the lack of pregnancy noted in animals treated with 125 and 250 mg/kg bw/day these findings cannot be discounted as being non-adverse in nature.

There were statistically significant increases (p<0.01 - p<0.001) in the number of abnormal sperm and sperm with a head only in animals treated with 250 mg/kg bw/day which may reflect sperm fragility in these animals and potentially contributed to the lack of pregnancy in female animals at 125 mg/kg bw/day and 250 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.

A No Observed Effect Level (NOEL) could not be established for reproductive toxicity as the reductions in implantation sites and the number of offspring born at 50 mg/kg bw/day could be considered adverse in nature.

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 eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 125 and 250 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.  Surviving adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and surviving 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

At 250 and 125 mg/kg bw/day, pregnancy was not achieved in any female; in view of this it was not possible to specifically assess effects of the test item on females during gestation or lactation at these dosages.

Mortality

One control female and one male animal treated with 125 mg/kg bw/day were found dead on Day 9.  One female treated with 250 mg/kg bw/day was found dead on Day 15.  Two male animals treated with 250 mg/kg bw/day were killed in extremis on Day 42 or 43, one female from this treatment group was killed in extremis on Day 51.  There were no further unscheduled deaths.

Clinical Observations

There were no clinical signs specifically related to systemic toxicity elicited by the test item for any animal which survived until the end of the treatment period.  

No significant clinical signs of toxicity were apparent prior to the animals being found dead during the study.  The two male animals treated with 250 mg/kg bw/day that were killed in extremis on Days 42 or 43 exhibited increased salivation, labored respiration, noisy respiration and hunched posture.  One of these animals also exhibited decreased respiratory rate and diarrhea, the other male animal also exhibited gasping respiration and lethargy.  The female animal treated with 250 mg/kg bw/day that was killed in extremis on Day 51 did not exhibit any clinical signs on Day 51 but had shown substantial body weight loss and had exhibited noisy respiration and decreased respiratory rate on Day 50.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters at 50, 125 or 250 mg/kg bw/day.

Functional Performance Tests

There were no treatment related changes in functional performance considered to be related to treatment at 50, 125 or 250 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 50, 125 or 250 mg/kg bw/day.

Body Weight

Males treated with 250 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment.  Recovery was evident during the second week, however, fluctuations were noted throughout the remainder of the treatment period such that overall body weight gain was lower than control by the end of the treatment period.    

Males treated with 125 mg/kg bw/day showed a slight reduction in body weight gain during the first week of treatment.  Recovery was generally evident thereafter, however, slight fluctuations were apparent during the remainder of the treatment period.  Overall body weight gain was lower than control at the end of treatment period.  No such effects were detected in male animals treated with 50 mg/kg bw/day.  

No adverse effects were detected in treated females during maturation.  Body weight gain during the first two weeks of gestation for females treated with 50 mg/kg bw/day was comparable to controls, however, a reduction in body weight gain was evident in these females during the final week of gestation.  During lactation, body weight gains were reduced in animals treated with 50 mg/kg bw/day, from Days 1 to 4, whilst slight signs of recovery

were subsequently evident body weight gains remained lower than control resulting in reductions in cumulative body weight gains throughout this phase of the study.  

Although females treated with 250 and 125 mg/kg bw/day were all non-pregnant, body weight gains during the first week post coitum were comparable to control females.  Subsequent body weight gains for these females was reduced; with the majority of females showing actual body weight losses.

Food Consumption

There were no adverse effects in male food consumption throughout the treatment period.

No effects were detected in food consumption for treated females during maturation.  Food consumption during gestation for females treated with 50 mg/kg bw/day was comparable to controls, however, food consumption was reduced in this treatment group during lactation from Days 4 to 7 and 7 to 14 respectively.  Although females treated with 250 and 125 mg/kg bw/day were all non-pregnant, food consumption during the first week post coitum was comparable to control females.  Food consumption then progressively reduced in these animals during the next two weeks of treatment.  

Food Conversion Efficiency

Reductions in food conversion efficiency were evident in animals of either sex treated at 250 mg/kg bw/day during the first week of treatment, this was noted in the males from this group during the final two weeks of treatment also, however, these generally followed the fluctuations in body weight gain seen in these animals.

Water Consumption

No effects were detected in water consumption for any treated male animal. Females treated with 250 mg/kg bw/day exhibited an increase in water consumption throughout maturation. No such effects were noted in female animals treated with 125 or 50 mg/kg bw/day.  

Reproductive Performance

Estrous Cycle

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.

Mating

No treatment-related effects were detected in mating performance.

