Formaldehyde, oligomeric reaction products with diethylenetriamine and phenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Formaldehyde, oligomeric reaction products with diethylenetriamine and phenol

EC Number:

500-129-7

EC Name:

Formaldehyde, oligomeric reaction products with diethylenetriamine and phenol

Cas Number:

55552-95-9

Molecular formula:

(C6H6O.C4H13N3.CH2O)x

IUPAC Name:

Formaldehyde, oligomeric reaction products with diethylenetriamine and 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 294 to 351g and were approximately eleven weeks old. The females weighed 200 to 236g and were approximately fourteen weeks old.

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

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 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 100 4 25 12 (25-36) 12 (37-48)
Intermediate 300 4 75 12 (49-60) 12 (61-72)
High 500/400# 4 125/100# 12 (73-84) 12 (85-96)
# Dose level and concentration was reduced from 500 mg/kg bw/day to 400 mg/kg bw/day on Day 22.

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

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Samples of three test item formulations were taken and analyzed for concentration of Formaldehyde, oligomeric reaction products with diethylenetriamine and phenol (EK111) at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Annex 2. The results indicate that the prepared formulations were within 9% of the nominal concentration.

Duration of treatment / exposure:

28 day

Frequency of treatment:

once per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Remarks:

400 mg/kg bw/day after Day 21

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

ix. 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.

x. 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.

xi. 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 is 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 500/400 mg/kg bw/day which survived until the end of the treatment period. No clinical signs were apparent in any animal treated with 100 mg/kg bw/day.

Animals of either sex treated with 500/400 mg/kg bw/day showed incidences of increased salivation (all males and eleven females) from Day 1 (males) and Day 2 (females) throughout the treatment period. Noisy respiration (all males and eight females) was also noted in these animals from Day 2 (males) until Day 38 and in female animals from Day 3 until Day 31. Isolated occurrences of stained ano-genital region (two females and one male) and fur stained by the test item (one male) were also noted.

Isolated occurrences of increased salivation (four animals) and noisy respiration (two animals) were noted in male animals treated with 300 mg/kg bw/day which were noted from Day 15 until Day 38. One female animal from this treatment group also exhibited increased salivation which was noted on Days 13 and 14 only.

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

Signs of noisy respiration and increased salivation were noted in all animals that were found dead or were killed in extremis during the study. One female which was found dead on Day 3 also exhibited decreased respiratory rate, labored respiration, prostration and lethargy prior to death. The male animal which was killed in extremis on Day 3 exhibited signs of decreased respiratory rate, labored respiration, prostration, lethargy and cyanosis. The female which was killed in extremis on Day 19 exhibited loss of forelimb function, stained snout, ptosis, prostration, pilo-erection, lethargy, hypothermia, hunched posture, dehydration and ataxia. The female which was killed in extremis on Day 39 exhibited stained ano-genital region, labored respiration, decreased respiratory rate, pilo-erection, lethargy, hunched posture and pallor of the extremities.

Description (incidence):

One male and one female animal treated with 500 mg/kg bw/day were killed in extremis or found dead on Day 3 respectively. Two males treated with 500 mg/kg bw/day were found dead on Day 16. One female treated with 500 mg/kg bw/day was killed in extremis on Day 19. One female and one male animal treated with 500/400 mg/kg bw/day were found dead on Day 32 or Day 41 respectively and a further female treated with 500/400 mg/kg bw/day was killed in extremis on Day 39.

Description (incidence and severity):

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

Males treated with 500/400 mg/kg bw/day showed a statistically significant (p<0.01) reduction in body weight gain during the first week of treatment, a further reduction was noted during Week 2 but without achieving statistical significance. Recovery was evident thereafter, during the third week body weight gain achieved statistical significance (p<0.01) by actually exceeding control. However, even though body weight gains exceeded control from Weeks 3 to 5 the overall body weight gain for these animals still remained 13% lower than control.

