Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A NOAEL of 300 mg/kg/d for reproductive toxicity was identified from an oral OECD 422 study in rats. Treatment with TMPTA at dose levels of 30, 100 and 300 mg/kg/day revealed no treatment-related effects in relation to reproduction (mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites). Further, there were no morphological findings in the reproductive organs of either sex that could be attributed to TMPTA and spermatogenic staging profiles were normal for all males examined. Based on these results, a NOAEL of 300 mg/kg bw/d for reproductive toxicity and for developmental toxicity could be derived from this screening study.

Additional supportive data: In a 90-day NTP study (NTP, Battelle Columbus Laboratories, 2005) effects of repeated dermal exposure of up to 12 mg/kg TMPTA on reproductive organs, sperm parameters (e.g. sperm count, sperm motility, and abnormalities) were examined in rats and mice. No effect on reproductive parameters was recorded, except for a slight decrease in left testis weight for the high dose rats (12 mg/kg bw), which did not result in reduced sperm production or histopathologic alterations and was thus rated as incidental and not biologically relevant.Thestudy is though, limited by the relatively low maximum tolerated dose of only 12 mg/kg based on skin lesions.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-02 to 2015-04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed in accordance to OECD testing guideline with no deviation to study plan (See attached study report in section 7.5.1 for further information)
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany (This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. WIL Research Europe B.V. has general and reproduction/developmental historical data in this species from the same strain and source).
- Age at study initiation: Approximately 10-12 weeks
- Weight at study initiation: 220 g (female); 320 (male)
- Fasting period before study: no

- Housing:
- Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
- Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
- Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were in dividually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Lactation Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the
dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.

- Diet (ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF®Spezialdiäten GmbH, Soest, Germany).
- Water (ad libitum): Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment.
- Other: Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hitemp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: NA
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400, specific gravity 1.125
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance and vehicle. No correction was made for the purity/composition of the test substance. Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Concentration in vehicle: 6, 20, 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: Polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany).
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Detection of mating was not confirmed for animal no. 59 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose preparations were taken at the Test Facility on two occasions during the treatment period (formulations were prepared and sampled on 04 and 31 March 2015). The samples were dispatched on dry ice to ABL where they were analyzed to assess accuracy of preparation (all groups) and homogeneity (lowest and highest concentration). In addition, stability in vehicle over 5 hours at room temperature protected from light was determined on samples (lowest and highest concentration) taken on 04 March 2015.

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Pups were not dosed directly but were potentially exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Details on study schedule:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Remarks:
Doses / Concentrations:
0, 30, 100, 300 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the outcome of the previous dose range finding study with Wistar Han rats (Project 507633). TMPTA was administered by oral gavage, once daily for 14 consecutive days at dose levels of 100, 300 or 1000 mg/kg/day (5 animals/sex/dose level). Concurrent controls (5 animals/sex) received the vehicle, polyethylene glycol 400, alone. The most relevant effects in this 14-days dose range finding study included lower body weights in males at 1000 mg/kg/day, local toxicity at the stomach in both males and females from 100 mg/kg/day onwards, and slightly higher liver weights in females at 1000 mg/kg/day. Based on these data, the dose levels selected for the current, definitive study (Project 507632) were 30, 100 and 300 mg/kg/day.

- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: none included
- Post-exposure recovery period in satellite groups: NA
Positive control:
None included in the study design
Parental animals: Observations and examinations:
Parental animals (F0):

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
Each male and female was also observed for signs of toxicity immediately following dosing and at approximately 1 hour following dose administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals. Once prior to start of
treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. These clinical observations were at least conducted immediately (0-30 min) after dosing, i.e. on the peak period of anticipated effects after dosing based on results of the dose range finding study (Project 507633). In this pilot study salivation was noted shortly after dosing. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.


BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION:Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study day 28 for males and lactation day 5 for females)
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters were examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLATELET), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count, Percent (RETIC), Absolute (RETIC ABSOLUTE), Mean Platelet Volume (MPV), Red cell distribution width (RDW), Hemoglobin Distribution Width (HDW), Differential leukocyte count: (Percent and absolute): Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Platelet estimatea, Red cell morphology (RBC MORPHOLOGY).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study day 28 for males and lactation day 5 for females)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters were examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Bile acids.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: FOB assessments were recorded for 5 animals/sex/group. The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).

The following tests were performed:
- hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength were recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming,
weaving or movements of the head.

Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
Each litter was examined to determine the following, if practically possible:
- Mortality / Viability: The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter. If possible, defects or cause of death were
evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
Parental animals (F0)

SACRIFICE: All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. Females that delivered were euthanized on lactation day 5 or within 24 hours of total litter loss; the numbers of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post mating day 25 (females with evidence of mating) or post-cohabitation day 25 (females with no evidence of mating).

GROSS PATHOLOGY: Yes
Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulfide solution for detection of early implantation loss. A gross necropsy was conducted on all animals including the female that was found dead during gestation; the numbers of corpora lutea and implantation sites were recorded and recognizable fetuses were examined externally for gross abnormalities. Necropsies included examination of the external surface, all orifices, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

ORGAN WEIGHTS: from F0 animals at the scheduled necropsies, the following organs were weighed: Adrenal glands, Ovaries with oviducts, Brain, Spleen, Epididymides, Testes, Heart, Thymus gland, Kidneys, Thyroids with parathyroids, Liver.

HISTOPATHOLOGY: Yes
At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin: Adrenal glands (2), Lymph node (Axillary, Mesenteric, Mandibular), Aorta, Bone with marrow (sternebrae), Bone marrow smear ( not placed in formalin), Brain (Cerebrum level 1, Cerebrum level 2, Cerebellum with medulla/pons), Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Coagulating glands, Prostate gland, Eyes with optic nerve (2) (in Davidson’s solution), Mandibular salivary glands (2), Gastrointestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary gland, Spinal cord (cervical), Spleen, Testes with epididymides (2) (fixed in Bouin’s solution), Thymus gland, Thyroids [with parathyroids, if present (2)], Heart, Trachea, Kidneys (2), Urinary bladder, Liver (sections of 2 lobes), Uterus with cervix and vagina (in 10% ammonium sulfide solution), Lungs (including bronchi, fixed by inflation with fixative), All gross lesions.

