Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

1
Reference substance name:
Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and diethylenetriamine
EC Number:
500-263-6
EC Name:
Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and diethylenetriamine
Cas Number:
77138-45-5
Molecular formula:
(C15H16O2.C4H13N3.CH2O)x
IUPAC Name:
Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and diethylenetriamine
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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on 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 100 4 25 12 (25-36) 12 (37-48)
Intermediate 300 4 75 12 (49-60) 12 (61-72)
High 500 4 125 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:
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared every two weeks and stored at approximately 4 ºC in the dark.

Samples of three test item formulations were taken and analyzed for concentration of Formaldehyde, oligomeric reaction products with 4,4’-isopropylidenediphenol and diethylenetriamine (EK110) at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Annex 2. The results indicate that the prepared formulations were within 14% of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once per day
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). 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 tworandomly 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).
Doses / concentrationsopen allclose all
Dose / conc.:
0 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)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Serial Observations
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately be fore 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 an d 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

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 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.
Oestrous cyclicity (parental animals):
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 observations:
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.
Statistics:
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.

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).
Reproductive indices:
Mating Performance and Fertility

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

ii. Fertility Indices

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

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

There were no clinical signs specifically related to systemic toxicity elicited by the test item for males or females treated with 300 or 500 mg/kg bw/day which survived until the end of the treatment period.

All animals of either sex treated with 500 mg/kg bw/day showed incidences of increased salivation from Day 1 until Day 44 (males) and Day 54 (females). Noisy respiration was also noted in all of these animals from Day 1 until Day 41 (males) and Day 54 (females). Ataxia was noted in seven males and four female animals on Days 2 and 3. Isolated occurrences of pilo-erection (one male), hunched posture (two males and two females), staining around the snout (two females) and sneezing (one female) were also noted. Each of these findings persisted for a maximum of two days only. Due to the isolated nature of these findings it is considered that they were not adverse in nature and probably reflected the irritant nature of the test item. One female animal treated with 500 mg/kg bw/day exhibited a scab on Day 52, due to the isolated nature of this finding it was considered to be unrelated to treatment with the test item.

All males and eleven female animals treated with 300 mg/kg bw/day also showed incidences of increased salivation from Day 2 (males) or Day 17 (females) until Day 44 (males) and Day 59 (females). Noisy respiration was also noted in all male animals from Day 1 until Day 44 and in nine females from Day 2 until Day 37. Isolated occurrences of hunched posture (one male and one female) and ataxia (one male and one female) were also noted. Each of these findings persisted for a maximum of two days only. Due to the isolated nature of these findings it is considered that they were not adverse in nature and probably reflected the irritant nature of the test item.

There were no observations apparent in animals of either sex treated with 100 mg/kg bw/day which could be specifically related to systemic toxicity of the test item.

An isolated occurrence of increased salivation was noted in one male animal treated with 100 mg/kg bw/day on Day 37 and occasional instances were noted in five females from Days 30 to 53. Three female animals also exhibited noisy respiration from Day 37 until Day 52. Increased salivation is commonly observed in this type of study and is generally considered to be due to an irritant/unpalatable nature of the test item and/or formulation. As such this observation is considered not to be specifically related to systemic toxicity of the test item.

Signs of noisy respiration may be indicative of some reflux related to the irritancy of the test item (Damsch et al 2011) and cannot be considered to be related specifically to toxicity of the test item.

The male animal which was found dead on Day 17 did not show any clinical signs of toxicity the day prior to death. The female animal which was found dead on Day 40 exhibited signs of noisy respiration on the day prior to death.
Description (incidence):
One male and one female animal treated with 500 mg/kg bw/day were found dead on Day 17 or Day 40 respectively.
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.

Male animals across all treatment groups exhibited statistically significant (p<0.05) reductions in body weight gain during the first week of treatment with ten and six animals treated with 500 and 300 mg/kg bw/day respectively exhibiting actual body weight losses. For male animals treated with 500 mg/kg bw/day (with the exception of Weeks 3 and 4) body weight gains remained lower than control with four and three animals exhibiting actual body weight losses during Weeks 5 and 6 respectively. This resulted in an overall reduction in body weight gain of 52% when compared to control animals.

