Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 24, 2017 to September 11, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 24, 2017 to September 11, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The deviations were considered to have no impact on the outcome of the study
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Crl:WI Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany, from SPF colony.
Housing conditions: Standard laboratory conditions
Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Number of animals: 48 male, 48 female rats, 12 animals/sex/group, 4 groups.
Age of animals: Young adult rats, approximately 10-11 weeks old at start and 12-13 weeks old at mating.
Body weight range: Males: 341 – 467 g, females: 242 – 300 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
Acclimation period: 6 d
Animal health: Only healthy animals were used for the test, as certified by the clinical Veterinarian. Females were nulliparous and non-pregnant.
Room numbers: 609, 639
Cage type: Type II polycarbonate
Bedding & nesting: LIGNOCEL® ¾ S certified wooden chips (batch number: 03018170329, expiry date: 29 March 2020 and batch number: 03018170104, expiry date: 04 January 2020) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. ARBOCEL® nest building material (batch number: 05072170228, expiry date: 28 February 2020 and batch number: 05072160415, expiry date: 15 April 2019) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding and nest building material were archived with the raw data.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.3 – 23.1°C (target range 22 ± 3°C)
Relative humidity: 32 – 70% (target range 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).

Environmental parameters (temperature and relative humidity) were continuously measured, minimum and maximum values were recorded twice a day during the study.

Food and water supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (batch number: 285 17890, expiry date: 31 August 2017 and batch number: 262 21592, expiry date: 31 January 2018), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided the analytical certificate for the batch used, which is archived with the raw data. Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A. u. 36., Hungary). The quality control results are retained with the raw data in the archives at Citoxlab Hungary Ltd.

Animal identification
Each adult/parental animal (P Generation) was identified by a unique number within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at Citoxlab Hungary Ltd. During the pre-exposure period, animals were identified with temporary numbers only. After this 2-week period, a randomisation was performed based on the body weights and the selected animals received their final animal numbers, as follows: This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Identification of the new-borns (Offspring, F1 Generation) was performed by ink marking of the digit-tips up to one day after birth.

Randomisation
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day -1). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.
Route of administration:
oral: gavage
Details on route of administration:
The dose levels were selected by the Sponsor in consultation with the Study Director based on available acute oral toxicity data (LD50 > 5000 mg/kg in rats) and information from a 14-day Dose Range Finding study in the rat (Citoxlab study code 17/014-220PE [3]). In the DRF study, there was no toxicity at 1000 mg/kg bw/day. The aim of this study is to use a maximum of 1000 mg/kg bw/day to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Based on the results from these preliminary studies, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study. The oral route was selected as it is one of the possible routes of human exposure.
Vehicle:
corn oil
Details on oral exposure:
The dosing solutions were administered to the test substance or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight. Dosing of both sexes began after 6 d of acclimatisation and pre-exposure period (14 d), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy. The first day of dosing of each animal was regarded as Day 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test substance formulations for concentration and homogeneity was performed using an HPLC-UV method in the Analytical Laboratory of Citoxlab Hungary Ltd. Top, middle and bottom duplicate samples were taken from test substance formulations three times during the study (during the first and last weeks and approximately midway during the treatment), one set to analyse (which could be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement. Acceptance criteria of the concentration analysis were set according to the analytical method validation, expected to be at 100 ± 15 % of the nominal concentration. The measured concentrations of Di-Trimethylolpropane Tetraacrylate evaluated for each test item-dose group varied between 100 % and 102 % of the nominal contents. The RSD was below 10% in each case. No test substance was detected in the control samples. These results were within the acceptable ranges (85% - 115%) and were considered suitable for the study purposes.
Duration of treatment / exposure:
Males were dosed for 28 d (14 d pre-mating and 14 d mating/post-mating) and then euthanized and subjected to necropsy examination. Females were dosed for 14 d pre-mating, during the mating period, through gestation and until the day before the necropsy (13-d post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.
Frequency of treatment:
Daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
55 male and 55 female Wistar rats were used in the pre-treatment period. At the end of the pre-treatment period, 48 females showing regular oestrus cycles and 48 males were selected and allocated to treatment groups. Animals were assigned to groups before the start of the treatment according to the following Experimental design:
Group designation: Control, low dose, mid dose, high dose
Dose level (mg/kg bw/day): 0, 100, 300, 1000
Concentration (mg/mL): 0, 20, 60, 200
Dose volume (mL/kg bw): 5
Animal numbers: Male 1001-1012 and Female: 1501-1512 (control), Male: 2001-2012 and Female: 2501-2512 (low dose), Male: 3001-3012 and Female: 3501-3512 (mid dose), Male: 4001-4012 and Female: 4501-4512 (high dose)
The control group was treated with the vehicle only (corn oil).
Observations and examinations performed and frequency:
Clinical observations
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily*, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment. On gestation day GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat). All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable. More detailed examinations were performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were performed outside the home cage in a standard arena, at similar day times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
*Note: No general clinical observations were made on those days when detailed clinical observations were made.