Microscopic examination was performed on all tissues listed above from all animals in the control and 300 mg/kg/day groups. In addition, the liver, stomach, and all gross lesions from all animals at all dosage levels were examined microscopically. All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands). In addition,


Postmortem examinations (offspring):
Pups surviving to planned termination were killed by decapitation on Day 5 of lactation. Pups found dead during the weekend were fixed in identified containers containing 70% ethanol (from Klinipath, Duiven, The Netherlands). All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
- Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnancy) / Total No. of Males (Females) Used for Mating x 100
- Male Fertility Index (%) = No. of Males Siring a Litter / Total No. of Males Used for Mating x 100
- Male Copulation Index (%) = No. of Males Siring a Litter / No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy) x 100
- Female Fertility Index (%) = No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating x 100
- Female Conception Index (%) = No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Confirmed Pregnancy) x 100
Offspring viability indices:
- Mean Live Litter Size = Total No. of Viable Pups on PND 0 / No. of Litters with Viable Pups on PND 0
- Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter) / No. of Litters Per Group x 100
- Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N) / No. of Litters Per Group
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See below
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY:
There were two unscheduled deaths in this study: One control female (no. 46) had to be euthanized on Day 22 post-coitum. Before her death, hunched posture and piloerection were noted. She already had delivered 10 pups (1 dead and 9 alive), and an additional 2 pups (1 dead and 1 alive) were found in her uterus at necropsy. Other necropsy findings included many dark-red foci on the lungs and caecum, and watery-clear fluid in the thoracic cavity. The dark red focus/foci in the caecum correlated to a moderate necrotizing enteritis and was the vehicle alone, there was no relation to treatment with the test substance. One high dose female (no. 79) treated at 300 mg/kg/day was found dead on Day 0 post-coitum (i.e. after 16 days of treatment). When performing vaginal lavage, she was noted with labored respiration and piloerection. Shortly thereafter she was found dead. There were no indications for a bad condition of this female the days before her death. Gross findings at necropsy included irregular surface of the forestomach, many, dark-red foci on the thymus, and uterus containing fluid with its left horn containing reddish fluid. The likely cause of death was a gavage accident as evidenced by the microscopically marked (acute) ulceration in the trachea. Both these deaths were considered incidental and not related to treatment with the test substance.
There were no adverse effects on clinical appearance noted with treatment up to 300 mg/kg/day. Salivation seen after dosing at 30, 100 and 300 mg/kg/day was considered not toxicologically relevant, considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste and/or irritating properties of the test substance rather than a sign of systemic toxicity. One female at 30 mg/kg/day (no. 55) had swelling of the right axillary region during the last week of the study. At necropsy, a tan hard nodule was found which turned out to be an adenoma of the mammary gland. This rare benign neoplasm was considered to be a spontaneous finding with no relation to treatment. Rales were noted for two females at 100 mg/kg/day (nos. 62 and 65), and two males (nos. 33 and 37) and one female (no. 77) at 300 mg/kg/day over 1-7 days during the treatment period. In addition, female no. 62 had slight lethargy, hunched posture, piloerection and a lean appearance for 1-2 days, together with reduced water consumption (subjective appraisal, taken from study daybook) and slight body weight loss on Day 17 post-coitum. Other incidental observations included piloerection or swelling of the throat region noted for one female each at 300 mg/kg/day during 2 and 5 days, respectively. At the limited incidence observed and because these signs were transient, they were not considered to be adverse.

BODY WEIGHT AND WEIGHT GAIN:
No toxicologically or statistically significant changes in body weights and body weight gain were noted.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
NA

FOOD EFFICIENCY:
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted. For males at 300 mg/kg/day, food consumption (absolute and relative) was slightly, but not statistically significantly, lower during the first week of treatment (Days 1-8 pre-mating). No toxicological significance was attributed to this as changes compared to the control group were only slight and occurred transiently.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
NA

OPHTHALMOSCOPIC EXAMINATION:
NA

HAEMATOLOGY:
There were no differences noted in haematological parameters between control and treated rats that were considered to be related to treatment with TMPTA.
For males at 300 mg/kg/day, the percentage of reticulocytes was statistically significantly higher compared to the concurrent control group. This finding was not considered to be toxicologically relevant as all individual values remained within the normal range of biological variation. At the individual level, a relatively high reticulocyte count with a concurrent lower red blood cell count and a high value for red blood cell distribution width (RDW) was found for one control female (no. 48). This finding correlated with a relatively high spleen weight (absolute and relative to body weight) and markedly increased extramedullary hematopoiesis in the spleen at the microscopic level. Since this was a female in the control group, a relation to treatment with the test substance could be excluded.

CLINICAL CHEMISTRY:
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. The following statistically significant changes in clinical biochemistry parameters were noted for treated rats as compared to controls: decreased potassium at 30 mg/kg/day (males), decreased calcium at 30 and 100 mg/kg/day (males), and increased calcium at 300 mg/kg/day (females). No toxicologically relevance was attached to these findings as values remained within the normal range of biological variation, the differences from the control group were not accompanied by relevant changes in other clinical biochemistry parameters and/or corroborative histopathological changes, and/or a dose-related response was absent. At the individual level, relatively high concentrations of urea, bile acids and/or inorganic phosphate were found for two females at 300 mg/kg/day (nos. 72 and 74). In the absence of corroborative histopathological findings, it was not considered to be toxicologically relevant.

URINALYSIS:
NA

NEUROBEHAVIOUR:
Hearing ability, pupillary reflex, static righting reflex and grip strength were not affected by treatment. The slightly lower grip strength of the fore- and hindlegs recorded for females at 300 mg/kg/day was not considered to be toxicologically relevant as changes were relative slight and values remained within the normal range of biological variation. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS:
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. Statistically significant changes comprised lower absolute adrenal weight in males at 30 mg/kg/day, higher relative heart weight in males at 300 mg/kg/day, and higher absolute and relative thymus weight in females at 100 mg/kg/day, when compared to control. These changes were considered to be of no toxicological relevance as they remained within the range considered normal for rats of this age and strain and/or occurred in the absence of a treatment-related distribution. The relatively high ovaries weight for one female at 300 mg/kg/day (no. 75) was caused by a wateryclear cyst (10x15 mm) on her left ovary. This cyst was considered to be a chance finding and not treatment-related. It is seen incidentally in female rats of this age and strain.