Males treated with 300 mg/kg bw/day showed a statistically significant (p<0.01) reduction in body weight gain during the first week of treatment, good signs of recovery were noted during Week 2, however, body weight gains were still reduced when compared to control but without achieving statistical significance. This trend of improvement continued into the third week with body weight gains statistically significantly (p<0.05) exceeding control. Body weights then appeared to fluctuate over the remainder of the treatment period. Overall body weight gain for these animals was 23% lower than control.

There was no adverse effect of treatment on body weight development in male animals treated with 100 mg/kg bw/day. A slight reduction in body weight gain was apparent during Week 2, however, during the remainder of the study body weight development was comparable to or actually exceeded controls, achieving statistical significance (p<0.05) during Week 3. Overall body weight gain for these animals was slightly lower than control.

There were no adverse effects on body weight development in treated females during maturation. Females treated with 500 mg/kg bw/day exhibited slight reductions (without achieving statistical significance) in body weight gain during Week 1, however, recovery was evident during the second week. There were also considered to be no adverse effects on body weight gain during the gestation period for any of the treated female animals. During the lactation phase of the study females treated with 500/400 exhibited comparable body weight development to control animals from Day 1 to Day 4 and then actually exceeded control from Days 4 to 7. Reductions in body weight gain were then noted in these animals during the second week of lactation which achieved statistical significance (p<0.001). Overall body weight gain for these animals was found to be 19% lower than control. There was considered to be no adverse effect of treatment on body weight development during lactation in females treated with 300 or 100 mg/kg bw/day. Females treated with 300 mg/kg bw/day exhibited a reduction in body weight gain during the second week of lactation which achieved statistical significance (p<0.01), however, as overall body weight gain remained comparable to control this was considered not to be of toxicological 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.

Male animals treated with 500/400 mg/kg bw/day showed slight reductions in food consumption when compared to control during the first two weeks of treatment and then during the final week.

Male animals treated with 300 mg/kg bw/day showed slight reductions in food consumption when compared to control during the first two weeks of treatment.

There were no effects in food consumption in male animals treated with 100 mg/kg bw/day.

There were no adverse effects noted in food consumption for treated females during maturation, gestation or lactation. Females treated with 500/400 mg/kg bw/day exhibited slightly reduced food consumption during the final week of lactation which did not achieve statistical significance. These differences were considered to be due to one female animal which was only raising one offspring and therefore had substantially lower litter demand, this effect is therefore considered not to be toxicologically significant as food consumption is comparable to control in all other animals at this dosage.

Description (incidence and severity):

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

At 500/400 or 300 mg/kg bw/day, food conversion efficiency for males appeared inferior to control during the pre-pairing phase of the study. Improvement was evident thereafter.

At 100 mg/kg bw/day, values for food conversion efficiency did not indicate any obvious effect of treatment.

Intergroup differences in food conversion efficiency for females during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 100, 300 or 500/400 mg/kg bw/day.

Description (incidence and severity):

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

There were no adverse 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.

There were no adverse effects of treatment on hematological parameters measured.

Statistically significant reductions in erythrocytes (p<0.05) and hematocrit (p<0.05) were noted in males treated with 500/400 mg/kg bw/day with reductions in hematocrit also being noted amongst males treated with 300 mg/kg bw/day.

Statistically significant increases in reticulocytes (p<0.01) were noted in animals of either sex across all treatment groups.

The normal control data ranges for erythrocytes were increased in control animals in two instances and decreased in two instances in male animals treated with 500/400 mg/kg bw/day. One control animal exhibited an extremely high hematocrit value with two animals treated with 500/400 mg/kg bw/day exhibiting values that were lower than the historical control, all values for animals treated with 300 mg/kg bw/day were within the expected parameters. All reticulocyte values for treated and control animals were within the normal historical control data range.

With the exception of reticulocytes, true dose relationships were not really apparent across the treatment groups for these parameters and as there were no histopathological correlates these findings are considered to be incidental and not toxicologically significant.

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 Appendix 22 and Appendix 23.