Body weight measurement
All adult animals were weighed with an accuracy of 1 g for randomisation purposes, then at least weekly during the pre-exposure period, on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 3, 7, 10, 14, 17 and 20 and on post-partal Days PPD0 (within 24 hours after parturition), PPD4, 7, 10, 13 and 14 (before termination). The body weight of the female animals measured on gestational Days GD3, 10 and 17 as well as PPD7 and PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

Food consumption measurement
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g weekly (on the days of body weight measurements).

Functional observation battery (FOB) and SMART
Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 26; females on PPD7-9). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength. To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots for the hind limbs was measured. Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs and the appropriate grip support. The results were tabulated with individual and mean data. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated using a scoring system, where score “0” was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal. Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for a 1-h observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.
Sacrifice and pathology:
Clinical pathology
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. For terminal blood sampling in all selected animals (5 males and 5 females/group), 3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Terminal procedures and macroscopic evaluation
At termination, the adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination. Gross necropsy was performed on all animals, irrespective of the date of death. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
Other examinations:
Haematology and blood clotting times
The following parameters were evaluated in animals selected:
RBC Red Blood Cell (erythrocyte) count, WBC White Blood Cell (leukocyte), Hgb Haemoglobin concentration, (g/dL), MCV Mean Corpuscular (erythrocyte), MCH Mean Corpuscular (erythrocyte), MCHC Mean Corpuscular (erythrocyte), RDW Red Cell (erythrocyte) volume (%), Plt Platelet (thrombocyte) count, MPV Mean Platelet Thrombocyte volume (fL), RETIC % Reticulocyte count (%), Neutrophil (%), Cell differentiation based on myeloperoxidase activity, LY % Lymphocyte (%), MO % Monocyte (%), BA % Basophil (%), EO % Eosinophil (%), LUC % Large Unstained Cells (%), Coagulations (APTT Activated Partial Thromboplastin, PT Prothrombin Time (sec)).
Blood smears were prepared for all selected animals but not examined. The smears were stored/archived at Citoxlab Hungary Ltd.

Clinical chemistry
The following parameters were evaluated in animals selected:
Glucose Blood sugar concentration (mmol/L), T-BIL Total Bilirubin concentration (μmol/L), Urea Urea concentration (mmol/L), Chol. Cholesterol concentration (mmol/L), Creat. Creatinine concentration (μmol/L), Phos. Phosphorus concentration (mmol/L), Na+ Sodium concentration (mmol/L), K+ Potassium concentration (mmol/L), Ca++Calcium concentration (mmol/L), Cl- Chloride concentration (mmol/L), Tot. Prot. Total Protein concentration (g/L), Alb. Albumin concentration (g/L), A/G Alb/glob ration Calculated value, AST/GOT Aspartate Aminotransferase, activity (U/L), ALT/GPT Alanine Aminotransferase activity (U/L), GGT Gamma-Glutamyl transferase activity (U/L), γ-Glutamyl-p-nitroanilide + Glycylglycine, ALKP Alkaline Phosphatase activity (U/L), Bile acids (μmol/L).

Urinalysis
Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 h. The evaluation of the urine samples was performed as indicated in the table below.
LEU / Leukocyte, NIT / Nitrite, pH, PRO / Protein, GLU / Glucose, UBG / Urobilinogen, BIL / Bilirubin, KET / Ketones, BLD / ERY Blood/Erythrocytes, SG / Specific Gravity, SED / Sediment, VOL / Volume, Colour/appearance

Organ weight measurements
At the time of termination, body weight and the weight of the following organs from all adult animals were determined:
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported. In case microscopic examination, the retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