GROSS PATHOLOGY:
Test item-related macroscopic findings were present in the non-glandular part of the stomach (i.e. forestomach) in the form of irregular surface (correlating microscopic observation: squamous cell hyperplasia forestomach) in 5/10 males treated at 100 mg/kg/day, and 7/10 males and 7/10 females (including the female that died spontaneously) at 300 mg/kg/day. No macroscopic findings were noted in the forestomach of males and females treated at 30 mg/kg/day, and in females treated at 100 mg/kg/day. Incidental findings among control and treated animals were within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related trend. These necropsy findings were therefore considered to be unrelated to treatment.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS):
No data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS):
No data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
The relatively long mean precoital time recorded at 300 mg/kg/day was caused by one female (no. 71) that was not mated before Day 14. Delayed mating occurs on occasion in this type of study. Since mating of this female was followed by a normal pregnancy and birth of healthy pups, no toxicologically significance was attached to this isolated finding. The mean precoital time for the remaining 9 females in this high dose group was 2.1 days and thus within the normal range.

Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproduction toxicity (mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites) was observed up to the highest dose level tested (300 mg/kg/day).
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
There were no adverse effects on early postnatal pup development (including the number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio) with treatment up to 300 mg/kg/day and clinical signs, body weight and external macroscopic examination of the pups did not reveal treatment-related findings.

The relatively low percentage of male pups in the 30 mg/kg/day group, when compared to the control group, was mainly attributable to female no. 54, who had 3 males out of 15 pups in total. At this incidence and in absence of a treatment-related distribution, it was considered not to be toxicologically relevant.

Mortality:
Postnatal loss was statistically significantly higher at 100 mg/kg/day, when compared to controls. This was caused by the loss of in total 5 pups in three litters on Day 2 (missing: pup 6 in litter 62, pups 2, 3 and 4 in litter 66; spontaneously dead: pup 7 in litter 69). Pups missing were most likely cannibalised. As a consequence, the viability index was statistically significantly lower for this mid dose group. As the mortality incidence was within normal ranges and in the absence of a dose-related response, no toxicological significance was attached to this finding.

Clinical signs:
Incidental clinical symptoms of pups that went missing included absence of milk in the stomach and pale appearance. These were only seen for pups from litter no. 66. Clinical signs for surviving pups included blue staining or blue spot on the neck or abdomen, thickened area on the snout, scabbing of the snout or head, pale appearance, and absence of milk in the stomach. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Body weights:
No toxicologically relevant changes in body weights of pups were observed. A trend towards slightly lower body weights of pups (both sexes) was noted at 300 mg/kg/day. No toxicological relevance was attached to this finding, as changes compared to controls were slight (not reaching statistical significance) and values remained within the available historical range.

Macroscopy:
Incidental macroscopic findings of pups that were found dead included beginning or advanced autolysis, absence of milk in the stomach, tail apex missing and reddish discoloration of the left foreleg. The only macroscopic finding for one surviving pup (litter 80, pup 9) was scabbing of the head. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Dose descriptor:
NOAEL
Remarks:
Development
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Reproductive effects observed:
not specified

Reproductive performence:

There was one couple treated at 30 mg/kg bw/day (male 16 and female 56) that was not pregnant. No abnormalities were seen in the reproductive organs, which could account for their nonpregnancy.

Microscopic examination:

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined using PAS/haematoxylin staining methods.

Conclusions:
TMPTA was evaluated in a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test in accordance with OECD 422. TMPTA was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg/day. No effects were seen and a NOAEL of 300 mg/kg bw/d for reproductive toxicity and for developmental toxicity could be derived from this screening study.
Executive summary:

The reproductive and developmental toxicity of TMPTA was evaluated in a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test in accordance with OECD 422. TMPTA was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during gestation, and at least 4 days of lactation (for 41-55 days).

Treatment with TMPTA at dose levels of 30, 100 and 300 mg/kg/day revealed no treatment-related effects in relation to reproduction (mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites) and developmental toxicity

(gestation index and duration, parturition, maternal care and early postnatal pup development i.e mortality, clinical signs, body weight and macroscopy). Further, there were no morphological findings in the reproductive organs of either sex which could be attributed to TMPTA and spermatogenic staging profiles were normal for all males examined.

Based on these results, a NOAEL of 300 mg/kg bw/d for reproductive toxicity and for developmental toxicity could be derived from this screening study.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
High quality OECD 422 study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
Limited data is available for Trimethylolpropane Triacrlate (TMPTA) from a 90-day dermal study in rats and mice (NTP 2005). In this study no effects on reproductive organs, sperm parameters or estrous cylce were observed. But the study is limited by the relatively low maximum tolerated dose of only 12 mg/kg based on skin lesions.
Additional information

An OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study) using oral gavage in rats has been planned in order to fulfill testing requiremant at Annex X and a testing proposal has been made.

Effects on developmental toxicity

Description of key information

A study was designed to evaluate embryo/foetal and teratogenic effects of TMPTA in rats (Cytec, Hazleton 1983, Serota D. G.). The study was equivalent or similar to OECD Guideline No. 414. TMPTA at a single dose level of 500 mg/kg bw was administered by oral gavage to 22 pregnant rats from Day 6 through 15 of gestation. Control group consisting of 22 pregnant rats received corn oil on a comparable regimen. All rats were observed twice daily for signs of toxicity. Body weights were recorded at gestation Day 0, 6, 9, 12, 15 and 20. All surviving animals were sacrificed on gestation Day 20 for the scheduled Caesarean section. Maternal, ovarian, uterine, litter, and foetal data were evaluated. Compound-related maternal toxicity was observed, and these effects consisted of decreased survival, increased incidence of clinical signs, and an increased incidence of gross pathology findings. Apparent effects on food and water consumption values were observed. Several visceral and/or skeletal anomalies were noted but these are considered to be non-significant developmental variations. In conclusion, TMPTA produced substantial maternal toxicity at a dose level of 500 mg/kg bw/day but did not induce teratogenic or embryotoxic effects in rats.