For animals treated with 300 mg/kg bw/day recovery was evident during Weeks 2, 3 and 4 as body weight gains were comparable to control, however, a statistically significant reduction in body weight gain (p<0.05) was again apparent during Week 5, a further reduction in body weight gain but without achieving statistical significance was apparent during Week 6 with five animals exhibiting actual body weight losses. This resulted in an overall reduction in body weight gain of 26.9% when compared to control animals.

Fluctuations in body weight gains were noted in male animals treated with 100 mg/kg bw/day during the remainder of the treatment period, however, mean body weight gains generally remained closer to control than the other two treatment groups, however, an overall reduction in body weight gain of 16.9% was still noted in this group when compared to control.

During maturation, female animals treated with 500 mg/kg bw/day exhibited statistically significant reductions in body weight gains (p<0.05) during the first week of treatment with six of these animals exhibiting actual body weight losses. An improvement was noted during the second week as body weight gains were comparable to control.

Body weight gains in the other two treatment groups were comparable to control during Week 1 but both showed reductions in body weight gains during the second week of treatment but without achieving statistical significance.

There were no effects on body weight gain during the gestation or lactation periods for any of the treated female animals.
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.

Male animals treated with 500 mg/kg bw/day showed reductions in food consumption when compared to control during the first week of the treatment period. Recovery was evident thereafter, however, group means still remained lower than control throughout the remainder of the treatment period. A similar effect was noted in the male animals treated with 300 mg/kg bw/day, however, the reductions were not as marked as those seen at 500 mg/kg bw/day.

There were no adverse effects noted in food consumption for male animals treated with 100 mg/kg bw/day. Reductions in food consumption were noted during the first two weeks of the treatment period, improvements were noted during the final two weeks of treatment where food consumptions were comparable to control.

There were considered to be no adverse effects noted in food consumption for treated females during maturation, gestation or lactation. Female animals treated with 500 mg/kg bw/day exhibited slight reductions in food consumption during the first week of maturation.
Description (incidence and severity):
Food efficiency for males and females during the pre-mating phase is given in Table 10.

At 500 mg/kg bw/day, food conversion efficiency (where calculated) for males appeared inferior to control during the treatment period. A similar effect was noted in male animals treated with 300 mg/kg bw/day (with the exception of Week 2), however, the effect was less marked. At 100 mg/kg bw/day, food conversion efficiency for males appeared generally unaffected by treatment.

Food conversion efficiency for females during the pre-pairing phase showed a reduction in animals treated with 500 mg/kg bw/day during the first week and in animals treated with 100 or 300 mg/kg bw/day during the second week.
Description (incidence and severity):
Group mean daily water consumptions during the pre-pairing phase are given in Table 11.

There were no effects in water consumption for any treated animal.
Description (incidence and severity):
Group mean values and standard deviations for test and control group animals are given in Table 21 (statistically significant differences are indicated). Individual data are given in Appendices 19 to 21.

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

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

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

Lymphocytes were statistically significantly reduced (p<0.05) in females treated with 500 mg/kg bw/day. However, all values were found to be within the historical control data range. This finding is considered to have been overemphasized by one control value which was higher than the historical control data range. As such this finding is considered not to be of any toxicological significance.
Description (incidence and severity):
Group mean values and standard deviations for test and control group animals are given in Table 22 (statistically significant differences are indicated). Individual data are given in Appendices 22 and 23.

A statistically significant increase in bile acids (p<0.05) was noted in female animals treated with 500 mg/kg bw/day, two treated female animals exhibited values that were far greater than the historical control data range and were atypical for the group as a whole and as such artificially elevated this finding, however, due to the liver findings noted at histopathological examination an association with treatment cannot be discounted.