Statistics
Data was recorded on the appropriate forms from the relevant SOPs of Citoxlab Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS v.9, as appropriate. Numerical data obtained during the conduct of the study was subjected as appropriate to calculation of group means and standard deviations and was reported in the Final Report. The statistical evaluation of data (labelled as † in the lists below) was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis). In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data. In case of the SAS v9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate. If either of the Shapiro-Wilk or Levene tests showed significance on the data, then a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate. For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
Statistics:
SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related clinical signs were observed during the study. A 1-2 cm width nodule was seen in one Low dose female (#2505) from Day 10 until the end of the observation period. Cold to touch whole body, piloerection and slight discharge from vagina were recorded for one Low dose female (#2511) on Days 39-40. Increased salivation, piloerection and red discharge at the nose were recorded for one Mid dose female (#3504) on Days 35-36. A 1-2 cm width wound was seen at the left cheek of a High dose male (#4012) during Days 13-15, that became a crust on Days 16-18 and then a scar from Day 19 until the end of the observation period. Thin fur at the right cheek was seen in one High dose female (#4503) from Day 22 until Day 32. The following symptoms were recorded for the found dead animal (#1502) before death: Slightly decreased activity, cold to touch whole body, hunched back, piloerection, pale skin at the tail, all paws and both pinnae.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (#1502) in the Control group was found dead on Day 42 (on the day of parturition). The cause of death could not be identified at necropsy. There was no other mortality during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant body weight or body weight gain values in the test substance treated groups (males/females) when compared to the control at any occasion that could be ascribed to the test substance. The measured values were within the range commonly recorded for this strain and age. Occasional statistically significant differences were regarded as incidental and of no toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test substance related differences in the mean daily food consumption in any test substance treated group when compared to the controls. The measured values were within the normal range for this strain and age. Occasional statistically significant differences were regarded as incidental and of no toxicological significance.
Food efficiency:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
Significantly lower (p<0.05 or p<0.01) prothrombin time (PTT) was recorded in the male and female Low dose animals and in the female High dose group. The platelet count was also significantly lower (p<0.05) in the female Low and Mid dose groups. These differences were considered to be incidental, there was no relationship with dose and all recorded values were within or near the historical control ranges. These differences were considered not reflecting an effect of the test substance. Besides this, there were no other statistically significant values recorded in any of the dose groups compared to the control.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no statistically different results in any of the male groups compared to the control. The following statistically significant differences were recorded in the females: higher (p<0.01) glucose concentration in the High dose group, higher (p<0.05) albumin concentration in the Mid and High dose groups and higher (p<0.05 or p<0.01) albumin/globulin (A/G) ratio in the Mid and High dose groups. The albumin and A/G ratio values were all well within the historical range and did not show a dose response; hence these statistical differences are regarded as unrelated to treatment. For the glucose concentration, 2 of the 5 female individual values were outside the historical range, but in males all values were normal (the mean was slightly below the controls, indicating no effect on glucose). There was no other supporting evidence of any changes in these animals (histopathology, urinalysis, etc.) so the relationship between the 2 high glucose values and treatment was considered to be equivocal. Taking into account the available information, the glucose difference is not considered to be a clear adverse effect. Besides this, there were no other statistically significant values recorded in any of the dose groups compared to the control.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Compared to the control, there were no statistically significant values recorded in any of the dose groups.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted during the assessment of grip strength, foot splay or motor activity. At evaluation of the landing foot splay test, slightly lower values were recorded for males at 1000 mg/kg bw/day (High dose), when compared to control means. The differences attained statistical significance (p<0.05). When evaluating individual data, the values of 4 of 5 males were within the control values and the performance of one male only (#4005) was lower. All values were in the normal range. It should be noted that the control values were relatively high compared with historical data; it is considered that there was no effect of treatment. In females, the landing foot splay values were comparable with the control mean in all dose groups. All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity-level was high, with progressive reduction in activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals and the Control when evaluating the overall distance travelled (0-60 min, cm). The test item did not increase or decrease the normal locomotor activity.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Significantly higher (p<0.05) brain relative to bodyweight ratio in the female High dose group and significantly lower (p<0.05 or p<0.01) relative (to brain weight) kidney weights were recorded in the female dose groups. As the observed values were near the middle of the historical control ranges and there was a lack of any supporting evidence of any changes in these animals (histopathology, urinalysis, etc.), these statistical differences were considered to have no toxicological significance. Regarding the kidney weights, there was no clear dose response (Low, Mid and High groups had very similar values); there were no statistical differences in female absolute or body weight adjusted kidney weights. No similar finding was found in the males. Besides this, there were no statistically different results in any other of the dose groups compared to the control.
Gross pathological findings:
no effects observed
Description (incidence and severity):
(A) Terminal / Parental Generation (Males, Day 28, Females, PND14)
Macroscopic Findings: No treatment related macroscopic findings were noted at necropsy. The following incidental or background findings were seen in the terminally euthanized animals: small size of the right adrenal gland (#1001), depressed area of renal capsule of left kidney (#1508), diffuse dark red discoloration of the left mandibular lymph node (#4508), enlargement of the pituitary gland (#2011), focal red discoloration of the right testis (#1011), diffuse dark red discoloration of the thymus (#4501), subcutaneous nodule in the ventral thoracic area of the skin (#2505) and dilatation of the left uterine horn (#4508).

(B) Found dead / Parental Generation
Macroscopic Findings: The following findings were seen in the found dead animal (#1502): The dark red dry material at the urogenital and perinasal area and the multifocal dark red discoloration of all lobes of the non-collapsed lungs are considered as agonal or post mortal. The bilaterial dilatation of the uterine horns is considered as a background finding.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
(A) Terminal / Parental Generation (Males, Day 28, Females, PND14)
Microscopic Findings
No test substance-related findings were seen at dose levels up to 1000 mg/kg bw/day. Based on the low incidence and/or severity and/or distribution across control and dosed animals the following observations were considered incidental or a common background: slight atrophy of the zona fasciculata and zona reticularis in the right adrenal gland (#1001), slight luminal dilatation of the left horn of the uterine (#4508), minimal focal cyst in one side of the piriform cortex of the brain (#4005), minimal tubular basophilia in the cortex of the right kidney (#4005), slight multifocal casts in the cortex and medulla of both kidneys (#1002), minimal focal casts in the outer stripe of the right kidney (#1003), minimal focal casts in the inner and outer stripes of the right kidney (#4505), minimal tubular multifocal mineralization in the outer stripe of both kidneys (#1504), slight pyelonephritis in the right kidney (#4507), slight focal atrophy in the subcapsular cortex of the left kidney (#1508), minimal multifocal hepatocellular vacuolation in the liver (#1005), slight multifocal haemorrhage in the mandibular lymph node (#4508), minimal multifocal infiltration of the inflammatory cells in the interstitium and dorsolateral lobe of the prostate (#1001), minimal or slight extramedullary haematopoiesis in the spleen (#1503, 1504, 1506, 1511, 4001, 4002, 4005, 4504, 4506, 4507), minimal multifocal tubular degeneration or atrophy of the right testis (#4003), slight focal congestion in the interstitium of the right testis (#1011), slight single cyst in the thymus (#4505), slight multifocal haemorrhage in the thymus (#4501) and slight inflammation of the mucosa and submucosa of the urinary bladder (#4507). In addition, as a macroscopic lesion was seen in the pituitary gland of one Low dose male (#2011) and in the subcutis of one Low dose female (#2505), these organs were macroscopically examined and the following findings were recorded: Multiple cysts in the pars distalis section of the pituitary gland (#2011) and adenoma in the mammary gland (#2505). The microscopic findings correlated with the macroscopic lesions.