At 500/400 mg/kg bw/day male animals exhibited statistically significant decreases in total protein (p<0.01) and albumin (p<0.05). Three and four males exhibited total protein or albumin values which were lower than the historical control data range respectively. Due to the effects noted in the kidneys at histopathological examination a relationship to treatment cannot be discounted.

There were considered to be no further toxicologically significant effects detected in the blood chemical parameters examined.

In 500/400 mg/kg bw/day males, statistically significant reductions (p<0.05) in phosphorous, alkaline phosphatase and bilirubin were noted. These animals also exhibited statistically significant increases (p<0.05) in potassium and aspartate aminotransferase.

Male animals treated with 300 mg/kg bw/day exhibited reductions in phosphorous (p<0.05). Females treated with 500/400 mg/kg bw/day exhibited statistically significant increases in glucose and chloride concentration (p<0.01), phosphorous was statistically significantly reduced (p<0.05).

Females treated with 300 mg/kg bw/day also exhibited statistically significant increases in glucose (p<0.01) with statistically significant reductions (p<0.05) in phosphorous also apparent.

All of these findings were generally found to be within the normal historical control data ranges and as similar effects were generally not seen across the sexes (with the exception of phosphorous) and there were no histopathological correlates these findings are considered to be incidental and 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 changes in the behavioral parameters in animals of either sex at 100, 300 or 500/400 mg/kg bw/day that indicated any underlying behavioral effect of treatment. Isolated instances of hunches posture and/or noisy respiration in male animals treated with 100 or 300 mg/kg bw/day were considered to be due to the irritant nature of the test item and not indicative of systemic toxicity.

Functional Performance Tests
There were considered to be no treatment related changes in functional performance considered to be related to treatment at 100, 300 or 500/400 mg/kg bw/day.

Female animals treated with 500/400 mg/kg bw/day exhibited a statistically significantly lower (p<0.05) hind limb grip strength. As this decrease was only noted in one out of the three runs completed and there were no clinical signs observed to signify a neurotoxic effect of the test item, this finding was considered to be incidental and of no toxicological relevance.

Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 or 500/400 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 25 (statistically significant differences are indicated). Individual data are given in Appendix 26 and Appendix 27.

Male animals treated with 500/400 mg/kg bw/day showed a statistically significant increase (p<0.05) in kidney weights both absolute and relative to terminal body weight. One absolute value was outside of the normal historical control data range with four values for relative organ weights outside of these ranges also. Due to the findings noted at histopathological examination a relationship to treatment cannot be discounted.

No toxicologically significant effects were detected in the organ weights measured in female animals treated with 500/400 mg/kg bw/day or in animals of either sex treated with 100 or 300 mg/kg bw/day.

Male animals treated with 500 mg/kg bw/day also exhibited statistically significant reductions in prostate weights (p<0.05) both absolute and relative to terminal body weight. One absolute and one relative value was outside of the normal historical control data range. This is considered to have contributed to the apparent reduction in this organ weight. As there were no histopathological correlates it is considered that this apparent effect is incidental and not specifically related to treatment with the test item.

Female animals treated with 100 and 300 mg/kg bw/day showed statistically significant increases (p<0.05) in adrenal weights both absolute and relative to terminal body weight. Four and three values for absolute organ weights for animals treated with 100 and 300 mg/kg bw/day respectively were outside of the historical control range. Whereas one relative value for animals treated with 100 mg/kg bw/day was outside of the historical control range. As no such effects were noted in animals treated with 500/400 mg/kg bw/day and there were no histopathological correlates these effects are considered to be incidental and unrelated to treatment with the test item.

Description (incidence and severity):

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

Offspring
Macroscopic necropsy findings for offspring did not indicate any obvious effect of maternal treatment on offspring development at 100, 300 or 500/400 mg/kg bw/day.

Adults
Neither the type, incidence or distribution of necropsy finding for surviving animals indicated any obvious effect of treatment at 100, 300 or 500/400 mg/kg bw/day.