There were no adverse effects of treatment on the blood chemical parameters measured in any treated male animal or in females treated with 100 or 300 mg/kg bw/day.

Males treated with 500 mg/kg bw/day showed a statistically significant increase (p<0.05) in alanine aminotransferase. Four control animals exhibited values that were below the historical control data range. Values for the treated male animals did not show a continual pattern as two values were lower and one was higher than the historical control data range and in the absence of any histopathological correlates these intergroup differences were considered not to be of any toxicological significance. A statistically significant reduction (p<0.05) in alkaline phosphatase was noted in male animals treated with 500 mg/kg bw/day. All values were generally within the historical control data range and in the absence of any histopathological correlates these intergroup differences were considered not to be of any toxicological significance.

Female animals treated with 500 mg/kg bw/day exhibited statistically significant reductions in calcium, sodium and potassium (p<0.05). All calcium values in control and females treated with 500 mg/kg bw/day were below the historical control data range, all other findings were generally within the normal historical control data ranges and in the absence of any histopathological correlates these intergroup differences were considered not to be of any toxicological significance.

Male animals treated with 100 mg/kg bw/day also exhibited a statistically significant (p<0.05) increase in bile acids, however, all values were within the normal historical control data ranges and as no such findings were noted in the other two (higher) treatment groups this was considered to be incidental and not to be of any toxicological significance.
Description (incidence and severity):
Functional Observations
Summary incidence of behavioral assessment observations are given in Table 3 and group mean behavioral assessment scores are given in Table 4. Group mean functional performance test values and standard deviations are given in Table 5. 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
Noisy respiration was apparent in one male animal treated with 500 mg/kg bw/day on Days 26 and 33. There were no further changes in the behavioral parameters noted in any treated male animals throughout the treatment period. Two female animals treated with 500 mg/kg bw/day exhibited noisy respiration on Day 18 of gestation. Two female animals treated with 300 mg/kg bw/day exhibited noisy respiration on Day 4 or 12 of lactation. One female animal treated with 100 mg/kg bw/day exhibited hunched posture, pilo-erection and tip-toe gait on Day 18 of gestation. A further female animal from this group exhibited signs of noisy respiration on Day 12 of lactation. One control female also exhibited noisy respiration on Day 12 of lactation.

Functional Performance Tests
There were no inter-group differences in functional performance that were considered to be related to treatment at 100, 300 or 500 mg/kg bw/day.

Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores at 100, 300 or 500 mg/kg bw/day.
Description (incidence and severity):
A complete histopathology phase report is presented in Annex 1.

Premature Decedents
Significant erosion with hemorrhage was noted in the glandular stomach of the male animal treated with 500 mg/kg bw/day which was found dead on Day 17 and this was considered to be the cause of death for this animal.

No changes, which could account for the death of the female animal which was found dead on Day 40 were found at histopathology.

Terminal Kill
Stomach
Inflammatory cell infiltration in the glandular region was present in 4/5 males treated with 500 mg/kg bw/day. This occurred mainly in the submucosal area but spread into the mucosa and was of a moderate or mild severity. It was also present in 4/5 males treated with 300 mg/kg bw/day at a minimal or mild severity. One of the males treated with 300 mg/kg bw/day also had a mild erosion in the glandular region.

Spongiosis was present at the limiting ridge in one control male along with 5/5 and 3/5 males treated with 300 or 500 mg/kg bw/day respectively.

These changes are likely to indicate irritation at the point of contact rather than being due to a systemic effect of the test item.

Liver
Increased cytoplasmic rarefaction was present in one female from the control and one from groups treated with 100 or 300 mg/kg bw/day (mild) along with 5/7 in females treated with 500 mg/kg bw/day (mild or moderate). This is likely to represent increased glycogen deposition.

The increase in cytoplasmic rarefaction in the liver of females is indicative of a mild adaptive or metabolic response to the administration of the test item.

