Registration Dossier

Diss Factsheets

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

1
Reference substance name:
Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and m-phenylenebis (methylamine)
EC Number:
500-607-5
EC Name:
Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and m-phenylenebis (methylamine)
Cas Number:
161278-17-7
Molecular formula:
unspecified
IUPAC Name:
1-[3-(aminomethyl)phenyl]methanamine; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-(2-{3-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-hydroxyphenyl}propan-2-yl)phenol; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-[2-(3-{[({3-[({[5-(2-{3-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-hydroxyphenyl}propan-2-yl)-2-hydroxyphenyl]methyl}amino)methyl]phenyl}methyl)amino]methyl}-4-hydroxyphenyl)propan-2-yl]phenol; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-[2-(4-hydroxyphenyl)propan-2-yl]phenol; 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol
Test material form:
liquid

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 266 to 355g and were approximately eleven weeks old. The females weighed 190 to 231g 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 30 4 7.5 12 (25-36) 12 (37-48)
Intermediate 100 4 25 12 (49-60) 12 (61-72)
High 300 4 75 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 14 days when stored refrigerated. Formulations were therefore prepared weekly and stored at approximately 4 ºC in the dark.

Samples of test item formulations were taken on three occasions and analyzed for concentration of Formaldehyde, oligomeric reaction products with 4,4’-
isopropylidenediphenol and m-phenylenebis(methylamine) (EK 195) 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 acceptable ranges for the purpose of this study (89-104%).
Duration of treatment / exposure:
28 day
Frequency of treatment:
once per day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 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, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The tissues from five selected control and 500/400 mg/kg bw/day dose group animals and any animals dying during the study, were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 500/400 mg/kg bw/day animals and animals which did not mate or achieve a pregnancy were also processed. In addition, sections of testes from all control and 500/400 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

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

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

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS. A complete histopathology phase report is presented in Annex 1 and represents the consensus view of both pathologists.
Other examinations:
Data Evaluation
Data were processed to give summary incidence or group mean values and standard deviations where appropriate. All data were summarized in tabular form.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Number of male offspring x 100/Total number of offspring
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

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

Results and discussion

Results of examinations

Description (incidence and severity):
A summary incidence of daily clinical observations is given in Table 2. Individual data are presented in Appendix 1.

There were no clinical signs observed that indicated any systemic effect of treatment at dosages of 30, 100 and 300 mg/kg bw/day.

Animals dosed at 300 mg/kg bw/day displayed increased salvation from Day 9 and 6 of dosing onwards (males and females, respectively), for the majority of the study. All animals dosed at 100 mg/kg bw/day also showed increased salivation from Day 6 and 9 (males and females, respectively) onwards, albeit with a much lower frequency than animals dosed at 300 mg/kg bw/day. Increased salivation for animals dosed at 30 mg/kg bw/day was restricted to one female on one occasion only. Noisy respiration was noted in six males and ten females dosed with 300 mg/kg bw/day on a number of occasions throughout the study. Four males and five females dosed with 100 mg/kg bw/day showed noisy respiration on several occasions, as did six males and two females dosed with 30 mg/kg bw/day. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to represent an adverse effect of treatment.

Other clinical observations included one female dosed at 100 mg/kg bw/day with staining around the snout on Day 50, and one female dosed with 300 mg/kg bw/day showed chromodacryorrhea from Days 45 to 46, and then subsequently had staining around the eyes from Days 47 to 52 of treatment.
Description (incidence):
There were no unscheduled deaths on the study.
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.

At 300 mg/kg bw/day males generally showed slightly lower body weight gains during the treatment period, resulting in 16% lower overall body weight gains in relation to the concurrent controls. These males achieved statistically significantly lower (p<0.01) body weight gains after one week of treatment. No adverse effects of treatment were noted in males treated with 100 mg/kg bw/day, however, during the mating period (Weeks 3 and 4) slightly lower body weight gains in relation to controls were noted which influenced the slightly lower overall body weight gains (7%).

No such effects were evident in males treated with 100 mg/kg bw/day or in any treated females during pre-pairing or gestation phases.

Females treated with 300 mg/kg bw/day showed an increase (p<0.01) in body weight gains in relation to controls following two weeks of treatment. An increase in body weight is considered not to reflect an adverse effect of treatment.

