Registration Dossier

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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 11 October 2017and xx month 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
EDA-BADGE-EDA
Cas Number:
854009-15-7
Molecular formula:
C25H40N4O4
IUPAC Name:
EDA-BADGE-EDA
Constituent 2
Chemical structure
Reference substance name:
EDA-BADGE-EDA-BADGE-EDA
Molecular formula:
C48H72N6O8
IUPAC Name:
EDA-BADGE-EDA-BADGE-EDA
Constituent 3
Chemical structure
Reference substance name:
EDA-BADGE α-glycol
Molecular formula:
C23H34N2O5
IUPAC Name:
EDA-BADGE α-glycol
Constituent 4
Reference substance name:
reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
IUPAC Name:
reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
Constituent 5
Reference substance name:
Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
IUPAC Name:
Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine
Test material form:
liquid: viscous
Specific details on test material used for the study:
Identification : 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylene diamine
Physical State/Appearance: Yellow/brown extremely viscous liquid
Chemical Name: Pheno1,4,4'-(1-methylethylidene)bis-,polymer with N-(2-aminoethyl)-1,2 -ethanediamine and (chloromethyl)oxirane
Purity : UVCB substance, purity not applicable
Batch Number : BBF01102V1
Label : 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylene diamine Batch BBF01102V1 Expiry date 01.01.2021
Date Received : 30 August 2016
Storage Conditions: In the dark at room temperature
Expiry Date : 01 January 2021

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST strain
Details on species / strain selection:
Wistar Han™:RccHan™:WIST strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. 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 267 to 357g, and were approximately eleven weeks old. The females weighed 180 to 229g, and were approximately fourteen weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of four 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 is 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; there were short term deviations from these targets that 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 exposure:
Test Item Preparation and Analysis
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 showed the formulations to be stable for at least twenty one days. Formulations were therefore prepared weekly and stored at approximately 4 ºC in the dark.
Samples of test item formulations were taken on two occasions and analyzed for concentration of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylene diamine at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are detailed below. The results indicate that the prepared formulations were within ± 16% of the nominal concentration.

Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL) Animal Numbers
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 60 4 15 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.
Details on mating procedure:
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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Preparation of Calibration Standards
Stock solutions of test item in dilution solvent (methanol) were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument parameters section below.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solution of 1.075 mg/mL by serial dilution covering the concentration range 0.05375 mg/mL to 0.2150 mg/mL.

Preparation of Test Samples
The formulaitons recieved were diluted with diluiton solvent (Methanol). An aliquot of test item formulation was accurately weighed into a volumetric flaska dn brough to volume with dilution solvent, this was then shaken and ultrasonicated to dissolve. Where necessary sample solutions were further diluted with dilution solvent to achieve the worming concnetration.

Preparation of Accuracy and Precision Samples
Samples of PEG 400 were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations.
The concentration of Test Item in the final solution was quantified by HPLC using UV detection as detailed in the instrument parameters section below.

Instrumentation Parameters

HPLC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: Synergi polar RP (150 x 4.6 mm id) at 40ºC
Mobile phase: Eluent A: 100 mM Ammonium formate + 0.1% formic acid in water
Eluent B: 0.1% formic acid in acetonitrile
Time %B
0 85
12 15
12.1 85
15 85
Flow-rate 0.8 mL/min
UV detector wavelength: 230 nm
Injection volume: 25 L
Retention time: ~ 5.4 mins

Data Evaluation and Calculations
The peak area response for Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms was measured.

Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The linearity of detector response over the calibration standard concentration range.
The method accuracy (recovery) and precision, by analyzing five recovery samples at nominal concentrations of 2.5 mg/mL and 50 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

RESULTS
Method Validation
The analytical procedure was successfully validated for Test Item in PEG 400 with respect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision. Results are summarized below.
The specificity of the analytical method was demonstrated by the absence of a peak at the characteristic retention time for Test Item in the control sample chromatogram.
The data was found to have a linear correlation within the calibration range of 0.05375 mg/mL to 0.2150 mg/mL. The R² fit of the calibration curve to the data was 0.9947, and was considered to be acceptable.
Method accuracy (recovery) and precision were confirmed and results indicated that a mean recovery value of 98% (CV=2.26%, n=5) was obtained for 2.5 mg/mL and 105% (CV=2.26%, n=5) was obtained for 50 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

Concentration of Dose Formulaitons
Samples of test item formulations were taken on two occasions and analyzed for concentration of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylene diamine at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 16% of the nominal concentration.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The concentrations of the test item in the prepared formulations were within ± 16% of the nominal concentration.