Two control females and three females treated with 500/400 mg/kg bw/day exhibited pale livers. One female treated with 100 mg/kg bw/day exhibited a dark area on the liver. As there were no histopathological correlates these findings were considered to be incidental and unrelated to treatment with the test item. Two control females and three females treated with 500/400 mg/kg bw/day exhibited pale livers.

The male animal treated with 500 mg/kg bw/day which was humanely killed on Day 3 exhibited gaseous distension of the cecum which was also discoloured red, the liver was dark and the stomach was discolored red/brown and had gaseous distension with a thin non-glandular region.

The female animal treated with 500 mg/kg bw/day which was found dead on Day 3 exhibited gaseous distension of the cecum which was also discolored red, the stomach was discolored red/brown with red fluid filled contents and had gaseous distension with a thin non-glandular region and the liver was dark.

One male animal treated with 500 mg/kg bw/day which was found dead on Day 16 exhibited a cecum which was enlarged, fluid filled and reddened, gaseous distension was noted in the duodenum, ileum, jejenum and stomach. The stomach had a raised limiting ridge, the glandular region was thickened and also contained a red colored fluid and was stained red/brown, a dark liver was also apparent.

The other male animal treated with 500 mg/kg bw/day which was found dead on Day 16 exhibited dark adrenals, gaseous distension of the duodenum, ileum, jejenum, dark liver and lungs. The stomach was also discolored red/brown with the glandular region being thickened and a thin non-glandular region.

One female animal treated with 500 mg/kg bw/day which was humanely killed on Day 19 exhibited a cecum which was fluid filled with gaseous distension, gaseous distension was also noted in the ileum, jejenum and stomach. The stomach also had red patches, with sloughing and thickening of the glandular region and also a thin non-glandular region. The lungs were also found to be dark.

One female animal treated with 500/400 mg/kg bw/day which was found dead on Day 32 exhibited gaseous distension of the duodenum, ileum, jejenum and stomach. The liver, lungs, and pancreas were found to be dark and the spleen had a dark area. The jejenum was discoloured red and the stomach also showed sloughing of the glandular region and a thin non-glandular region.

The female animal which treated with 500/400 mg/kg bw/day was humanely killed on Day 39 exhibited gaseous distension throughout the gastro-intestinal tract, an increased pelvic space in both kidneys with both also being fluid filled.

The male animal treated with 500/400 mg/kg bw/day which was found dead on Day 41 exhibited red adrenals, dark liver, lungs and thymus. The stomach was also discolored red/brown with a thin non-glandular region being apparent.

Description (incidence and severity):

A complete histopathology phase report is presented in Annex 1.

Premature Decedents
Ulceration or erosion with hemorrhage was present in the glandular stomach and considered to be the cause of death in 6/8 premature decedents. This change occurred predominantly in the antral area. Inflammatory change and reactive hyperplasia, with or without erosion of the mucosa, in parts of the intestine were present in 3/8 animals. Changes were noted in the trachea of 4/8 animals.

The deaths of eight animals are considered to be related to the administration of the test item. Significant changes were noted in the glandular stomach along with clinical signs including gasping or laboured respiration, indicating respiratory distress. Whilst the primary cause of death in six of the animals was considered to be the significant stomach changes it is considered likely that the respiratory signs along with gaseous distension of the gastro-intestinal tract are indicative of some reflux related to the irritancy of the test item (Damsch et al 2011). Histological change in the trachea is supportive of this.

Terminal Kill

Kidneys
Multifocal basophilic tubules, graded as minimal or mild, were present in all Group 4 males. These changes were low grade but did correlate with an increase in organ weight.

Stomach
Ulceration in the glandular region, focal and minimal, was present in one Group 4 male. This occurred in the antral area and was surrounded by an area of mucosal hyperplasia.

Cecum
Reactive hyperplasia of the mucosa was noted in a Group 4 female (minimal inflammatory infiltrate/fibrosis with thickening and basophilia in the mucosa).

Duodenum
Reactive hyperplasia with neutrophilic infiltration was present in one Group 4 female. No other changes which could be related to the administration of the test item were noted and are considered to be incidental.