Uterus
Pigment deposition was present at the involuting implantation sites of all applicable females treated with 500 mg/kg bw/day (10) and 4/12 treated with 300 mg/kg bw/day. Whilst pigment is seen at a low level at most involuting implantation site this was more intense and orange in colour. The accumulation of this pigment is of unknown significance and may be indicative of accumulation of test item or a metabolite in the area. There was no indication that pathological damage or other changes were caused and there was no evidence on any effect on fertility or pup survival.

No other changes were noted which could be related to the administration of the test item and all are considered to be incidental.

Non-Productive Matings
Animal 24F (Control) paired with 12M had inflammatory changes in the uterus that are likely to have resulted in failure to produce a litter.

Animal 44F (treated with 100 mg/kg bw/day) and 32M showed no changes to account for the lack of pregnancy.

Animal 48F (treated with 300 mg/kg bw/day) suffered total litter loss. This animal had significant kidney changes which were likely to have had a functional effect on the animal and may have contributed to the inability to raise a litter.

Animal 92F (treated with 500 mg/kg bw/day) and male 84 showed no changes at histopathology to account for the lack of mating.

There were no test item-related microscopic findings noted 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 follicles and corpora lutea in the ovaries.
Description (incidence and severity):
Thyroid Hormone Analysis
Group mean T4 (Thyroxine) in adult males and offspring at Day 13 of age are presented in Table 26. A complete Thyroid Hormone Analysis phase report is presented in Annex 3.

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100, 300 or 500 mg/kg bw/day.

Reproductive function / performance (P0)

Description (incidence and severity):
A summary of estrous cycle assessment is presented in Table 12. Individual data are given in Appendix 10.

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle. All females generally showed regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.
Description (incidence and severity):
Mating
A summary of adult performance is presented in Table 1. A summary incidence for mating performance is presented in Table 13. Individual data are given in Appendix 11.

No treatment-related effects were detected in mating performance.

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

No treatment-related effects were detected in fertility.

One control female and one female treated with 100 mg/kg bw/day were non-pregnant following positive evidence of mating. One female treated with 500 mg/kg bw/day did not mate with her respective partner. Positive evidence of mating was not apparent in two animals from the 100 and 300 mg/kg bw/day treatment groups but both of these animals were subsequently found to be pregnant.

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

Gestation lengths for controls and treated female were between 22 and 23½ days. Overall, the distribution of gestation lengths for treated females was considered to be essentially similar to control. However, statistically significantly longer gestation lengths (p<0.05-p<0.01) were apparent across all treatment groups when compared to control. As all gestation lengths were within the historical control data range this was considered not to be of any toxicological
significance.

Two female animals from the 100 and 300 mg/kg bw/day treatment groups did not show positive evidence of mating but were subsequently found to be pregnant, the gestation lengths for these animals could therefore not be determined.

Litter Responses
There were 1, 1, 0 and 1 females (that survived to parturition) that failed to achieve pregnancy in the control, 100, 300 and 500 mg/kg bw/day dosage groups respectively. Additionally, one female animal treated with 100 mg/kg bw/day exhibited a total litter loss prior to Day 13. The following assessment is generally made using the 11, 10, 12 and 10 litters which were reared to Day 13 of 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 14, 16 and 17. Individual data are given in Appendices 12, 14 and 15.

There was no adverse effect on litter size and offspring survival from implantation to birth and subsequently to termination on Day 13 of age in any treatment group. Sex ratio across all treatment groups were comparable to controls and did not indicate any selective effect of maternal treatment on survival for either sex. Live birth index and offspring viability in treated females was also comparable to controls.

For females treated with 100 mg/kg bw/day there was a statistically significant reduction (p<0.05) in the percentage of male offspring when compared to control on Day 4 after cull and on Day 7. However, as all values were within the historical control data range this was considered not to be of any toxicological significance.

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 15, 18, 19 and 20. Individual values and observations are given in Appendices 13, 16, 17 and 18.

There was no detrimental 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 100, 300 or 500 mg/kg bw/day.