A NOAEL of 300 mg/kg/d for reproductive and developmental toxicity screening was identified from an oral OECD 422 study in rats (WIL research 2015). Treatment with TMPTA at dose levels of 30, 100 and 300 mg/kg/day revealed no treatment-related effects in relation to reproduction (mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites) and developmental toxicity (gestation index and duration, parturition, maternal care and early postnatal pup development i.e. mortality, clinical signs, body weight and macroscopy).

In a new study, the prenatal developmental toxicity of TMPTA was examined in rabbits according to OECD 414 at doses of 10, 30, 100 and 130 mg/kg/day (Peter, B 2016). Dose levels were based on findings from a range finding study in which mated rabbits were exposed by oral gavage from Day 6 to Day 28 post-coitum, inclusive, at dose levels of 0, 50, 100 and 200 mg/kg bw/day. Clear toxicity was observed at 200 mg/kg bw/day, one female had to be euthanized on Day 16 and another on Day 23 post-coitum due to severe toxicity. Additional toxicologically relevant findings in this group consisted of clinical signs (lethargy, pale and lean appearance, piloerection and/or hunched posture), significant body weight loss and reduced food consumption. No macroscopic abnormalities were noted at necropsy. At 100 mg/kg bw/day, there was only a single female with signs of toxicity. In the first part of the study, eighty-nine mated female New Zealand White rabbits were assigned to four groups. Groups 1, 2 and 3 consisted of 22 females, and Group 4 of 23 females. The test item, TMPTA, was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 10, 30 and 100 mg/kg bw/day (Groups 2, 3 and 4, respectively). Rabbits of the control group received the vehicle, 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, only.

 

In the first part of this study (Groups 1-4), possible treatment-related developmental findings were observed at the high dose of 100 mg/kg bw/day. To investigate this further, it was deemed necessary to add an extra group. To test the feasibility of a higher dose level for this extra group, an extra group of 6 mated females was added to the dose range finding study to test a dose of 150 mg/kg bw/day. Animals were treated and observed under the same conditions as for the previous groups. At 150 mg/kg bw/day, faeces abnormalities, body weight loss, reduced food and/or water consumption were noted. In addition, one female had an early delivery. Based on these results, a dose of 150 mg/kg bw/day was judged too high. Therefore, it was decided to select a dose of 130 mg/kg bw/day for the additional group in the definitive study.

 

For the second part of the study, forty-four mated female New Zealand White rabbits were assigned to two groups (Groups 6 and 7) of 22 animals each. The test item, TMPTA, was administered once daily

by oral gavage from Days 6 to 28 post-coitum at 130 mg/kg bw/day (Group 7). Rabbits of the concurrent control group (Group 6) received the vehicle, 1% aqueous carboxymethyl cellulose with 0.1% Tween-80, only. Accuracy, homogeneity and stability of formulations were demonstrated by analyses. No relevant maternal toxicity was observed up to and including 130 mg/kg bw/day. There was no test-item related mortality following treatment up to and including 130 mg/kg bw/day. Reduced faeces production, varying in duration and severity, was observed in females of both treated and control groups. The incidence was slightly higher at 100 and 130 mg/kg bw/day.

There was a trend towards slightly lower food consumption (absolute and relative to body weight) from Days 13-23 post-coitum in the 100 mg/kg bw/day group and from Days 13-29 post-coitum in the 130 mg/kg bw/ day, which resulted in slightly reduced body weight gains from Days 16-23 post-coitum and Days 16-29 post-coitum, respectively, compared to the concurrent control group. As these changes in food consumption and body weight were relatively slight (not always reaching statistical significance), they were considered as non-adverse. No test-item related macroscopic abnormalities were noted following treatment up to and including 130 mg/kg bw/day.

At 130 mg/kg bw/day, a trend towards slightly lower mean foetal body weights (male, female and combined) was noted when compared to its concurrent control group. Mean combined foetal body weights were 37.8 and 39.3 grams, respectively. This change occurred secondary to the slightly reduced food consumption and slightly lower body weight gains observed for dams in this highest dose group as compared to the concurrent control group. However, as examination of the foetal skeletons did not reveal any signs for growth delay and considering the fact that foetal body weights remained within the available historical range, it was regarded as non-adverse.

At 100 mg/kg/day an increased litter proportion of visceral malformations of the eye, liver oedema and ascites were observed. As the visceral malformations of the eye, liver oedema and ascites represented rare findings just above the available historical data set from WIL Research, the study was extended with a further dose level of 130 mg/kg/day to further evaluate these findings. No such findings were observed at 130 mg/kg/day.

There were no effects on litter size, sex ratio, and no increase in foetal abnormalities (external, visceral and skeletal malformations and variations) was seen following treatment at 130 mg/kg bw/day. No developmental toxicity was observed at 10, 30 and 100 mg/kg bw/day. Based on the results in this prenatal developmental toxicity study the No Observed Adverse Effect Levels (NOAELs) for maternal and developmental toxicity were at least 130 mg/kg bw/day.Data from a previous dose range finding study revealed insufficient tolerance (including an early delivery) at 150 mg/kg bw/day and unscheduled deaths at 200 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 February 2015 - 30 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Storage condition of test material: At room temperature protected from light
Species:
rabbit
Strain:
other: Charles River, Chatillon sur Chalaronne, France
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Age at delivery: 17-19 weeks
- Weight at study initiation (Day 0 post coitum): 2995 to 4574 gram
- Fasting period before study: no
- Housing: Females were individually housed in labelled cages with perforated floors (Ebeco, Germany) and shelters (Ebeco, Germany).
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Italy). In addition, pressed hay (Tecnilab-BMI bv, The Netherlands) and wooden sticks (Swedish aspen wood, The Netherla nds) were provided during the study period.
- Water: Free access to tap-water - Acclimation period: At least 5 days prior to pairing

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24 - Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 27 March 2015 To: 02 May 2016
Route of administration:
oral: gavage
Vehicle:
other: 1% Aqueous carboxymethyl cellulose with 0.1% Tween-80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visuall y acceptable level. Adjustment was made for specific gravity of the test substance. No correction was made for the purity/composition of the test substance.