Non-Productive Matings
Two females from Group 3 (64 and 68) were non-pregnant. Examination of the tissues from these animals and their male partners (52 and 56) did not reveal any changes which are likely to have affected their fertility.

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

Description (incidence and severity):

Thyroid Hormone Analysis
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 500/400 mg/kg bw/day.

A statistically significant increase in Thyroxine (T4) was noted in male offspring from females treated with 300 mg/kg bw/day. As no such effects were noted in female offspring or from offspring of either sex at the higher dosage, this was considered to be incidental and unrelated to treatment with the test item.

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

Estrous cycles during the pre-pairing phase of the study were unaffected by treatment at 100, 300 or 500/400 mg/kg bw/day.

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

No treatment-related effects were detected in mating performance.

One female treated with 300 mg/kg bw/day did not mate with her partner. As this was only in relation to one pair of animals this was considered to be incidental and unrelated to treatment with the test item.

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.

Fertility as assessed by the number of females that achieved pregnancy, was unaffected by treatment at 100, 300 or 500/400 mg/kg bw/day.

Two females treated with 300 mg/kg bw/day found to be non-pregnant (one of these animals had shown positive evidence of mating). Histopathological examinations of these females and their respective male partners did not reveal any microscopic changes which could account for the lack of fertility, therefore, these were considered incidental and unrelated to treatment.

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

Gestation length appeared unaffected by treatment at 100, 300 or 500/400 mg/kg bw/day. Gestation lengths for controls and treated females were between 22 and 23 days. Statistical significance was achieved for animals treated with 300 and 500/400 mg/kg bw/day (p<0.05 and p<0.01 respectively) as gestation lengths were slightly longer than control. However, as all gestation lengths were within the historical control data range these findings were considered not to be of any toxicological importance. One female did not show positive evidence of mating but was subsequently found to be pregnant, the gestation length for this animal could therefore not be determined.

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

There was considered to be no 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 was also comparable to controls and did not indicate any selective effect of maternal treatment on survival for either sex.

At 500/400 mg/kg bw/day, the mean number of post-implantations was lower than control although this value failed to attain statistical significance. The number of implantations was not particularly low at this dosage and this difference appears to be incidental and unrelated to maternal treatment. All values were within the normal historical control data range. The mean total number of offspring born was slightly lower than control but consistent with the previous slightly lower mean number of implantations at this dosage. Mean live litter size on Day 1 was lower than control due to what would appear to be inferior offspring survival, as indicated by an apparent inferior mean live birth index at this dosage compared to control. Subsequent offspring survival from Day 4 to termination on Day 13 was identical to control and was therefore unaffected by maternal treatment.

The apparent reductions on total number of offspring born, live litter size on Day 1 and inferior live birth index at this dosage compared to control were accentuated by one female which only gave birth to five live offspring out of a litter of fifteen (with only one of these surviving to Day 1 post partum). With this animal removed from Group means, live birth index, litter size and offspring viability on Day 1 drew more in line with control. As such, it is considered that these apparent effects had been accentuated by this one animal and as this was noted in isolation it cannot be attributed to treatment with the test item and is more than likely due to normal biological variation.

At 300 mg/kg bw/day a statistically significant reduction (p<0.05) in offspring viability was noted on Day 4, however, as this was not noted in animals treated at 500/400 mg/kg bw/day this was considered to be incidental and of no 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 and group mean ano-genital distance and visible nipple counts in male offspring are given in Tables 15, 18 , 19 and 20. Individual values and observations are given in Appendices 13, 16, 17 and 18.

There was no effect of maternal treatment on litter weights, offspring body weight on Day 1 or subsequent body weight gains to termination at 100, 300 or 500/400 mg/kg bw/day.

As a consequence of the slightly lower litter size from animals treated with 500/400 mg/kg bw/day, litter weights on Days 1, 4, 7, and 13 post partum appeared markedly reduced (in the first instance) when compared to controls but without achieving statistical significance. These findings were overemphasised by one animal which only gave birth to one live offspring. Slightly reduced litter weights were still apparent when this animal was removed from the group means but these were considered to reflect the slightly lower litter size at this dosage and were considered to be unrelated to maternal treatment with the test item.