The clinical signs apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any effect of maternal treatment on offspring development in any treatment group.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
500 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 (P0)

Critical effects observed:
no

Results: F1 generation

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of Formaldehyde, oligomeric reaction products with 4,4’-isopropylidenediphenol and diethylenetriamine (EK110) to rats by gavage, at dose levels of 100, 300 and 500 mg/kg bw/day, resulted in the early deaths of one male and one female animal treated with 500 mg/kg bw/day. Significant erosion with hemorrhage was noted in the glandular stomach of the male animal treated with 500 mg/kg bw/day which was found dead on Day 17 and this was considered to be the cause of death for this animal.

In general, it is considered that the effects noted in the animals at 500 mg/kg bw/day were considered to be the result of gastric irritancy caused by the test item rather than attributable to true systemic toxicity. However, due to the two deaths noted a No Observed Adverse Effect Level (NOAEL) cannot be established at this dose level.

A No Observed Adverse Effect Level (NOAEL) can be established at 300 mg/kg bw/day for animals of either sex because the findings in general do not reflect true systemic toxicity as the effects noted are considered to be in relation to the irritant nature of the test item.

The No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.
Executive summary:

Introduction

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

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

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately 6 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 100, 300 and 500 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).

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 females and offspring on Days 14 and 13 post partum respectively.  Any female which did not produce a pregnancy was terminated around the same time as littering females.  Any

female which did not show positive evidence of mating 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

One male and one female animal treated with 500 mg/kg bw/day were found dead on Day 17 or Day 40 respectively.

Clinical Observations

There were no clinical signs specifically related to systemic toxicity elicited by the test item for any treated males or females.

All animals of either sex treated with 500 mg/kg bw/day showed incidences of increased salivation throughout the treatment period.  Noisy respiration was also noted in all of these animals throughout the majority of the treatment period.  

All males and eleven female animals treated with 300 mg/kg bw/day showed incidences of increased salivation from Day 2 (males) or Day 17 throughout the treatment period.  Noisy respiration was also noted in all male animals and in nine females throughout the majority of

the treatment period.  

An isolated occurrence of increased salivation was noted in one male animal treated with 100 mg/kg bw/day and occasional instances were noted in five females towards the end of the treatment period.  Three female animals also exhibited noisy respiration towards the end of the treatment period.

The male animal which was found dead on Day 17 did not show any clinical signs of toxicity the day prior to death.  The female animal which was found dead on Day 40 exhibited signs of noisy respiration on the day prior to death.

Behavioral Assessment

Noisy respiration was apparent in one male animal treated with 500 mg/kg bw/day on Days 26 and 33.  There were no further changes in the behavioral parameters noted in any treated male animals throughout the treatment period.

Two female animals treated with 500 mg/kg bw/day exhibited noisy respiration on Day 18 of gestation.  Two female animals treated with 300 mg/kg bw/day exhibited noisy respiration on Day 4 or Day 12 of lactation.

One female animal treated with 100 mg/kg bw/day exhibited hunched posture, pilo-erection and tip-toe gait on Day 18 of gestation.  A further female animal from this group exhibited signs of noisy respiration on Day 12 of lactation.  One control female also exhibited noisy

respiration on Day 12 of lactation.

Functional Performance Tests

There were no inter-group differences in functional performance that were considered to be related to treatment at 100, 300 or 500 mg/kg bw/day.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores at 100, 300 or 500 mg/kg bw/day.

Body Weight

Male animals across all treatment groups exhibited reductions in body weight gain during the first week of treatment with animals treated with 500 and 300 mg/kg bw/day respectively exhibiting actual body weight losses.  

Male animals treated with 500 mg/kg bw/day generally showed body weight gains which were lower than control during the remainder of the treatment period, this resulted in an overall reduction in body weight gain of 52% when compared to control animals.

Male animals treated with 300 mg/kg bw/day showed signs of recovery during the next three weeks, however, reductions in body weight gains were again apparent during the final two weeks of treatment.  An overall reduction in body weight gain of 26.9% when compared to

control animals was noted in these animals.