There was no adverse effect of treatment in body weight development during lactation in any treated female. During the lactation phase the 100 and 300 mg/kg bw/day females showed slightly lower body weight gains in relation to controls, although this did not attain statistical significance.
Description (incidence and severity):
Group mean food consumptions are given in Table 9 and are presented graphically in Figure 3 and Figure 4. Individual values for females during gestation and lactation are presented in Appendix 9. Food efficiency for males and for females during the pre-mating phase is given in Table 10.

There were no effects evident for food consumptions or food conversion efficiency in treated males or in treated females during maturation, gestation or lactation.
Description (incidence and severity):
Daily visual assessment of water consumption did not reveal any significant intergroup differences.
Description (incidence and severity):
Group mean values and standard deviations for test and control group animals are given in Table 20 (statistically significant differences are indicated). Individual data are given in Appendix 19 and Appendix 20.

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

Reticulocyte count was statistically significantly higher (p<0.01) for animals of either sex from all treatment groups compared to controls in a dose related manner for males only. The majority of individual values were above the background control ranges, however, with no histopathological correlates this finding was considered to be of no toxicological significance.

Mean corpuscular hemoglobin concentration was significantly lower (p<0.05) for males dosed with 300 mg/kg bw/day compared to controls. All individual values were within the background control ranges and with no histopathological correlates this finding was considered to be of no toxicological significance.
Description (incidence and severity):
Group mean values and standard deviations for test and control group animals are given in Table 21 (statistically significant differences are indicated). Individual data are given in Appendices 22 to 23.

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

Females treated with 300 mg/kg bw/day showed a statistically significant increase (p<0.05) in levels of urea compared to controls. All females showed values within the background control ranges, and one of the individual control values was below the background control range. With no histopathological correlates, this finding was considered to be of no 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 adverse changes in the behavioural parameters at 30, 100 and 300 mg/kg bw/day.

A number of animals dosed at 100 and 300 mg/kg bw/day showed sporadic instances of noisy respiration during these behavioral assessments.

Functional Performance Tests
There were no changes in functional performance considered to be related to treatment at 30, 100 and 300 mg/kg bw/day.

Females treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in overall activity, in the absence of any clinical signs of neurotoxicity, this intergroup difference was considered not to be of toxicological importance.

Females treated with 30 or 100 mg/kg bw/day showed a statistically significant increase (p<0.01) in activity during the last 20% activity for the activity motor assessments. In the absence of any clinical signs of neurotoxicity and as no such effects were noted in animals treated with 300 mg/kg bw/day, the intergroup difference was considered not to be of toxicological importance.

Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores at 30, 100 and 300 mg/kg bw/day.
Description (incidence and severity):
Group mean absolute and body weight-relative organ weights and standard deviations for test and control group animals are presented in Table 24 (statistically significant differences are indicated). Individual data are given in Appendix 26 and Appendix 27.

No toxicologically significant effects were detected in the organ weights measured in animals of either sex treated with 30, 100 and 300 mg/kg bw/day.

All treated males showed statistically significant lower (p<0.05) liver weights for both absolute and body weight-relative values. The majority of individual values were within the background control ranges and without any histopathological correlates this finding was considered to be of no toxicological significance.

Males at 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in seminal vesicle weights both absolute and relative to terminal body weight values. The majority of individual values were within the background control ranges and without any histopathological correlates this finding was considered to be of no toxicological significance.
Description (incidence and severity):
A summary incidence of necropsy findings for offspring and adults is given in Tables 22 and 23. Individual data are given in Appendices 24 and 25.

Offspring
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 30, 100 or 300 mg/kg bw/day.

Adults
There were no treatment-related macroscopic abnormalities detected.

In isolation one control male had a mass located on the right epididymis, which was identified as a sperm granuloma.
Description (incidence and severity):
A complete histopathology phase report is presented in Annex 1.

Mesenteric Lymph Node
Histiocyte aggregates were noted in two and three females treated with 100 and 300 mg/kg bw/day, respectively and two males at 300 mg/kg bw/day.

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 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
Female 18 and Male 6 (control), Female 44 and Male 32 (30 mg/kg bw/day), Female 67 and Male 55 (100 mg/kg bw/day) showed no changes to account for the infertility.