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

Ano-genital distance in offspring on Day 1 post partum and visible nipple count for male offspring on Day 13 post partum appeared unaffected by maternal treatment at 100, 300 or 500/400 mg/kg bw/day.

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 diethylenetriamine and phenol (EK111) to Wistar Han™:RccHan™:WIST strain rats for approximately six weeks (males) and up to 8 weeks (females) (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 100, 300 and 500 mg/kg bw/day resulted in the early deaths/terminations of three males and two female animals treated with 500 mg/kg bw/day. This dose level was reduced to 400 mg/kg bw/day on Day 22, however, this reduction still resulted in the early deaths/terminations of one male and two female animals. The primary cause of death in six of these animals was considered to be the significant stomach changes and it is considered likely that the respiratory signs along with gaseous distension of the gastro-intestinal tract are indicative of some reflux related to the irritancy of the test item (Damsch et al 2011). Histological change in the trachea is supportive of this. The microscopic stomach changes identified in male animals at 500/400 mg/kg bw/day were considered to be the result of gastric irritancy rather than attributable to true systemic toxicity.

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.

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 (reduced to 400

mg/kg bw/day on Day 22).  A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400 ). A dose volume of 4 mL/kg bw was employed.

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 assessment 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 all adult males and Day 13 post partum offspring were analyzed for Thyroxine (T4).

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

Surviving adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively.  Any female which did not show positive evidence of mating or 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.

Results

Adult Responses

Mortality

One male and one female animal treated with 500 mg/kg bw/day were killed in extremis or found dead on Day 3 respectively. Two males treated with 500 mg/kg bw/day were found dead on Day 16. One female treated with 500 mg/kg bw/day was killed in extremis on Day 19. One female and one male animal treated with 500/400 mg/kg bw/day were found dead on Day 32 or Day 41 respectively and a further female treated with 500/400 mg/kg bw/day was killed in extremis on Day 39.

Clinical Observations

There were no clinical signs specifically related to systemic toxicity elicited by the test item for males or females treated with 300 or 500/400 mg/kg bw/day which survived until the end of the treatment period. No clinical signs were apparent in any animal treated with 100 mg/kg bw/day.  

Signs of noisy respiration and increased salivation were noted in all animals that were found dead or were killed in extremis during the study. One female which was found dead on Day 3 also exhibited decreased respiratory rate, labored respiration, prostration and lethargy prior to death. The male animal which was killed in extremis on Day 3 exhibited signs of decreased respiratory rate, labored respiration, prostration, lethargy and cyanosis. The female which was killed in extremis on Day 19 exhibited loss of forelimb function, stained snout, ptosis, prostration, pilo-erection, lethargy, hypothermia, hunched posture, dehydration and ataxia. The female which was killed in extremis on Day 39 exhibited stained ano-genital region, labored respiration, decreased respiratory rate, pilo-erection, lethargy, hunched posture and

pallor of the extremities.

Behavioral Assessment

There were no changes in the behavioral parameters in animals of either sex at 100, 300 or 500/400 mg/kg bw/day that indicated any underlying behavioral effect of treatment.  

Functional Performance Tests

There were no treatment related changes in functional performance in animals of either sex at 100, 300 or 500/400 mg/kg bw/day.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 or 500/400 mg/kg bw/day.

Body Weight

Males treated with 500/400 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment, a further slight reduction was noted during Week 2.  Recovery was evident thereafter, even though body weight gains exceeded control from Weeks 3 to 5 the overall body weight gain for these animals still remained lower than control.

Males treated with 300 mg/kg bw/day showed a reduction in body weight gain during the first two weeks of treatment. Body weights then appeared to fluctuate over the remainder of the treatment period. Overall body weight gain for these animals was lower than control.