VEHICLE - Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch. 1% Aqueous carboxymethyl cellulose (carboxymethyl cellulose: BUFA, The Netherlands; water: Elix, Millipore S.A.S., France) with 0.1% Tween-80 (Merck-Schuchardt, Germany). - Amount of vehicle (if gavage): 2 mL/kg bw/day


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The delegated phase was performed by the Principal Investigator for Formulation Analysis (ABL). Samp les for analyses were taken in Study weeks 1 and 4. The samples were dispatched on dry ice to ABL w here they were analyzed to assess accuracy of preparation (all groups) and homogeneity (Group 2, 4 and 7). In addition, stability in vehicle over 5 hours at room temperature protected from light was determined on samples taken on 08 April 2015 (Group 2 and Group 4) and taken on 05 April 2016 (Group 7).
Details on mating procedure:
- Impregnation procedure: cohoused One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was est ablished by visual observation of mating. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
From Days 6 to 28 post-coitum, inclusive
Frequency of treatment:
Once daily for 7 days/week, approximately the same time each day with a maximum of 6 hours differe nce between the earliest and latest dose.
Duration of test:
Until Day 29 post-coitum
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
130 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
No. of animals per sex per dose 22 females/dose in groups 1, 2, 3, 6 and 7 (0, 10, 30, 130 and 0 mg/kg bw/day)

23 females/dose in group 4 (100 mg/kg b/wday). One female from this group died on the first treatment day due to an oral gavage accident. To compensate for her loss, a spare female was mater and added to group 4).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Groups 1-4 (0, 10, 30 and 100 mg/kg bw/day) Dose levels were based on a range finding study in which mated rabbits were exposed by oral gavage from Day 6 to Day 28 post-coitum, inclusive, at dose levels of 0, 50, 100 and 200 mg/kg bw/day. Clear toxicity was observed at 200 mg/kg bw/day. One female had to be euthanized on Day 16 and another on Day 23 post-coitum due to severe toxicity. Additional toxicologically relevant findings in this group co nsisted of clinical signs (lethargy, pale and lean appearance, piloerection and/or hunched posture), signifi cant body weight loss and reduced food consumption. No macroscopic abnormalities were noted at necr opsy. At 100 mg/kg bw/day, there was only a single female with signs of toxicity. These included a calm and lean appearance on Day 27 post-coitum, severely reduced food consumption from Days 13-16 and 23-26 post-coitum, and body weight loss (-11.3% after correction for gravid uterine weight). At necropsy, no macroscopic abnormalities were noted for the 100 mg/kg bw/day group. There was no toxicity at 50 mg/kg bw/day.
There were 2-5-6-5 pregnant females in the control, 50, 100, and 200 mg/kg bw/day groups, respectivel y. Since 2 females had to be euthanized preterm in the high dose group, only 3 litters were available for evaluation in this group. The only finding was a trend towards a lower mean fetal body weight (-17% as compared to the concurrent control group). There were no other developmental findings at either dose level. No external developmental malformations or variations were noted.

Groups 6 and 7 (0 and 130 mg/kg bw/day) Dose levels were based on the results from the additional group added to the dose range finding study. At 150 mg/kg bw/day, faeces abnormalities, body weight loss, reduced food and/or water consumption were noted. In addition, one female had an early delivery. Based on these results, a dose of 150 mg/kg bw/day was judged too high. Therefore, it was decided in consultation with the Sponsor to select a dose of 130 mg/kg bw/day for the additional Group 7.

NB. Please note that for computer technical reasons, the two additional groups had to be planned into the computer as Groups 6 and 7. No Group 5 was included in this study
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
-Time schedule for examination: Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 postcoitum.

WATER CONSUMPTION : Yes
Subjective appraisal was maintained during the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: liver sampling for groups 130 mg/kg bw/day and concurrent control group on Day 29 post-coitum