Fluctuations in body weight gains were noted in male animals treated with 100 mg/kg bw/day during the remainder of the treatment period, however, mean body weight gains generally remained closer to control than the other two treatment groups.  An overall reduction in body

weight gain of 16.9% was still noted in this group when compared to control.

During maturation, female animals treated with 500 mg/kg bw/day exhibited reductions in body weight gains during the first week of treatment.  An improvement was noted during the second week as body weight gains were comparable to control.  Body weight gains in the other two treatment groups were comparable to control during Week 1, however, both groups showed reductions in body weight gains during the second week of treatment.

There were no effects on body weight gain during the gestation or lactation periods for any of the treated female animals.

Food Consumption

Male animals treated with 500 mg/kg bw/day showed reductions in food consumption when compared to control during the first week of the treatment period.  Recovery was evident thereafter, however, group means still remained slightly lower than control throughout the

remainder of the treatment period.  A similar effect was noted in the male animals treated with 300 mg/kg bw/day, however, the reductions were not as marked as those seen at 500 mg/kg bw/day.  

Male animals treated with 100 mg/kg bw/day exhibited reductions in food consumption when compared to control during the first two weeks of the treatment period, improvements were noted during the final two weeks of treatment where food consumptions were comparable to control.

There were no adverse effects noted in food consumption in any treated female during maturation, gestation or lactation.

Food Conversion Efficiency

At 500 mg/kg bw/day, food conversion efficiency (where calculated) for males appeared inferior to control during the treatment period.  A similar effect was noted in male animals treated with 300 mg/kg bw/day (with the exception of Week 2), however, the effect was less

marked.  At 100 mg/kg bw/day, food conversion efficiency for males appeared generally unaffected by treatment.

Food conversion efficiency for females during the pre-pairing phase of the study was reduced in animals treated with 500 mg/kg bw/day during the first week and in animals treated with 100 or 300 mg/kg bw/day during the second week.

Water Consumption

There were no effects in water consumption for any treated animal.

Reproductive Performance

Estrous Cycles

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

Mating

No treatment-related effects were detected in mating performance.

Fertility

No treatment-related effects were detected in fertility..

Gestation Lengths

Gestation lengths for controls and treated females were between 22 and 23½ days.  Overall, the distribution of gestation lengths for treated females was considered to be essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no adverse effect on litter size and offspring survival from implantation to birth and subsequently to termination on Day 13 of age in any treatment group.  Sex ratios across all treatment groups were comparable to controls and did not indicate any selective effect of

maternal treatment on survival for either sex.  Live birth index and offspring viability in treated females was also comparable to controls.

Offspring Growth and Development

There was no detrimental 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 100, 300 or 500 mg/kg

bw/day.

Laboratory Investigations

Hematology

Male animals treated with 500 mg/kg bw/day and 300 mg/kg bw/day exhibited increases in neutrophils.  Due to the effects noted in the stomach at histopathological examination an association with treatment cannot be discounted.

There were no further effects which could be associated with treatment on the hematological parameters measured.

Blood Chemistry

An increase in bile acids was noted in female animals treated with 500 mg/kg bw/day.

There were no adverse effects of treatment on the blood chemical parameters measured in any treated male animal or in females treated with 100 or 300 mg/kg bw/day.  

Pathology

Necropsy

Offspring

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 in any treatment group.

Adults

The male animal treated with 500 mg/kg bw/day which was found dead on Day 17 exhibited gaseous distension of the cecum, duodenum, ileum (including peyers patches) jejunum and stomach.  The stomach also exhibited red patches on the glandular region and a raised limiting ridge.  The female animal treated with 500 mg/kg bw/day which was found dead on Day 40 exhibited enlarged and red adrenals, gaseous distension was noted in the cecum, colon, duodenum, ileum (including peyers patches), jejunum and stomach.  The stomach was

also enlarged and fluid filled and also had red patches on the glandular region, the non-glandular region also appeared thin.