(B) Found dead / Parental Generation
Microscopic Findings
The following observations were recorded in the found dead animal (#1502): signs of proestrus in the uterus, slight diffuse congestion of all lobes of the lungs, moderate extramedullary haematopoiesis in the spleen and minimal focal erosion or ulcer in the pylorus of the stomach.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Thyroid gland weights and thyroid hormone levels
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the adult male or PND13 pup dose groups. The thyroid gland weights of the PND13 pups were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test substance. The measurement of the thyroid hormone levels in the PND4 pups and adult females was not performed as it was not deemed necessary by the Study Director.
Details on results:
Kindly refer the attachments in attached background material section of IUCLID for detailed results tables.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no treatment related adverse effects at highest tested dose
Key result
Critical effects observed:
no
Conclusions:
Under the study conditions, the NOAEL for systemic effects in female and male wistar rats, was considered to be at the highest tested dose i.e., 1000 mg/kg bw/day.
Executive summary:

A study was conducted to determine the repeated dose toxicity of the test substance, di-TMPTTA (UVCB) using combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, according to OECD 422 Guideline, in compliance with GLP. Male and female Wistar rats (48 animals each sex) were treated daily with test substance in corn oil (100, 300 or 1000 mg/kg bw/day) by oral gavage for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 d in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day PPD13. Parameters measured during the study included signs of morbidity and mortality twice daily, daily general observation or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment, such as functional observation battery (FOB) including measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. The thyroxine (T4) levels in the Day 13 pups and adult males were also assessed. For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the control and high dose groups. During the treatment period, no test substance related mortality, clinical adverse effects, or changes in neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry or urinalysis parameters was observed. No test substance-related macroscopic findings were recorded in any of the dose groups at necropsy; no microscopic effects were seen at histopathology. There were no test substance-related differences among groups in the weights of organs measured when compared to controls. Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the adult male dose groups. Under the study conditions, the NOAEL for systemic effects in female and male wistar rats, was considered to be at the highest tested dose i.e., 1000 mg/kg bw/day (Weisz, 2018).

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The deviations were considered to have no impact on the outcome of the study
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species and strain: Crl:WI Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany, from SPF colony.
Housing conditions: Standard laboratory conditions
Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals and environmental conditions:
Number of animals: 48 male, 48 female rats, 12 animals/sex/group, 4 groups.
Age of animals: Young adult rats, approximately 10-11 weeks old at start and 12-13 weeks old at mating.
Body weight range: Males: 341 – 467 g, females: 242 – 300 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
Acclimation period: 6 d
Animal health: Only healthy animals were used for the test, as certified by the clinical Veterinarian. Females were nulliparous and non-pregnant.
Room numbers: 609, 639
Cage type: Type II polycarbonate
Bedding & nesting: LIGNOCEL® ¾ S certified wooden chips (batch number: 03018170329, expiry date: 29 March 2020 and batch number: 03018170104, expiry date: 04 January 2020) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. ARBOCEL® nest building material (batch number: 05072170228, expiry date: 28 February 2020 and batch number: 05072160415, expiry date: 15 April 2019) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding and nest building material were archived with the raw data.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.3 – 23.1°C (target range 22 ± 3°C)
Relative humidity: 32 – 70% (target range 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).

Environmental parameters (temperature and relative humidity) were continuously measured, minimum and maximum values were recorded twice a day during the study.

Food and water supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (batch number: 285 17890, expiry date: 31 August 2017 and batch number: 262 21592, expiry date: 31 January 2018), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided the analytical certificate for the batch used, which is archived with the raw data. Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A. u. 36., Hungary). The quality control results are retained with the raw data in the archives at Citoxlab Hungary Ltd.

Animal identification
Each adult/parental animal (P Generation) was identified by a unique number within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at Citoxlab Hungary Ltd. During the pre-exposure period, animals were identified with temporary numbers only. After this 2-week period, a randomisation was performed based on the body weights and the selected animals received their final animal numbers, as follows: This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Identification of the new-borns (Offspring, F1 Generation) was performed by ink marking of the digit-tips up to one day after birth.