There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.
Description (incidence and severity):
Thyroid Hormone Analysis
A complete Thyroid Hormone Analysis phase report is presented in Annex 3. Mean serum T4 concentrations are presented in Table 25.

Evaluation of thyroid hormone (T4) in adult males and offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 30, 100 or 300 mg/kg bw/day.

For adult males receiving 100 or 300 mg/kg bw/day, T4 blood levels were statistically significantly lower (p<0.05 - p<0.01) than control. At 100 and 300 mg/kg bw/day, all individual values were within the historical control range. In view of the lack of any supporting effect on thyroid weights or histopathology, the observed differences from control were considered to reflect atypically high control values (two values above the historical control range) rather than any effect of treatment.

Reproductive Performance
Estrous Cycles
A summary incidence of estrous cycle assessment is presented in Table 11. 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 most females showing regular cycles over the pre-pairing phase of the study. Two females treated with 100 mg/kg bw/day exhibited short periods of extended estrus.

There were also no intergroup differences (considered to be related to treatment with the test item) in the stage of estrus on the day of necropsy. One non-pregnant animal treated with 100 mg/kg bw/day exhibited early estrus on the day of necropsy.

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

There were no treatment-related effects detected in mating performance. The majority of females showed evidence of mating within four days of pairing, with the exception of one 30 mg/kg bw/day female which completed mating after twelve days of pairing.

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

Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.

One female (No. 18, 44 and 67 (male partner no. 6, 32 and 55, respectively)) each from the control, 30 and 100 mg/kg bw/day dose groups were found to be non-pregnant after showing positive evidence of mating. No histopathological changes were noted to account for the lack of fertility.

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

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 eleven females each from control, 30, 10 and 300 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 13 of age. One female dosed with 300 mg/kg bw/day had a total litter loss. The following assessment of litter response is based on the eleven litters reared to termination on Day 13 of lactation/age.

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

There was no obvious effect of maternal treatment on the number of implantations or post-natal survival of the offspring from birth to termination (Day 13 of age) at 30, 100 or 300 mg/kg bw/day. Post-implantation loss was slightly increased for all treated animals and litter size was slightly decreased for animals dosed with 300 mg/kg bw/day. However, neither of these parameters attained statistical significance. These effects were predominantly influenced by one litter (No. 88) having a smaller litter resulting in a wide standard deviation (SD) for the group mean. The individual value was outside the background control ranges. Therefore, this was considered to be the result of biological variation and unrelated to treatment.

There were no treatment-related intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated groups when compared with controls.

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

There were no adverse effects noted in any treated litters.
At 300 mg/kg bw/day litter weights, offspring body weight and body weight gain were slightly lower in relation to controls particularly between Days 7 and 13 post partum and without attaining statistical significance. This outcome was considered to be the result of biological variation, as isolated litters had lower body weight gains during this time in relation to controls and other litters in this group, rather than an effect of treatment. There was no detrimental effect of treatment with the test item indicated by ano-genital distance on Day 1 post partum or visible nipple count in male offspring on Day 13 post partum at 30, 100 or 300 mg/kg bw/day.

Clinical signs detected in pups from treated dose groups included cold, bruising, small, missing and found dead. Such findings are often observed in this type of study and were considered not to be related to treatment with the test item.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 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 Formaldehyde, oligomeric reaction products with 4,4’-isopropylidenediphenol and m-phenylenebis(methylamine) (EK 195) to rats by gavage, at dose levels of 30, 100 and 300 mg/kg bw/day, resulted in lower body weight gains, particularly in males (16%) compared to controls primarily due to the irritant nature of the test item rather than a true systemic toxic effect and microscopic changes including histiocyte aggregates present in the mesenteric lymph nodes in both males and females treated with 300 mg/kg bw/day and females at 100 mg/kg bw/day at a minimal severity. Without any additional pathological changes this finding was considered to be non adverse. Therefore, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day for animals of either sex.

The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 300 mg/kg bw/day, due to the lower offspring weights seen at this dose level.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints, and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016).

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

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to nine weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 100 and 300 mg/kg bw/day.  A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) over the same period.  