One female treated with 100 mg/kg bw/day, three females treated with 300 mg/kg bw/day and one male and two females treated with 500 mg/kg bw/day exhibited pale livers.

Four females treated with 500 mg/kg bw/day exhibited pale and mottled livers.  One female treated with 100 mg/kg bw/day and one female treated with 500 mg/kg bw/day exhibited a thickened glandular region of the stomach.

There were no further findings noted amongst surviving animals at necropsy that were considered to be specifically related to treatment with the test item.

Organ Weights

There were no toxicologically significant effects detected in the organ weights measured in any treated animal.

Histopathology

Premature Decedents

Significant erosion with hemorrhage was noted in the glandular stomach of the male animal treated with 500 mg/kg bw/day which was found dead on Day 17 and this was considered to be the cause of death for this animal.  No changes which could account for the death of the female animal which was found dead on Day 40 were found at histopathology.

Terminal Kill

Stomach

Inflammatory cell infiltration in the glandular region was present in 4/5 males treated with 500 mg/kg bw/day.  This occurred mainly in the submucosal area but spread into the mucosa and was of a moderate or mild severity.  It was also present in 4/5 males treated with

300 mg/kg bw/day at a minimal or mild severity.  One of the males treated with 300 mg/kg bw/day also had a mild erosion in the glandular region.  Spongiosis was present at the limiting ridge in one control male along with 5/5 and 3/5 males treated with 300 or 500 mg/kg bw/day respectively.

Liver

Increased cytoplasmic rarefaction was present in one female from the control and one from groups treated with 100 or 300 mg/kg bw/day (mild) along with 5/7 in females treated with 500 mg/kg bw/day (mild or moderate).  This is likely to represent increased glycogen

deposition and represents a mild adaptive or metabolic response.

Uterus

Pigment deposition was present at the involuting implantation sites of all applicable females treated with 500 mg/kg bw/day (10) and 4/12 treated with 300 mg/kg bw/day.  Whilst pigment is seen at a low level at most involuting implantation site this was more intense and

orange in colour.  There was no associated pathology.  No other changes were noted which could be related to the administration of the test item and all are considered to be incidental.  

Non-Productive Matings

Animal 24F (Control) paired with 12M had inflammatory changes in the uterus that are likely to have resulted in failure to produce a litter.

Animal 44F (treated with 100 mg/kg bw/day) and 32M showed no changes to account for the lack of pregnancy.  

Animal 48F (treated with 300 mg/kg bw/day) suffered total litter loss.  This animal had significant kidney changes which were likely to have had a functional effect on the animal and may have contributed to the inability to raise a litter.  

Animal 92F (treated with 500 mg/kg bw/day) and male 84 showed no changes at histopathology to account for the lack of mating.

There were no test item-related microscopic findings noted 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 follicles and corpora lutea in the ovaries.

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100, 300 or 500 mg/kg bw/day

Conclusion

The oral administration of Formaldehyde, oligomeric reaction products with 4,4’-isopropylidenediphenol and diethylenetriamine (EK110) to rats by gavage, at dose levels of100, 300 and 500 mg/kg bw/day, resulted in the early deaths of one male and one female animal treated with 500 mg/kg bw/day.  Significant erosion with hemorrhage was noted in the glandular stomach of the male animal treated with 500 mg/kg bw/day which was found dead on Day 17 and this was considered to be the cause of death for this animal.  

In general, it is considered that the effects noted in the animals at 500 mg/kg bw/day were considered to be the result of gastric irritancy caused by the test item rather than attributable to true systemic toxicity.  However, due to the two deaths noted a No Observed Adverse

Effect Level (NOAEL) cannot be established at this dose level.

A No Observed Adverse Effect Level (NOAEL) can be established at 300 mg/kg bw/day for animals of either sex because the findings in general do not reflect true systemic toxicity as the effects noted are considered to be in relation to the irritant nature of the test item.

The No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was considered to be 500 mg/kg bw/day.