There was no adverse effect of treatment on body weight development in male animals treated with 100 mg/kg bw/day. Overall body weight gain for these animals slightly lower than control.

There were no adverse effects on body weight development in treated females during maturation or gestation.  

During the lactation phase of the study females treated with 500/400 exhibited comparable or superior body weight development to control animals during the first week. Reductions in body weight gain were then noted in these animals during the second week. Overall body weight gain for these animals was found to be lower than control.  

There was no adverse effect of treatment on body weight development during lactation in females treated with 300 or 100 mg/kg bw/day.

Food Consumption

Male animals treated with 500/400 and 300 mg/kg bw/day showed slight reductions in food consumption when compared to control during the first two weeks of treatment. A further reduction was then noted in males treated with 500/400 mg/kg bw/day during the final week.

There was no effects in food consumption in male animals treated with 100 mg/kg bw/day.  

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

Food Conversion Efficiency

At 500/400 and 300 mg/kg bw/day, marked differences in food conversion efficiency for males compared to the control during the pre-pairing phase of the study were observed. This was reflected in reductions in food consumption. At 100 mg/kg bw/day, food conversion efficiency for males appeared unaffected by treatment.

Food conversion efficiency for females during the pre-pairing phase of the study appeared unaffected by treatment at 100, 300 or 500/400 mg/kg bw/day.

Water Consumption

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

Reproductive Performance

Estrous Cycles

Estrous cycles during the pre-pairing phase of the study were unaffected by treatment at 100, 300 or 500/400 mg/kg bw/day.

Mating

No treatment-related effects were detected in mating performance at at 100, 300 or 500/400 mg/kg bw/day.

Fertility

Fertility as assessed by the number of females that achieved pregnancy, was unaffected by treatment at 100, 300 or 500/400 mg/kg bw/day.

Gestation Lengths

Gestation length appeared unaffected by treatment at 100, 300 or 500/400 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no 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 was also comparable to controls and did not indicate any selective effect of maternal treatment on survival for either sex.

Offspring Growth and Development

There was considered to be no effect of maternal treatment on litter weights, offspring body weight on Day 1 or subsequent body weight gains to termination at 100, 300 or 500/400 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.

Ano-genital distance in offspring on Day 1 post partum and visible nipple count for male offspring on Day 13 post partum appeared unaffected by maternal treatment at 100, 300 or 500/400 mg/kg bw/day.

Laboratory Investigations

Hematology

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

Blood Chemistry

Male animals treated with 500/400 mg/kg bw/day showed decreases in total protein and albumin.  

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

Pathology

Necropsy

Offspring

Macroscopic necropsy findings for offspring did not indicate any obvious effect of maternal treatment on offspring development at 100, 300 or 500/400 mg/kg bw/day.

Adults

Neither the type, incidence or distribution of necropsy findings for surviving animals indicated any obvious effect of treatment at 100, 300 or 500/400 mg/kg bw/day.

The male animal treated with 500 mg/kg bw/day which was humanely killed on Day 3 exhibited gaseous distension of the cecum which was also discolored red, the liver was dark and the stomach was discolored red/brown and had gaseous distension with a thin non-glandular region.  

The female animal treated with 500 mg/kg bw/day which was found dead on Day 3 exhibited gaseous distension of the cecum which was also discolored red, the stomach was discolored red/brown with red fluid filled contents and had gaseous distension with a thin non-glandular region and the liver was dark.  

One male animal treated with 500 mg/kg bw/day which was found dead on Day 16 exhibited a cecum which was enlarged, fluid filled and reddened, gaseous distension was noted in the duodenum, ileum, jejenum and stomach. The stomach had a raised limiting ridge, the glandular region was thickened and also contained a red colored fluid and was stained red/brown, a dark liver was also apparent.  

The other male animal treated with 500 mg/kg bw/day which was found dead on Day 16 exhibited dark adrenals, gaseous distension of the duodenum, ileum, jejenum, dark liver and lungs. The stomach was also discolored red/brown with the glandular region being thickened and a thin non-glandular region.  