OTHER: Yes
-Blood sampling for groups 130 mg/kg bw/day and concurrent control group on Day 28 post-coitum (last day of dosing).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter from dose groups 100 and 130 mg/kg bw/day and control groups
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test)
based on a pooled variance estimate was applied for the comparison of the treated groups and the co
ntrol group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal
distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of
viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and
sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations
(external, visceral and skeletal), and each particular external, visceral and skeletal malformation or vari
ation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differe
nces. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used
to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead
fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
For each litter the following calculations were performed:
Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites) / number of corpora lutea) x 100
Post-implantation loss (%) = ((number of implantation sites - number of live fetuses) / number of implantation sites) x 100
Viable fetuses affected/litter (%) = (number of viable fetuses affected/litter / number of viable fetuses/litter) x 100
Historical control data:
Historical control data included for Study: date range 2011-2015
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Description (incidence and severity)
None of the clinical signs seen for surviving animals in this study were considered to be adverse.
Reduced faeces production, varying in duration and severity, was observed in females of both treated and
control groups. The incidence was slightly higher at 100 and 130 mg/kg bw/day. The number of females
with reduced faeces production was 13 and 14 females in the two control groups, 13 females at 10 mg/
kg bw/day, 14 females at 30 mg/kg bw/day, 18 females at 100 mg/kg bw/day and 19 females at 130 mg/
kg bw/day. For females with a more severely reduced faeces production (moderate to severe) also often
reduced intake of food and water was noted.
Incidental findings noted for control and/or treated animals included pale feces, diarrhea, thickened area
of the throat region, broken teeth, a lean or pale appearance, piloerection, a hunched posture, alopecia,
scars, swelling, scabbing or a wound on various body areas.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were 5 unscheduled deaths, though none of these were attributable to treatment related toxicity.
One control female was euthanized on Day 18 post-coitum due to a poor condition. She was noted with
lethargy, pale and lean appearance, piloerection and reduced feces production prior to euthanasia. From
start of treatment (Day 6 post-coitum) she had lost 18% of her weight, which was reflected in the finding
of emaciation at the macroscopic examination. As this was a control female, this death was not treatment
related.
Three females died due to complications during dosing: one female from dose group 30 mg/kg bw/day
was found dead on Day 27 post-coitum, and two females from dose group 100 mg/kg bw/day were found
dead on Days 6 and 22 post-coitum, respectively. Two of these animals had labored respiration during or
immediately after the dosing procedure. When found dead, one female was noted with blood around her
mouth and nose, and had reduced feces production from Day 19 post-coitum onwards. At necropsy, the t
wo females from dose group 100 mg/kg bw/day were found with hemorrhagic/clotted blood and foamy co
ntents in the trachea. They also had grey-white fluid in the lungs and reddish and/or grey-white discolorati
on of the lungs. There were no macroscopic findings seen for animal from dose group 30 mg/kg bw/day.
As one female (100 mg/kg bw/day) died very early in the study (Day 6 post-coitum) she could be
replaced by a reserve animal. However, this latter female was found dead on the morning of Day 21 postcoitum.
Most likely she aborted during the night and died from associated complications. Her cage was
covered with blood, but no uterine contents or fetuses were found. She had 13 fetuses in her uterus who
all were dead. There were no further necropsy findings. This female did not consume any food on Day
20 post-coitum. Throughout treatment, however, she had no clinical signs and her body weights, weight
gain, and food consumption were normal. Taken together, her death was considered to be secondary to
complications during the abortion and was not directly attributable to treatment with the test substance
itself. At this single occurrence it was considered a chance finding and thus not related to treatment with
TMPTA.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 130 mg/kg/day, slightly reduced body weight gains were noted from Days 16-29 post-coitum onwards
compared to the concurrent control group (not statistically significant on Day 29 post-coitum). Body
weight gain corrected for weight of the gravid uterus was slightly lower in this group compared to the con
current controls (-6.8% versus -6.2%; not reaching statistical significance). Because of the slight and nonsignificant
difference, this finding was not considered as adverse.
There were no toxicologically relevant effects on body weights or weight gains with treatment up to and
including 100 mg/kg bw/day.
A trend towards slightly lower body weight gains was noted for females at 100 mg/kg bw/day from Day
16 post-coitum onwards, though the difference from controls was not statistically significant. Females at
30 and 10 mg/kg bw/day also had lower body weight gains from Day 20 post-coitum onwards, though the
differences from controls were slight and not statistically significant. Terminal body weight corrected for
(gravid) uterine weight was unaffected by treatment up to and including 100 mg/kg bw/day.
A slightly, but statistically significantly lower body weight gain was recorded for the 10 mg/kg/day group
as compared to its concurrent control (Group 1) on Day 0 post-coitum. This was by no mean related to
the test item, since treatment did not start before Day 6 post-coitum .
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 130 mg/kg bw/day, there was a trend towards slightly lower food consumption (absolute and relative
to body weight) from Days 13-29 post-coitum onwards compared to the concurrent control group, but not
reaching statistical significance. This finding was considered treatment-related, but not adverse as the
differences from controls were slight (reaching no statistical significance) and transient.
There were no toxicologically relevant effects on food consumption noted with treatment up to 100 mg/kg/
day.
At 100 mg/kg bw/day, there was a trend towards lower food consumption (absolute and relative to body
weight) from Days 13-23 post-coitum. Differences from controls were relatively slight, reaching statistical
significance for relative food consumption from Days 16-20 post-coitum only. In addition, statistically
significantly reduced food consumption was noted at 10 mg/kg bw/day from Days 3-9 post-coitum
(absolute and relative to body weight). As the differences from controls were transient and/or occurred in
the absence of a dose-related trend, these were not considered to be toxicologically relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant macroscopic findings seen at the scheduled necropsy with trea
tment up to and including 130 mg/kg bw/day. One female at this dose level was noted emaciated. However,
when correcting her terminal body weight for gravid uterus weight it was almost the same (- 0.2%) as
at start of treatment (Day 6 post-coitum). Moreover, also one control female in Group 1 was noted
emaciated at necropsy. Taken together, this necropsy finding was not considered to be related to treatment.
Incidental macroscopic findings noted for control and/or treated animals were within the background
range of findings that are encountered among rabbits of this age and strain, were observed for individual
animals only and did not show a dose-related trend. These necropsy findings were therefore considered
to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One 100 mg/kg bw/day treated female was found dead on Day 21 post-coitum most likely due to complications caused by abortion.
Thirteen dead fetuses were found in her uterus.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A significant increase in pre-implantation loss was found in the 130 mg/kg bw/day group compared to its
concurrent control. As treatment is started after implantation, this is in no way related to treatment. In the
same group, the slightly higher post-implantation loss compared to the concurrent control (9.2 vs 3.6%
per litter, respectively) could be contributed to one female in this group with 100% of late resorptions
only. This female had only 3 implantation sites. When excluding her, the post-implantation loss per litter
was within the normal range (i.e. 4.9% per litter).
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
One female in the 130 mg/kg bw/day showed 100% of late resorptions, which is considered to be incide
ntal (within the range of normal biological variation).
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
At 130 mg/kg bw/day, one female had 3 late resorptions
Dead fetuses:
no effects observed
Description (incidence and severity):
One dead fetus was found in Group 1. As this group was treated with the vehicle alone, this finding was
by no means related to treatment with TMPTA.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
There was one non-pregnant female in the first control group and the 30 and 100 mg/kg bw/day groups.
In the second control group (concurrent control for dose group at 130 mg/kg bw/day), 4 females were
found non-pregnant. At 130 mg/kg bw/day, one female had 3 late resorptions only. All remaining females
were pregnant with viable fetuses at Day 29 post-coitum, but one female from the first control group
was euthanized, and one female at 30 mg/kg bw/day and three females at 100 mg/kg bw/day (including
the one with abortion mentioned above) were found dead before scheduled necropsy on Day 29 post-coit
um. Consequently, the number of litters with viable fetuses was 20, 22, 20 and 19 in the first control group