Fertility

All of the females treated with 250 and 125 mg/kg bw/day and two females treated with 50 mg/kg bw/day were non-pregnant following positive evidence of mating.

Gestation Lengths

Overall, the distribution of gestation lengths for females treated with 50 mg/kg bw/day was essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Females treated with 50 mg/kg bw/day showed a reduction in the number of implantation sites.  Of the litters born, litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum were lower for females treated with 50 mg/kg bw/day when compared to control litters.  Live birth index and offspring viability in females treated with 50 mg/kg bw/day was comparable to controls.  Sex ratio was also comparable to controls and did not indicate any selective effect of maternal treatment on survival for either sex.  

Offspring Growth and Development

As a consequence of the reduced litter size, litter weights on Days 1, 4, 7, and 13 post partum were reduced when compared to controls.  However, offspring body weights and body weight gains at birth and subsequently on Days 1, 4, 7, and 13 post partum actually exceeded control litters.

Ano-genital distance in offspring on Day 1 post partum and visible nipple count for male offspring on Day 13 post partum appeared unaffected by maternal treatment at 50 mg/kg bw/day.

Laboratory Investigations

Hematology

There were no toxicologically significant effects detected in the hematological parameters

examined.

Blood Chemistry

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

Pathology

Necropsy

Offspring

The necropsy findings apparent for offspring on the study were typical for the age observed.  Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment at 50 mg/kg bw/day.

Adults  

One control female that was found dead on Day 9 exhibited gaseous distension of the cecum, a dark area on the liver, reddened lungs, torn esophagus, dark spleen, sloughing of the stomach and a dark area in the non-glandular region of the stomach.  The male animal treated with 125 mg/kg bw/day which was also found dead Day 9 exhibited gaseous distension throughout the gastro-intestinal tract, yellow colored contents in the colon and jejunum, dark liver, reddened lungs, sloughing of the stomach and a raised limiting ridge in the stomach.  One female treated with 250 mg/kg bw/day which was found dead on Day 15 exhibited gaseous distension throughout the gastro-intestinal tract (excluding the duodenum), a blue

duodenum, a dark area on the liver, the stomach showed sloughing and a raised limiting ridge and a thin non-glandular region was also apparent.  The two male animals treated with 250 mg/kg bw/day that were killed in extremis on Day 42 or 43 generally exhibited gaseous distension throughout the gastro-intestinal tract and a raised limiting ridge in the stomach.  The male that was killed in extremis on Day 42 also exhibited a reddened cecum, yellow colored contents in the colon, small epididymides, small seminal vesicles, small levator ani-bulbocavernous muscle, small testes, small liver and sloughing of the non-glandular region of the stomach.  The male that was killed in extremis on Day 43 also exhibited an enlarged cecum and a thin non-glandular region of the stomach.  The female from this treatment group which was killed in extremis on Day 51 exhibited gaseous distension throughout the gastro-intestinal tract, sloughing of the stomach and the non-glandular region of the stomach was thin and the limiting ridge was raised.

One female treated with 50 mg/kg bw/day, one female treated with 125 mg/kg bw/day and five males and four females treated with 250 mg/kg bw/day exhibited a thin non-glandular region of the stomach and a raised limiting ridge.  One male treated with 125 mg/kg bw/day and two males treated with 250 mg/kg bw/day exhibited a raised limiting ridge in the stomach.  One male and one female treated with 250 mg/kg bw/day exhibited raised white patches on the non-glandular region of the stomach.  One female treated with 250 mg/kg bw/day exhibited gaseous distension of the cecum and ileum. There were no further findings that were considered to be directly related to treatment with the test item.

Thyroid Hormone Analysis

Levels of thyroxine (T4) in adult males did not indicate any obvious effect of treatment or indication of endocrine disruption at 50, 125 or 250 mg/kg bw/day.

Levels of thyroxine (T4) in offspring at Day 13 of age did not indicate any obvious effect of maternal treatment or indication of endocrine disruption at 50 mg/kg bw/day.

Sperm Analysis

Analysis of sperm concentration, motility and morphology revealed reductions in sperm motility in animals treated with 250 and 125 mg/kg bw/day, a reduction in concentration was also noted in males treated with 250 mg/kg bw/day.  There were increases in the number of abnormal sperm and sperm with a head only in animals treated with 250 mg/kg bw/day.  No such findings were apparent in males treated with 125 or 50 mg/kg bw/day.  Homogenisation resistant spermatid counts did not reveal any adverse effect of treatment in any treatment group.

