Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: suspension
Details on test material:
- Name of test material (as cited in study report): TMPTA
- Physical state: Clear, colorless liquid
- Analytical purity: 80.2 %
- Lot/batch No.: 41095133SP01
- Expiration date of the lot/batch: 30 September 2015
- Storage condition of test material: At room temperature protected from light

Test animals

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.

Administration / exposure

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.
Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

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).

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.