Duration of treatment / exposure:
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 10, 30 and 60 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.
Frequency of treatment:
Daily
Details on study schedule:
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). 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, 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 or 45.
xi. On Day 13 post partum, where possible, blood sampling to produce serum samples for assessment of thyroid hormones was performed on two randomly selected offspring (one male and one female) per litter. Where possible, a further two randomly selected offspring (one male and one female) per litter were sampled to produce plasma samples. Thyroid/parathyroid samples were also retained from one male and one female from each litter where litter sizes allowed. All surviving offspring were killed and examined externally; where external observations were detected an internal necropsy was performed.
xii. 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 surviving 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 produce a pregnancy was also sacrificed and examined macroscopically around the same time as littering females. In addition, blood samples to produce both serum and plasma were taken from all adult animals at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
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). 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, 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 or 45.
xi. On Day 13 post partum, where possible, blood sampling to produce serum samples for assessment of thyroid hormones was performed on two randomly selected offspring (one male and one female) per litter. Where possible, a further two randomly selected offspring (one male and one female) per litter were sampled to produce plasma samples. Thyroid/parathyroid samples were also retained from one male and one female from each litter where litter sizes allowed. All surviving offspring were killed and examined externally; where external observations were detected an internal necropsy was performed.
xii. 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 surviving 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 produce a pregnancy was also sacrificed and examined macroscopically around the same time as littering females. In addition, blood samples to produce both serum and plasma were taken from all adult animals at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Examinations

Parental animals: Observations and examinations:
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 during the working week (except for females during parturition 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.

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 and 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.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

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

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.

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++)







Oestrous cyclicity (parental animals):
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.
Litter observations:
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.
Postmortem examinations (parental animals):
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 serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
Where possible, serum samples were taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. Where possible, plasma samples were also taken from two randomly allocated offspring per litter (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.

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 following partial fixation with Parathyroid)
Ovaries Uterus (weighed with Cervix)
Pituitary (weighed following partial fixation)
On Day 13 of age, where possible, for one male and one female offspring per litter, the thyroid/parathyroids were retained in buffered 10% 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
Esophagus gland)
Eyes * Skin
Glans Penis Spleen
Gross lesions Stomach
Heart Testes ♦
Ileum (including peyer’s patches) Thyroid/Parathyroid
Jejunum Trachea
Kidneys Thymus
LABC (levator ani-bulbocavernous) muscle Urinary bladder
Liver Uterus & Cervix (with oviducts)
Lungs (with bronchi)# Vagina
Lymph nodes (mandibular and mesenteric)
Tissues were dispatched to the Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues from five selected control and 60 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 60 mg/kg bw/day animals and any animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 60 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 mesenteric lymph node from animals of either 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.
Statistics:
See below
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 (%) = No. animals mated / No. animals paired x 100%
Pregnancy Index (%) = No. pregnant females / No. animals mated x 100%

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 (%) = No. females delivering live offspring / No. pregnant females x 100%
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).
i. Implantation Losses (%)
Group mean percentile post-implantation loss was calculated for each female/litter as follows:

Post–implantation loss (%) = No. implantation sites - total No. offspring born / No. implantation sites x100

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

Live Birth Index (%) = No. offspring alive on Day 1/ No offspring born x 100

Viability Index 1 (%) = No. offspring alive on Day 4/ No offspring alive on Day 1 x 100

Viability Index 2 (%) = No. offspring alive on Day 13/ No offspring alive on Day 4 x 100

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, 7 and 13 post partum, using the following formula:

No. Male offsrping / Total No. offspring x 100%

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed that were considered to be indicative of systemic toxicity.
Animals of either sex from all treatment groups showed instances of increased salivation and noisy respiration, with fewer incidences in the lower dosage groups. Noisy respiration was also observed in five control males. Increased salivation is often observed when administering test formulations via the oral gavage route and is generally considered to reflect unpalatability or slight irritancy of the test item formulations rather than any systemic effect of treatment. The incidences of noisy respiration observed were considered to reflect isolated difficulties with dosing particular animals on occasions rather than any effect of treatment.
Incidental findings that were considered unrelated to treatment included staining of the snout and decreased respiratory rate for one and two males, respectively at 60 mg/kg bw/day, scab formation in one female at 60 mg/kg bw/day, pilo-erection and staining around the snout in one male at 10 mg/kg bw/day, exophthalmos in one female at 10 mg/kg bw/day and generalised fur loss in two females at 10 mg/kg bw/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
On Day 3 of treatment, one control female (No.18) was found dead, the animal previously exhibited clinical signs of noisy respiration, hunched posture, pilo-erection, staining around the eyes and snout, plus non-weight bearing on the front right forelimb. At necropsy, findings were clear fluid filled thoracic cavity, dark liver, red discolouration of the lungs and a tear in the esophagus. It was therefore considered that this death was associated with dosing trauma.
There were no further unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no adverse effects on body weight development detected for males or for females during pre-pairing, gestation or lactation phases.
During the final week of treatment for males, all treatment groups showed a statistically significant increase (p<0.05) in body weight gains in relation to controls. As an increase in body weight gain is not considered to be adverse, this finding was considered to be of no toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no adverse effects on food consumption or food conversion efficiency for males or females during pre-pairing, gestation or lactation phases.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual assessment of water consumption did not reveal any significant intergroup differences.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes identified in the hematological parameters measured.
Females from all treatment groups showed a statistically significant increase (p<0.05) in hematocrit and reticulocyte count. Females treated with 60 mg/kg bw/day also showed a statistically significant reduction (p<0.05) in mean corpuscular hemoglobin concentration. All individual values were within historical control ranges and a true dose related response was not evident in hematocrit values. Therefore, in the absence of any histology correlates, the intergroup differences were considered not to be of toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the blood chemical parameters measured.
Males from all treatment groups showed a statistically significant increase (p<0.05) in phosphorus concentrations and at 60 mg/kg bw/day, males also showed an increase (p<0.05) in sodium concentration in relation to controls. Individual values for each parameter were within the historical control range and with no histopathological correlates these findings were considered to be of no toxicological significance.
Females from all treatment groups showed a statistically significant increase (p<0.05) in bile acid compared to control values. With the exception of one 30 mg/kg bw/day female, the remaining individual values were within the historical control range and with no histopathological correlates these findings were considered to be of no toxicological significance.

Thyroid Hormone Analysis
An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioral Assessments
There were no treatment-related changes in the behavioral parameters measured.
Noisy respiration was observed in one control, one 10 mg/kg bw/day and two 60 mg/kg bw/day males. Generalised fur loss, pallor of the extremities and exophthalmos was evident in one 10 mg/kg bw/day female. These findings are consistent with the clinical signs evident and do not indicate any underlying neurological effect.

Functional Performance Tests
There were no treatment-related changes detected in the functional performance measurements.
Statistical analysis of the data did not reveal any significant intergroup differences.

Sensory Reactivity Assessments
Sensory reactivity assessments did not indicate any adverse effects of treatment.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Premature Decedent
One control female (No 18) died early in the study. It had marked atrophy in the thymus and marked inflammatory changes surrounding the esophagus, thus confirming a dosing accident as the cause of death.
Mesenteric Lymph node
Histiocyte aggregates of minimal severity were present in three males at 30 mg/kg bw/day and three males at 60 mg/kg bw/day. In females minimal histiocyte aggregates were present in one female each from the control and 10 mg/kg bw/day dose group and in four females at 60 mg/kg bw/day at minimal or mild severity.
The changes evident in the mesenteric lymph nodes are known to occur in response to the oral administration of some test items and occurred at a minimal or mild severity. Without further evidence of pathological change, necrosis or abscess formation, it is considered not to affect the functionality of the lymph node and generally is considered to be non-adverse.
Non-Productive Matings
One female, 95 (60 mg/kg bw/day) was non-pregnant. It was paired with male 83 which showed debris in the epididymis and mild tubular degeneration in the testes which may have had an effect on fertility.
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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
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 estrous cycle on the day of necropsy.
Description (incidence and severity):
Mating
There were no treatment-related effects were detected on mating performance.
The majority of females mated within four days of pairing. One 30 mg/kg bw/day female completed mating after eight days of pairing and a further one 30 mg/kg bw/day female completed mating after thirteen days of pairing.