Randomisation
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day -1). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The dose levels were selected by the Sponsor in consultation with the Study Director based on available acute oral toxicity data (LD50 > 5000 mg/kg in rats) and information from a 14-day Dose Range Finding study in the rat (Citoxlab study code 17/014-220PE [3]). In the DRF study, there was no toxicity at 1000 mg/kg bw/day. The aim of this study is to use a maximum of 1000 mg/kg bw/day to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Based on the results from these preliminary studies, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study. The oral route was selected as it is one of the possible routes of human exposure. The dosing solutions were administered to the test substance or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight. Dosing of both sexes began after 6 d of acclimatisation and pre-exposure period (14 d), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy. The first day of dosing of each animal was regarded as Day 0.
Details on mating procedure:
Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male from the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 5 days. A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test substance formulations for concentration and homogeneity was performed using an HPLC-UV method in the Analytical Laboratory of Citoxlab Hungary Ltd. Top, middle and bottom duplicate samples were taken from test substance formulations three times during the study (during the first and last weeks and approximately midway during the treatment), one set to analyse (which could be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement. Acceptance criteria of the concentration analysis were set according to the analytical method validation, expected to be at 100 ± 15 % of the nominal concentration. The measured concentrations of Di-Trimethylolpropane Tetraacrylate evaluated for each test item-dose group varied between 100 % and 102 % of the nominal contents. The RSD was below 10% in each case. No test substance was detected in the control samples. These results were within the acceptable ranges (85% - 115%) and were considered suitable for the study purposes.
Duration of treatment / exposure:
Males were dosed for 28 d (14 d pre-mating and 14 d mating/post-mating) and then euthanized and subjected to necropsy examination. Females were dosed for 14 d pre-mating, during the mating period, through gestation and until the day before the necropsy (13-d post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.
Frequency of treatment:
Males were dosed for 28 d (14 d pre-mating and 14 d mating/post-mating) and then euthanized and subjected to necropsy examination. Females were dosed for 14 d pre-mating, during the mating period, through gestation and until the day before the necropsy (13-d post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
55 male and 55 female Wistar rats were used in the pre-treatment period. At the end of the pre-treatment period, 48 females showing regular oestrus cycles and 48 males were selected and allocated to treatment groups. Animals were assigned to groups before the start of the treatment according to the following Experimental design:
Group designation: Control, low dose, mid dose, high dose
Dose level (mg/kg bw/day): 0, 100, 300, 1000
Concentration (mg/mL): 0, 20, 60, 200
Dose volume (mL/kg bw): 5
Animal numbers: Male 1001-1012 and Female: 1501-1512 (control), Male: 2001-2012 and Female: 2501-2512 (low dose), Male: 3001-3012 and Female: 3501-3512 (mid dose), Male: 4001-4012 and Female: 4501-4512 (high dose)
The control group was treated with the vehicle only (corn oil).

Examinations

Parental animals: Observations and examinations:
Clinical observations
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily*, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment. On gestation day GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat). All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable. More detailed examinations were performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were performed outside the home cage in a standard arena, at similar day times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
*Note: No general clinical observations were made on those days when detailed clinical observations were made.

Body weight measurement
All adult animals were weighed with an accuracy of 1 g for randomisation purposes, then at least weekly during the pre-exposure period, on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 3, 7, 10, 14, 17 and 20 and on post-partal Days PPD0 (within 24 h after parturition), PPD4, 7, 10, 13 and 14 (before termination). The body weight of the female animals measured on gestational Days GD3, 10 and 17 as well as PPD7 and PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

Organ weight measurements
At the time of termination, body weight and the weight of the following organs from all adult animals were determined:
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.

Food consumption measurement
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g weekly (on the days of body weight measurements).

Functional observation battery (FOB) and SMART
Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 26; females on PPD7-9). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength. To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots for the hind limbs was measured. Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs and the appropriate grip support. The results were tabulated with individual and mean data. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated using a scoring system, where score “0” was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal. Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for a 1-h observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.

Haematology and blood clotting times
The following parameters were evaluated in animals selected:
RBC Red Blood Cell (erythrocyte) count, WBC White Blood Cell (leukocyte), Hgb Haemoglobin concentration, (g/dL), MCV Mean Corpuscular (erythrocyte), MCH Mean Corpuscular (erythrocyte), MCHC Mean Corpuscular (erythrocyte), RDW Red Cell (erythrocyte) volume (%), Plt Platelet (thrombocyte) count, MPV Mean Platelet Thrombocyte volume (fL), RETIC % Reticulocyte count (%), Neutrophil (%), Cell differentiation based on myeloperoxidase activity, LY % Lymphocyte (%), MO % Monocyte (%), BA % Basophil (%), EO % Eosinophil (%), LUC % Large Unstained Cells (%), Coagulations (APTT Activated Partial Thromboplastin, PT Prothrombin Time (sec)).
Blood smears were prepared for all selected animals but not examined. The smears were stored/archived at Citoxlab Hungary Ltd.

Clinical chemistry
The following parameters were evaluated in animals selected:
Glucose Blood sugar concentration (mmol/L), T-BIL Total Bilirubin concentration (μmol/L), Urea Urea concentration (mmol/L), Chol. Cholesterol concentration (mmol/L), Creat. Creatinine concentration (μmol/L), Phos. Phosphorus concentration (mmol/L), Na+ Sodium concentration (mmol/L), K+ Potassium concentration (mmol/L), Ca++Calcium concentration (mmol/L), Cl- Chloride concentration (mmol/L), Tot. Prot. Total Protein concentration (g/L), Alb. Albumin concentration (g/L), A/G Alb/glob ration Calculated value, AST/GOT Aspartate Aminotransferase, activity (U/L), ALT/GPT Alanine Aminotransferase activity (U/L), GGT Gamma-Glutamyl transferase activity (U/L), γ-Glutamyl-p-nitroanilide + Glycylglycine, ALKP Alkaline Phosphatase activity (U/L), Bile acids (μmol/L).