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

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

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

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

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

Vaginal smears were also performed in the morning on the day of termination for all treated females.  Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively.  Any female which did not produce a pregnancy was terminated around the same time as littering females.  All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed from all control and high dose animals (Group 1 and 4) as well as any gross lesions from Groups 1 to 4 was performed in the first instance.  As there were treatment-related findings in the mesenteric lymph nodes, examination of this tissue was subsequently extended to include relevant animals from the low and intermediate dose groups.  All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Results

Adult Responses

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

There were no clinical signs observed that indicated any systemic effect of treatment at dosages of 30, 100 and 300 mg/kg bw/day.

Behavioral Assessment

There were no adverse changes in the behavioral parameters at 30, 100 and 300 mg/kg bw/day.

Functional Performance Tests

There were no changes in functional performance considered to be related to treatment at 30, 100 and 300 mg/kg bw/day.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores at 30, 100 and 300 mg/kg bw/day.

Body Weight

At 300 mg/kg bw/day males generally showed slightly lower body weight gains during the treatment period, resulting in 16% lower overall body weight gains in relation to the concurrent controls.  

No adverse effects on body weight development were noted in male animals treated with 100 mg/kg bw/day.  

No effects of treatment on body weight development were noted in male animals treated with 30 mg/kg bw/day.  

No adverse effects of treatment on body weight development were noted in any treated female during maturation, gestation or lactation.

Food Consumption

There were no effects evident for food consumptions or food conversion efficiency in treated males or in treated females during maturation, gestation or lactation.

Water Consumption

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

Reproductive Performance

Estrous Cycles

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 (considered to be related to treatment with the test item) in the stage of estrus on the day of necropsy.

Mating

No treatment-related effects were detected in mating performance.

Fertility

No treatment-related effects were detected in fertility.

Gestation Lengths

Gestation lengths were essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no obvious effect of maternal treatment on the number of implantations or post-natal survival of the offspring from birth to termination (Day 13 of age) at 30, 100 or 300 mg/kg bw/day.  Post-implantation loss was increased for all treated animals.  Litter size was slightly lower for animals dosed with 300 mg/kg bw/day in relation to controls.

Offspring Growth and Development

There were no adverse effects noted in any treated litters.  

At 300 mg/kg bw/day litter weights, offspring body weight and body weight gain were slightly lower in relation to controls particularly between Days 7 and 13 post partum.  There was no detrimental effect of treatment with the test item indicated by ano-genital distance on Day 1 post partum or visible nipple count in male offspring on Day 13 post partum at 30, 100 or 300 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

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 30, 100 or 300 mg/kg bw/day.

Adults

No toxicologically significant effects were detected in animals of either sex treated with 30, 100 and 300 mg/kg bw/day.

Thyroid Hormone Analysis

At 300 and 100 mg/kg bw/day adult males showed statistically significantly lower T4 concentrations (p<0.01 and p<0.05, respectively) in relation to the concurrent control, in a dose related manner.  There were no histopathological correlates to the liver or thyroid and a review of historical control data from previous studies suggests this finding is considered to be the result of higher biological variation within the control males rather than an effect of treatment.

Organ Weights

No toxicologically significant effects were detected in the organ weights measured in animals of either sex treated with 30, 100 and 300 mg/kg bw/day.

Histopathology

Treatment-related changes were confined to histocyte aggregates in the mesenteric lymph nodes in two males at 300 mg/kg bw/day and two and three females treated with 100 and 300 mg/kg bw/day, respectively.  This finding is known to occur in response to the oral administration of some test items and occurred at a minimal severity and is generally considered to be a non-adverse finding.

Conclusion

The oral administration of Formaldehyde, oligomeric reaction products with 4,4’-isopropylidenediphenol and m-phenylenebis(methylamine) (EK 195) to rats by gavage, at dose levels of 30, 100 and 300 mg/kg bw/day, resulted in lower body weight gains,

particularly in males (16%) compared to controls primarily due to the irritant nature of the test item rather than a true systemic toxic effect and microscopic changes including histiocyte aggregates present in the mesenteric lymph nodes in both male and females treated with 300 mg/kg bw/day and females at 100 mg/kg bw/day at a minimal severity.  Without any additional pathological changes this finding was considered to be non adverse.  Therefore, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day for animals of either sex.

The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 300 mg/kg bw/day, due to the lower offspring weights seen at this dose level.