One female animal treated with 500 mg/kg bw/day which was found dead on Day 19 exhibited a cecum which was fluid filled with gaseous distension, gaseous distension was also noted in the ileum, jejenum and stomach. The stomach also had red patches, with sloughing and thickening of the glandular region and also a thin non-glandular region. The lungs were also found to be dark.  

One female animal treated with 500/400 mg/kg bw/day which was humanely killed on Day 32 exhibited gaseous distension of the duodenum, ileum, jejenum and stomach. The liver, lungs, and pancreas were found to be dark and the spleen had a dark area. The jejenum was discolored red and the stomach also showed sloughing of the glandular region and a thin non-

glandular region.  

The female animal which was treated with 500/400 mg/kg bw/day was humanely killed on Day 39 exhibited gaseous distension throughout the gastro-intestinal tract, an increased pelvic space in both kidneys with both also being fluid filled.  

The male animal treated with 500/400 mg/kg bw/day which was found dead on Day 41 exhibited red adrenals, dark liver, lungs and thymus. The stomach was also discolored red/brown with a thin non-glandular region being apparent.

Organ Weights

Male animals treated with 500/400 mg/kg bw/day showed increases in kidney weights both absolute and relative to terminal body weight. No further toxicologically significant effects were detected in these animals.  

No toxicologically significant effects were detected in the organ weights measured in female animals treated with 500/400 mg/kg bw/day or in animals of either sex treated with 100 or 300 mg/kg bw/day.

Histopathology

Premature Decedents

Ulceration or erosion with hemorrhage was present in the glandular stomach and considered to be the cause of death in 6/8 premature decedents. This change occurred predominantly in the antral area.  Inflammatory change and reactive hyperplasia, with or without erosion of the mucosa, in parts of the intestine were present in 3/8 animals. Changes were noted in the trachea of 4/8 animals.  

Terminal Kill

Kidneys

Multifocal basophilic tubules, graded as minimal or mild, were present in all Group 4 males.

Stomach

Ulceration in the glandular region, focal and minimal, was present in one Group 4 male.  This occurred in the antral area and was surrounded by an area of mucosal hyperplasia.

Cecum

Reactive hyperplasia of the mucosa was noted in a Group 4 female (minimal inflammatory infiltrate/fibrosis with thickening and basophilia in the mucosa).

Duodenum

Reactive hyperplasia with neutrophilic infiltration was present in one Group 4 female.

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

Non-Productive Matings

Two females from Group 3 (64 and 68) were non-pregnant. Examination of the tissues from these animals and their male partners (52 and 56) did not reveal any changes which are likely to have affected their fertility.

Thyroid Hormone Analysis

Levels of thyroxine (T4) in adult males did not indicate any obvious effect of treatment or indication of endocrine disruption at 100, 300 or 500/400 mg/kg bw/day.

Levels of thyroxine (T4) in offspring at Day 13 of age did not indicate any obvious effect of maternal treatment or indication of endocrine disruption at 100, 300 or 500/400 mg/kg bw/day.

Conclusion

The oral administration of Formaldehyde, oligomeric reaction products with diethylenetriamine and phenol (EK111) to Wistar Han™:RccHan™:WIST strain rats for  approximately six weeks (males) and up to 8 weeks (females) (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 100, 300 and 500 mg/kg bw/day resulted in the early deaths/terminations of three males and two female animals treated with 500 mg/kg bw/day. This dose level was reduced to 400 mg/kg bw/day on Day 22, however, this reduction still resulted in the early deaths/terminations of one male and two female animals. The primary cause of death in six of these animals was considered to be the significant stomach changes and it is considered likely that the respiratory signs along with gaseous distension of the gastro-intestinal tract are indicative of some reflux related to the irritancy of the test item (Damsch et al 2011).  Histological change in the trachea is

supportive of this.  The microscopic stomach changes identified in male animals at 500/400 mg/kg bw/day were considered to be the result of gastric irritancy rather than attributable to true systemic toxicity.  

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.