Urinalysis
Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 h. The evaluation of the urine samples was performed as indicated below.
LEU / Leukocyte, NIT / Nitrite, pH, PRO / Protein, GLU / Glucose, UBG / Urobilinogen, BIL / Bilirubin, KET / Ketones, BLD / ERY Blood/Erythrocytes, SG / Specific Gravity, SED / Sediment, VOL / Volume, Colour/appearance
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded. Live pups were counted, sexed, weighed individually within 24 h of parturition (PND0) and on PND4 and PND13, with accuracy of 0.01g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded. The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0). Presence of nipples/areolae in male pups were recorded on PND13 (individual records were maintained). All pups were examined externally at weighing on PND4. One male and one female pup (where possible) was allocated randomly for culling for blood sampling on PND4. All pups were culled on PND13. Dams were sacrificed on PND14 after fasting overnight.

Postmortem examinations (parental animals):
Clinical pathology
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. For terminal blood sampling in all selected animals (5 males and 5 females/group), 3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Terminal procedures and macroscopic evaluation
At termination, the adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination. Gross necropsy was performed on all animals, irrespective of the date of death. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs. The number of implantation sites and of corpora lutea were recorded in the females as applicable.

Tissue preservation and microscopic evaluation
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, testes with epididymides in Bouin’s solution, and all other organs in 10% buffered formalin solution. In case microscopic examination, the retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. For the adult animals, a detailed histological examination was performed as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• on the selected list of retained organs in one additional Control female (#1502) that was found dead,
• the pituitary gland of one Low dose male (#2011) and the subcutis from the ventral thoracic area of one Low dose female (#2505), because a macroscopic finding (abnormality) was seen,
• retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups.
Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Postmortem examinations (offspring):
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities. The anaesthetic product was diluted for pups’ euthanasia as required.
Statistics:
SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis)
Reproductive indices:
Data was recorded to provide information on parameters to include:

(A) Parental Males
- Number of pairings
- Number of fertile pairings
- Number of infertile males
- Male mating index
- Male fertility index