Organ Weights

No toxicologically significant effects were detected in the organ weights measured in animals of either sex treated with 50, 125 and 250 mg/kg bw/day.  

Histopathology

Premature Decedents

In Animal 13F (Control) many of the tissues were autolyzed and no histopathological changes to account for death were noted, but necropsy confirmed a dosing error.

Animal 52 M (Group 3) and 75M (Group 4) showed minor changes in the thymus indicative of stress.  Animals 88 and 92 F (Group 4) both showed minimal or mild hyperplasia in the non-glandular stomach and stress related changes were present in animal 92.  Animal 77M showed notable stress related change, mild hyperplasia in the non-glandular stomach and changes in the reproductive tract including tubular atrophy in the testes.  None of these animals had any indication of a cause of death and it is likely after taking into account the clinical and necropsy findings that reflux may be occurring and histological examination of the nasopharynx, likely to be definitive, was not possible.

Terminal Kill

Stomach

Changes in the non-glandular region of the stomach, namely diffuse or focal (limiting ridge) hyperplasia, were present in 4/7 males and 2/5 females in Group 4 at a minimal or mild severity.  In Group 3 minimal hyperplasia, focal (limiting ridge), was present in 2/5 males and one female in Group 3.  No changes were apparent in Group 2 animals.

Trachea

Metaplasia, minimal or mild was present in the trachea of one male and three females in Group 4 only.  This is indicative of a reaction to irritation possibly caused by reflux or inadvertent deposition of an irritant from the end of the gavage tube.

No other changes which could be related to the administration of the test item were noted and all are considered to be incidental.  There was no histological change to account for the increase in weight of the kidneys and this may be related to functional change.

Non-Productive Matings

No pregnancies occurred in Group 3 and 4 animals and 2/12 matings in Group 2.  With the exception of animal 77M (Group 4 premature decedent), which showed moderate degeneration in the testes there were no changes in males or females which could account for the lack of pregnancy.  Females showed an expected range of cyclical stages. 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.

Conclusion

The oral administration of Isophthalic acid, oligomeric reaction products with 1-chloro-2,3 -epoxypropane and phthalic anhydride (EA100) to Wistar Han™:RccHan™:WIST strain rats for approximately six weeks (males) and up to eight weeks (females) (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 50, 125 and 250 mg/kg bw/day resulted in the early deaths/terminations of two males and two female animals treated with 250 mg/kg bw/day and one male animal treated with 125 mg/kg bw/day.  The death of these animals was considered to be potentially related to the administration of the test item. The clinical signs noted, including gasping or laboured respiration, indicate respiratory distress.  However, in the absence of significant lung lesions, these signs, along with gaseous distension of the gastro-intestinal tract are indicative of reflux related responses to the gavage procedure, combined with the irritancy or consistency of the test item (Damsch et al 2011).

Histological change in the trachea in animals surviving to termination is supportive of this. The changes in the non-glandular stomach of animals surviving to termination are likely to reflect a minor irritant effect of the test item on the non-glandular mucosa where there is prolonged exposure due to the unique structure of the rodent stomach.  Whilst this is an adverse change in the animals affected it is not generally considered to be significant to man as the corresponding anatomical area is not present.  The changes in the trachea of some animals treated with 250 mg/kg bw/day is also likely to reflect the irritant effect of the test item, due to reflux or accidental deposition during dosing.

A No Observed Adverse Effect Level (NOAEL) can be established at 50 mg/kg bw/day for animals of either sex because the findings in general (reductions in body weight gain and food consumption during lactation) do not reflect true systemic toxicity as the effects noted are considered to be potentially related to the irritant nature of the test item.

Analysis of sperm concentration, motility, morphology and homogenisation resistant spermatid counts revealed reductions in sperm motility, but without achieving statistical significance in animals treated with 250 and 125 mg/kg bw/day.  A reduction in concentration was also noted in animals treated with 250 mg/kg bw/day but without achieving statistical significance.  Due to the lack of pregnancy noted in animals treated with 125 and 250 mg/kg bw/day these findings cannot be discounted as being non-adverse in nature.  

There were statistically significant increases (p<0.01 - p<0.001) in the number of abnormal sperm and sperm with a head only in animals treated with 250 mg/kg bw/day which may reflect sperm fragility in these animals and potentially contributed to the lack of pregnancy in female animals at 125 mg/kg bw/day and 250 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.  

A No Observed Effect Level (NOEL) could not be established for reproductive toxicity as the reductions in implantation sites and the number of offspring born at 50 mg/kg bw/day could be considered adverse in nature.