Fertility
Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.
One high dose pair (Female 95 and Male 83) did not achieve pregnancy following positive evidence of mating. Male 83 showed debris in the epididymis and mild tubular degeneration in the testes which may have had an effect on fertility.

Gestation Length
Gestation lengths were between 22 and 24 days and the distribution of gestation lengths for test item-treated females appeared to be similar to controls.

Litter Responses
In total 11, 12, 12, and 11 females from control, 10, 30, and 60 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 13 of age. 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
There was no effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at any dose level.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Key result
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: P1 (second parental generation)

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs detected in pups from all treated groups included small size, cold, weak, no milk in the stomach, physical injury, found dead or missing. Such findings are often seen in this type of study and were considered unlikely to be treatment-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights, ano-genital distance on Day 1 post partum or visible nipple count in male offspring on Day 13 post partum at any dose level.
Gross pathological findings:
no effects observed
Description (incidence and severity):
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 adverse effect of maternal treatment on offspring development at 10, 30 or 60 mg/kg bw/day.
Other effects:
no effects observed
Description (incidence and severity):
Litter Responses
Offspring Litter Size, Sex Ratio and Viability
There was no effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at any dose level.
Offspring Growth and Development
There was no effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights, ano-genital distance on Day 1 post partum or visible nipple count in male offspring on Day 13 post partum at any dose level.
The clinical signs and 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 on offspring development at 10, 30 or 60 mg/kg bw/day.

Effect levels (F1)

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F2 generation

Details on results (F2)

Not applicable

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
60 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylene diamine to rats by gavage, at dose levels of 10, 30 and 60 mg/kg bw/day, resulted in histiocyte aggregates present in the mesenteric lymph nodes in both male and females treated with 60 mg/kg bw/day and males treated with 30 mg/kg bw/day at a minimal to mild severity. Without any additional pathological changes this finding was considered to be non adverse. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 60 mg/kg bw/day.
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 60 mg/kg bw/day.
Executive summary:

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 10, 30 and 60 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 12post 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 13post 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 14post 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

On Day 3 of treatment, one control female was found dead. The clinical and necropsy observations along with the histopathology findings suggested the death may be associated with dosing trauma.

There were no further unscheduled deaths.

Clinical Observations

There were no clinical signs observed that were considered to be indicative of systemic toxicity. 

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests

There were no treatment-related changes detected in the functional performance parameters.

Sensory Reactivity Assessments

There were no treatment-related changes detected in sensory reactivity.

Body Weight

There were no adverse effects on body weight development detected for males or for females during pre-pairing, gestation or lactation phases.

Food Consumption

There were no adverse effects on food consumption or food conversion efficiency for males or for females during pre-pairing, gestation or lactation phases.


Water Consumption

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

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 estrous cycle on the day of necropsy.

Mating

There were no treatment-related effects detected on mating performance.

Fertility

There were no treatment-related effects detected in conception rates.

Gestation Lengths

Gestation lengths were essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at any dose level.

Offspring Growth and Development

There was no effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights, ano-genital distance on Day 1post partumor visible nipple count in male offspring on Day 13post partumat any dose level.

The clinical signs and 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 on offspring development at 10, 30 or 60 mg/kg bw/day.


Laboratory Investigations

Hematology

There were no toxicologically significant changes identified in the hematological parameters measured.

Blood Chemistry

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

Pathology

Necropsy

Adults

There were no treatment-related abnormalities detected at necropsy.

Thyroid Hormone Analysis

An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Organ Weights

There were no treatment-related changes detected in the organ weights measured.

Histopathology

Treatment-related findings were as follows:

Mesenteric Lymph node

Histiocyte aggregates were present in three males each treated with 30 or 60 mg/kg bw/day at minimal severity. In females, minimal histiocyte aggregates were present in one female each from the control and 10 mg/kg bw/day dose group at minimal severity and in four females treated with 60 mg/kg bw/day at minimal or mild severity.

Conclusion

The oral administration of 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylene diamine to rats by gavage, at dose levels of 10, 30 and 60 mg/kg bw/day, resulted in histiocyte aggregates present in the mesenteric lymph nodes in both male and females treated with 60 mg/kg bw/day and males treated with 30 mg/kg bw/day at a minimal to mild severity. Without any additional pathological changes this finding was considered to be non adverse. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 60 mg/kg bw/day.