(B) Parental Females
- Oestrus cycle data
- Number of pairings
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Number of non-mated females
- Female mating index
- Female fertility index
- Gestation index
- Duration of pregnancy (days)
- Number of corpora lutea / dams
- Number of implantations / dams
- Number of dams with live pups Day 0, 4 and 13
- Pre-implantation mortality
- Intrauterine mortality
- Total mortality (intra and extra uterine mortality)
Offspring viability indices:
- Mean pup body weight (per pup within the group and per litter) on PND0, 4 and 13
- Mean pup body weight gain (per litter) between PND0-4, PND4-13 and PND0-13
- Number of live births per litter, and number of viable pups per litter on postnatal Days 0, 4 and 13
- Survival Index of pups on postnatal Days 0, 4 and 13
- Sex ratio % (on postnatal Days 0, 4 and 13)
- Thyroid hormone analysis
- Anogenital distance, nipple retention
- Necropsy findings (macroscopic)
- Organ weights (thyroid glands, absolute and relative to the body weight)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No test substance related clinical signs were observed during the study. A 1-2 cm width nodule was seen in one Low dose female (#2505) from Day 10 until the end of the observation period. Cold to touch whole body, piloerection and slight discharge from vagina were recorded for one Low dose female (#2511) on Days 39-40. Increased salivation, piloerection and red discharge at the nose were recorded for one Mid dose female (#3504) on Days 35-36. A 1-2 cm width wound was seen at the left cheek of a High dose male (#4012) during Days 13-15, that became a crust on Days 16-18 and then a scar from Day 19 until the end of the observation period. Thin fur at the right cheek was seen in one High dose female (#4503) from Day 22 until Day 32. The following symptoms were recorded for the found dead animal (#1502) before death: Slightly decreased activity, cold to touch whole body, hunched back, piloerection, pale skin at the tail, all paws and both pinnae.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (#1502) in the Control group was found dead on Day 42 (on the day of parturition). The cause of death could not be identified at necropsy. There was no other mortality during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant body weight or body weight gain values in the test substance treated groups (males/females) when compared to the control at any occasion that could be ascribed to the test substance. The measured values were within the range commonly recorded for this strain and age. Occasional statistically significant differences were regarded as incidental and of no toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test substance related differences in the mean daily food consumption in any test substance treated group when compared to the controls. The measured values were within the normal range for this strain and age. Occasional statistically significant differences were regarded as incidental and of no toxicological significance.
Haematological findings:
no effects observed
Description (incidence and severity):
Significantly lower (p<0.05 or p<0.01) prothrombin time (PTT) was recorded in the male and female Low dose animals and in the female High dose group. The platelet count was also significantly lower (p<0.05) in the female Low and Mid dose groups. These differences were considered to be incidental, there was no relationship with dose and all recorded values were within or near the historical control ranges. These differences were considered not reflecting an effect of the test substance. Besides this, there were no other statistically significant values recorded in any of the dose groups compared to the control.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no statistically different results in any of the male groups compared to the control. The following statistically significant differences were recorded in the females: higher (p<0.01) glucose concentration in the High dose group, higher (p<0.05) albumin concentration in the Mid and High dose groups and higher (p<0.05 or p<0.01) albumin/globulin (A/G) ratio in the Mid and High dose groups. The albumin and A/G ratio values were all well within the historical range and did not show a dose response; hence these statistical differences are regarded as unrelated to treatment. For the glucose concentration, 2 of the 5 female individual values were outside the historical range, but in males all values were normal (the mean was slightly below the controls, indicating no effect on glucose). There was no other supporting evidence of any changes in these animals (histopathology, urinalysis, etc.) so the relationship between the 2 high glucose values and treatment was considered to be equivocal. Taking into account the available information, the glucose difference is not considered to be a clear adverse effect. Besides this, there were no other statistically significant values recorded in any of the dose groups compared to the control.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Compared to the control, there were no statistically significant values recorded in any of the dose groups.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted during the assessment of grip strength, foot splay or motor activity. At evaluation of the landing foot splay test, slightly lower values were recorded for males at 1000 mg/kg bw/day (High dose), when compared to control means. The differences attained statistical significance (p<0.05). When evaluating individual data, the values of 4 of 5 males were within the control values and the performance of one male only (#4005) was lower. All values were in the normal range. It should be noted that the control values were relatively high compared with historical data; it is considered that there was no effect of treatment. In females, the landing foot splay values were comparable with the control mean in all dose groups. All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity-level was high, with progressive reduction in activity in each 5- minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals and the Control when evaluating the overall distance travelled (0-60 min, cm). The test item did not increase or decrease the normal locomotor activity.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
(A) Terminal / Parental Generation (Males, Day 28, Females, PND14)
Microscopic Findings: No test substance-related findings were seen at dose levels up to 1000 mg/kg bw/day. Based on the low incidence and/or severity and/or distribution across control and dosed animals the following observations were considered incidental or a common background: slight atrophy of the zona fasciculata and zona reticularis in the right adrenal gland (#1001), slight luminal dilatation of the left horn of the uterine (#4508), minimal focal cyst in one side of the piriform cortex of the brain (#4005), minimal tubular basophilia in the cortex of the right kidney (#4005), slight multifocal casts in the cortex and medulla of both kidneys (#1002), minimal focal casts in the outer stripe of the right kidney (#1003), minimal focal casts in the inner and outer stripes of the right kidney (#4505), minimal tubular multifocal mineralization in the outer stripe of both kidneys (#1504), slight pyelonephritis in the right kidney (#4507), slight focal atrophy in the subcapsular cortex of the left kidney (#1508), minimal multifocal hepatocellular vacuolation in the liver (#1005), slight multifocal haemorrhage in the mandibular lymph node (#4508), minimal multifocal infiltration of the inflammatory cells in the interstitium and dorsolateral lobe of the prostate (#1001), minimal or slight extramedullary haematopoiesis in the spleen (#1503, 1504, 1506, 1511, 4001, 4002, 4005, 4504, 4506, 4507), minimal multifocal tubular degeneration or atrophy of the right testis (#4003), slight focal congestion in the interstitium of the right testis (#1011), slight single cyst in the thymus (#4505), slight multifocal haemorrhage in the thymus (#4501) and slight inflammation of the mucosa and submucosa of the urinary bladder (#4507). In addition, as a macroscopic lesion was seen in the pituitary gland of one Low dose male (#2011) and in the subcutis of one Low dose female (#2505), these organs were macroscopically examined and the following findings were recorded: Multiple cysts in the pars distalis section of the pituitary gland (#2011) and adenoma in the mammary gland (#2505). The microscopic findings correlated with the macroscopic lesions.

(B) Found dead / Parental Generation
Microscopic Findings: The following observations were recorded in the found dead animal (#1502): signs of proestrus in the uterus, slight diffuse congestion of all lobes of the lungs, moderate extramedullary haematopoiesis in the spleen and minimal focal erosion or ulcer in the pylorus of the stomach.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid gland weights and thyroid hormone levels
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the adult male or PND13 pup dose groups. The thyroid gland weights of the PND13 pups were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test substance. The measurement of the thyroid hormone levels in the PND4 pups and adult females was not performed as it was not deemed necessary by the Study Director.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Each female selected for the study showed regular oestrus cycle during the pre-exposure period.
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproductive ability assessment and indices
There were no differences between the control and test substance treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of Di-Trimethylolpropane Tetraacrylate. The mating indices were 100% in all groups. The male and female fertility indices were also 100% in all groups. The gestation index was 92, 92, 100 and 92% in the Control, Low, Mid and High dose groups, respectively. Test substance administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 5 days of pairing (cohabitation) for all females. The mean duration of mating was 2.8, 2.7, 3.0 and 2.5 d in Control, Low, Mid and High dose groups, respectively.