and the 10, 30 and 100 mg/kg bw/day groups, respectively; and 21 and 18 litters in the 130 mg/kg bw/ day
group and its concurrent control, respectively.
Other effects:
not examined
Details on maternal toxic effects:
No relevant maternal toxicity was observed up to and including 130 mg/kg bw/day. There was no test-i
tem related mortality following treatment up to and including 130 mg/kg bw/day.
Reduced faeces production, varying in duration and severity, was observed in females of both treated and
control groups. The incidence was slightly higher at 100 and 130 mg/kg bw/day.
There was a trend towards slightly lower food consumption (absolute and relative to body weight) from
Days 13-23 post-coitum in the 100 mg/kg bw/day group and from Days 13-29 post-coitum in the 130 mg/
kg bw/day, which resulted in slightly reduced body weight gains from Days 16-23 post-coitum and Days
16-29 post-coitum, respectively, compared to the concurrent control group. As these changes in food
consumption and body weight were relatively slight (not always reaching statistical significance), they
were considered as non-adverse.
No test-item related macroscopic abnormalities were noted following treatment up to and including 130
mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 130 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 130 mg/kg bw/day, a trend towards slightly lower mean fetal body weights (male, female and combin
ed) was noted when compared to its concurrent control group. Mean combined fetal body weights were
37.8 and 39.3 grams, respectively. This change, however, did not reach statistical significance and
although low, values remained within the available historical control range .
There were also no toxicologically relevant changes in fetal body weights at the lower dose levels tested
(10, 30 and 100 mg/kg bw/day). Mean fetal body weights in females, but not males were slightly lower
at 100 mg/kg/day as compared to controls (not statistically significant). Mean fetal body weights (both
sexes combined) were 41.0, 40.0, 40.1 and 40.1 grams in the first control, 10, 30 and 100 mg/kg bw/day
group, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 130 mg/kg bw/day, a trend towards slightly lower mean fetal body weights (male, female and combin
ed) was noted when compared to its concurrent control group. Mean combined fetal body weights were
37.8 and 39.3 grams, respectively. This change, however, did not reach statistical significance and
although low, values remained within the available historical control range .
There were also no toxicologically relevant changes in fetal body weights at the lower dose levels tested
(10, 30 and 100 mg/kg bw/day). Mean fetal body weights in females, but not males were slightly lower
at 100 mg/kg/day as compared to controls (not statistically significant). Mean fetal body weights (both
sexes combined) were 41.0, 40.0, 40.1 and 40.1 grams in the first control, 10, 30 and 100 mg/kg bw/day
group, respectively.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Litter sizes were not affected by treatment up to and including 130 mg/kg bw/day. Mean litter sizes were
8.7, 7.3, 8.5 and 8.1 for the first control, 10, 30 and 100 mg/kg bw/day group, respectively. The mean
litter size in the 130 mg/kg bw/day group was significantly lower compared to its concurrent control group
(8.0 versus 9.4 fetuses/litter), but remained within the normal range of biological variation.
One dead fetus was found in the control group. As this group was treated with the vehicle alone, this
finding was by no means related to treatment with TMPTA.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no treatment related effects on the sex ratio between control and treated animals.
Mean sex ratios (% males : % females) were 52:48, 54:46, 52:48 and 49:51 for respectively the first
control, 10 30 and 100 mg/kg bw/day groups, and 48:52 each for the 130 mg/kg bw/day group and its
concurrent control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter sizes were not affected by treatment up to and including 130 mg/kg bw/day. Mean litter sizes were
8.7, 7.3, 8.5 and 8.1 for the first control, 10, 30 and 100 mg/kg bw/day group, respectively. The mean
litter size in the 130 mg/kg bw/day group was significantly lower compared to its concurrent control group
(8.0 versus 9.4 fetuses/litter), but remained within the normal range of biological variation.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
There were no external developmental malformations or variations for fetuses up to and including 130
mg/kg bw/day that survived until planned necropsy.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology up to and including 130 mg/kg bw/day.
The only skeletal malformation seen at 130 mg/kg bw/day was a caudal vertebral anomaly observed in
one fetus. As this was an isolated finding which was also observed in historical controls (3 fetuses in 3
litters), it was considered to be a chance finding.
Fusion between sternebrae occurred in one control, one control from additional control group and two 100
mg/kg bw/day fetuses (litter proportions were 0.4%, 0.5% and 1.8% per litter in these respective dose gr
oups). However, this malformation is the most commonly one observed in historical controls (upper range
value is 2.4% per litter) and was therefore not considered to be toxicologically relevant.
At 100 mg/kg bw/day, sternum with supernumerary ossification site was observed in three fetuses out
of three litters, resulting in a mean litter proportion of 3.8% per litter, while a sternal supernumerary os
sification site was not observed in any of the control fetuses. Although the incidence of 3.8% is above
the upper historical control limit (1.6% per litter), this variation was not observed at 130 mg/kg bw/day.
Furthermore, for a related finding, namely supernumerary skull site, no dose-related increase in the mea
n litter proportion was observed. Additionally, general ossification parameters like unossified sternebra no
s. 5 and/or 6, unossified metacarpals and/or metatarsals, unossified tarsals, unossified hyoid body and/
or arches, and unossified skull bone line were unaffected by treatment. Taken together, the increased
incidence of sternum with supernumerary ossification site in the 100 mg/kg/day group was considered to
be of no toxicological relevance.
Remaining skeletal variations were not considered treatment-related as they occurred infrequently,
occurred at frequencies that were within the range of available historical control data or were observed in
control fetuses only.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to and including
130 mg/kg bw/day.
Treatment at 100 mg/kg bw/day resulted in a higher litter proportion of visceral malformations due to mal
formations of the eye, liver edema and ascites. The total percent per litter with visceral malformations was
1.3%, 0.6%, 1.7% and 4.3% per litter in the control, 10, 30 and 100 mg/kg bw/day group, respectively.
Three fetuses in the 100 mg/kg bw/day group had an eye malformation, while none was noted at lower
dose levels. A small eye occurred in two fetuses out of two litters and cataract was observed in one
fetus. The mean litter proportion for absent and/or small eyes in the high dose group was higher than
the maximum historical control value (1.5 versus 0.5% per litter) and cataract was not seen previously
among actual historical controls. Because small eyes and cataracts are anomalies of embryologically dist
inct tissues, they are not considered developmentally linked findings. It is highly unlikely that these ocular
findings reported at 100 mg/kg bw/day in the definitive study of TMPTA are related to treatment.
In two fetuses of 1 litter in the 100 mg/kg bw/day group unusual malformations was observed. Edema of
all liver lobes and ascites (accumulation of fluid in the abdomen) were noted for both fetuses. Of these
related malformations (portal hypertension causes ascites), only ascites was seen previously in a single
historical control fetus.
To investigate if the above findings were spontaneous in origin, an extra dose group of 130 mg/kg bw/day
was added to the study. The total percent per litter with visceral malformations was 1.5% and 1.2% per
litter in the concurrent control and 130 mg/kg bw/day group, respectively. Among the fetuses of the 130
mg/kg bw/day group there were none with an eye malformation, and no cases of liver edema or ascites
were observed. Therefore, it was considered that the eye and liver/abdomen malformations observed at
100 mg/kg bw/day had occurred by chance and were not related to treatment.
Any remaining visceral malformations and variations were not considered treatment related as they
occurred infrequently, did not follow a dose-related trend, occurred at frequencies that were within the
range of available historical control data or were observed in control fetuses only.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
At 130 mg/kg bw/day, a trend towards slightly lower mean fetal body weights (male, female and combined)
was noted when compared to its concurrent control group. Mean combined fetal body weights were
37.8 and 39.3 grams, respectively. This change occurred secondary to the slightly reduced food consumption
and slightly lower body weight gains observed for dams in this highest dose group as compared
to the concurrent control group. However, as examination of the fetal skeletons did not reveal any signs
for growth delay and considering the fact that fetal body weights remained within the available historical
range, it was regarded as non-adverse.
There were no effects on litter size, sex ratio, and no increase in fetal abnormalities (external, visceral and
skeletal malformations and variations) was seen following treatment at 130 mg/kg bw/day.
No developmental toxicity was observed at 10, 30 and 100 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 130 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
130 mg/kg bw/day (actual dose received)
Conclusions:
Based on the results in this prenatal developmental toxicity study the No Observed Adverse Effect Levels
(NOAELs) for maternal and developmental toxicity were at least 130 mg/kg bw/day. Data from a previous
dose range finding study revealed insufficient tolerance (including an early delivery) at 150 mg/kg bw/day
and unscheduled deaths at 200 mg/kg bw/day.
Executive summary:

TMPTA was investigated for prenatal developmental toxicity in rabbits in an study performed according to

OECD 414 guideline and in accordance with GLP principles. Dose levels were based on a range finding

study in which mated rabbits were exposed by oral gavage from Day 6 to Day 28 post-coitum, inclusive, at

dose levels of 0, 50, 100 and 200 mg/kg bw/day. Clear toxicity was observed at 200 mg/kg bw/day, while

at 100 mg/kg bw/day, there was only a single female with signs of toxicity. In the first part of the study,

eighty-nine mated female New Zealand White rabbits were assigned to four groups. Groups 1, 2 and 3

consisted of 22 females, and Group 4 of 23 females. The test item, TMPTA, was administered once daily

by oral gavage from Days 6 to 28 post-coitum at doses of 10, 30 and 100 mg/kg bw/day (Groups 2, 3 and 4,

respectively). Rabbits of the control group received the vehicle, 1% aqueous carboxymethyl cellulosewith

0.1% Tween-80, only.

In the first part of this study (Groups 1-4), possible treatment-related developmental findings were observed

at the high dose of 100 mg/kg bw/day. To investigate this further, it was deemed necessary to add an extra

group. To test the feasibility of a higher dose level for this extra group, an extra group of 6 mated females

was added to the dose range finding study to test a dose of 150 mg/kg bw/day. Animals were treated and

observed under the same conditions as for the previous groups. At 150 mg/kg bw/day, faeces abnormalities,

body weight loss, reduced food and/or water consumption were noted. In addition, one female had an early

delivery. Based on these results,a dose of 150 mg/kg bw/day was judged too high. Therefore, it was decided

to select a dose of 130 mg/kg bw/day for the additional group in the definitive study.

For the second part of the study, forty-four mated female New Zealand White rabbits were assigned to

two groups (Groups 6 and 7) of 22 animals each. The test item, TMPTA, was administered once daily

by oral gavage from Days 6 to 28 post-coitum at 130 mg/kg bw/day (Group 7). Rabbits of the concurrent

control group (Group 6) received the vehicle, 1% aqueous carboxymethyl cellulosewith 0.1% Tween-80,

only. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

No relevant maternal toxicity was observed up to and including 130 mg/kg bw/day. There was no test-item

related mortality following treatment up to and including 130 mg/kg bw/day. Reduced faeces production,

varying in duration and severity, was observed in females of both treated and control groups. The incidence

was slightly higher at 100 and 130 mg/kg bw/day.

There was a trend towards slightly lower food consumption (absolute and relative to body weight) from Days

13-23 post-coitum in the 100 mg/kg bw/day group and from Days 13-29 post-coitum in the 130 mg/kg bw/

day, which resulted in slightly reduced body weight gains from Days 16-23 post-coitum and Days 16-29

post-coitum, respectively, compared to the concurrent control group. As these changes in food consumption

and body weight were relatively slight (not always reaching statistical significance), they were considered

as non-adverse. No test-item related macroscopic abnormalities were noted following treatment up to and

including 130 mg/kg bw/day.

At 130 mg/kg bw/day, a trend towards slightly lower mean fetal body weights (male, female and combined)

was noted when compared to its concurrent control group. Mean combined fetal body weights were 37.8 and

39.3 grams, respectively. This change occurred secondary to the slightly reduced food consumption and

slightly lower body weight gains observed for dams in this highest dose group as compared to the concurrent

control group. However, as examination of the fetal skeletons did not reveal any signs for growth delay and

considering the fact that fetal body weights remained within the available historical range, it was regarded as

non-adverse. There were no effects on litter size, sex ratio, and no increase in fetal abnormalities (external,

visceral and skeletal malformations and variations) was seen following treatment at 130 mg/kg bw/day. No

developmental toxicity was observed at 10, 30 and 100 mg/kg bw/day.

Based on the results in this prenatal developmental toxicity study the No Observed Adverse Effect Levels

(NOAELs) for maternal and developmental toxicity were at least 130 mg/kg bw/day. Data from a previous

dose range finding study revealed insufficient tolerance (including an early delivery) at 150 mg/kg bw/day

and unscheduled deaths at 200 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
130 mg/kg bw/day
Species:
rabbit
Quality of whole database:
High qulality OECD 414 study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

An OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study) using oral gavage in rats has been planned in order to fulfill testing requiremant at Annex X and a testing proposal has been made.

Justification for classification or non-classification

The available data including the recent OECD 422 and OECD 414 studies indicate a lack of adverse effects on fertility and development. Thus, TMPTA is not to be classified for toxicity to reproduction.

Labelling reproductive toxicant:

CLP: no labelling