Evaluation of the pre-implantation and gestation periods
There was no effect of treatment noted during the pre-implantation and gestation periods. The mean duration of pregnancy was comparable in the control and test substance treated groups. The number of corpora lutea and implantation sites recorded in the High dose were slightly below the control values, but when taking into account the normal historical range, it was considered that there was no effect of test substance on this parameter. The historical control range for the corpora lutea number is 16.09±5.44 (mean±2SD) and for the implantation site number is 15.73±5.46 (mean±2SD). One female in the High dose group (#4508) had only 2 corpora lutea and 1 implantation site. The low implantation site number and corpora lutea count of this animal caused a significant change in the mean of the High dose group implantation site number and corpora lutea count. In case this animal is excluded from the calculations, the High dose group is not significantly different from the Control group in any of the parameters. The High dose group means without animal #4508 are shown. As the rest of the animals in the High dose group were comparable to the control group in all parameters, the gestation issue of this animal is considered to be incidental and not test substance related.

Details on results (P0)

Kindly refer the attachments in attached background material section of IUCLID for detailed results tables.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: 'see remark'

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of viable pups on PND0, 4 and 13 as well as pup survival indices on PND0, 4 and 13 were comparable to control values in each dose group. Overall, there were no treatment-related effects on pup mortality and on the viability of pups on PND0, 4 and 13. There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups. Occasional statistically significant differences in offspring mortality and viability parameters were regarded as incidental and of no toxicological significance. One female in the High dose group (#4508) did not deliver any pups. This female had also only 2 corpora lutea and 1 implantation site. These factors caused a significant change in the mean of the High dose group pups born parameter. In case this animal is excluded from the calculations, the High dose group is not significantly different from the Control group in any of the parameters. The High dose group means without animal #4508 are shown. Mortality data was similar across all groups and the mean litter body weights were actually slightly above control in the High dose, hence this difference in pups born is considered to be incidental and not test substance related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test substance related differences in the offspring body weights or weight gains in any test item treated group when compared to the controls. The measured values were within the range commonly recorded for this strain and age. When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND0, 4 and 13 showed no toxicologically significant differences compared to controls in the F1 generation.
Sexual maturation:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Terminal / F1 Generation (PND13)
No macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination on PND13.

Found dead / F1 Generation
On PND0, there were 5 Control, 13 Low dose, 3 Mid dose and 1 High dose pups found dead that remained intact (not cannibalized or autolysed). Those were subjected to necropsy with macroscopic examination. No test item-related macroscopic findings were seen. No other pups that died during the lactation period examined had any test substance-related macroscopic findings.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid gland weights and thyroid hormone levels
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the adult male or PND13 pup dose groups. The thyroid gland weights of the PND13 pups were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test substance. The measurement of the thyroid hormone levels in the PND4 pups and adult females was not performed as it was not deemed necessary by the Study Director.

Anogenital distance, nipple retention
There were no statistically significant differences in the test substance treated groups (males/females) when compared to the control at any occasion. In summary, there were no effects on the anogenital distance on PND0 or on the presence of nipples/areolae on PND13 that were ascribed to the test substance.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: 'see remark'

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the no observed adverse effect level (NOAEL) for the test substance is considered to be 1000 mg/kg bw/day for the reproduction as well as developmental toxicity in wistar rats.

Executive summary:

A study was conducted to determine the reproductive/developmental toxicity of the test substance, Di-trimethylolpropane tetraacrylate (100%) using combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, according to OECD 422 Guideline, in compliance with GLP. Male and female Wistar rats (48 animals each sex) were treated daily with test substance in corn oil (100, 300 or 1000 mg/kg bw/day) by oral gavage for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 d in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day PPD13. Parameters measured during the study included signs of morbidity and mortality twice daily, daily general observation or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment, such as functional observation battery (FOB) including measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. The thyroxine (T4) levels in the Day 13 pups and adult males were also assessed. The reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the control and high dose groups. During the treatment period, no test substance related mortality, clinical adverse effects, or changes in neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry or urinalysis parameters was observed. No test substance-related macroscopic findings were recorded in any of the dose groups at necropsy; no microscopic effects were seen at histopathology. There were no test substance-related differences among groups in the weights of organs measured when compared to controls. Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the adult male dose groups. Each female selected for the study showed regular oestrus cycle during the pre-exposure period. Test substance administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 5 d of pairing (cohabitation) for all females. There was no effect of treatment noted during the pre-implantation and gestation periods. The mean duration of pregnancy was comparable in the control and test substance treated groups. The number of viable pups on PND0, 4 and 13 as well as pup survival indices on PND0, 4 and 13 were comparable to control values in each dose group. Overall, there were no treatment-related effects on pup mortality and on the viability of pups on PND0, 4 and 13. There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups. Occasional statistically significant differences in offspring mortality and viability parameters were regarded as incidental and of no toxicological significance. There were no test substance related differences in the offspring body weights or weight gains in any test substance treated group when compared to the controls. The measured values were within the range commonly recorded for this strain and age. When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND0, 4 and 13 showed no toxicologically significant differences compared to controls in the F1 generation. There were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test substance. There were no effects on the anogenital distance on PND0 or on the presence of nipples/areolae on PND13 that were ascribed to the test substance. No test substance related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. There were no adverse effects on the F1 offspring viability, clinical signs, development or at observations following euthanasia. Under the study conditions, the no observed adverse effect level (NOAEL) for the test substance is considered to be 1000 mg/kg bw/day for the reproduction as well as developmental toxicity in wistar rats (Weisz, 2018).