2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Oral administration of Di-Trimethylolpropane at dose levels up to and including 1000 mg/kg/day was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters, spleen cell immunophenotyping or pathology on either the F0 or F1 generation. In addition the reproductive performance and fertility of the F0 animals and the survival/development of the F1 offspring up to weaning were unaffected by treatment at all dose levels. Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 2019 to 10 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: Preliminary Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

Adopted 25 June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals: 10 weeks.
- Basis for dose level selection: Based on results derived from a previously performed 90 day toxicity study, a combined repeated dose toxicity screening study and a deveopmental toxicity study
- Exclusion of extension of Cohort 1B: No information indicate the genotoxicity, endocrine disruption related effect of this substance. No information indicate the substance leads to significant exposure or specific metabolism pattern that would require extended exposure. Therefore, extension of cohort 1B to include the F2 generation was not performed.
- Exclusion of developmental neurotoxicity Cohorts 2A/2B and Cohort 3: No indication of the developmental neurotoxicity or immunotoxicity of this substance was seen from existing information. Cohort 2A/2B and cohort 3 are considered not needed at this moment.

- Route of administration
Based on the information provided in the technical dossier and/or in the chemical safety report, ECHA agreed that the oral route - which is the preferred one as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 4.1, October 2015) Chapter R.7a, section R.7.5.4.3 - was the most appropriate route of administration. More specifically, even though the information indicated that human exposure to the registered substance by the inhalation route is likely, the exposure concentrations reported in the chemical safety report for the inhalation route are low. Hence, the test was performed by the oral route.
- Other considerations
The Sprague Dawley rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for reproductive toxicity testing. The total number of animals used in this study was considered to be the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier)l: Perstorp Holding AB (Sponsor)
Lot/Batch number: 190100256 (exp. 06 Feb 2022)
- Purity: 99.2%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15 to 25°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Homogeneity and stability was confirmed in corn oil formulations at nominal concentrations of 5 mg/mL and 250 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for 5 days and refrigerated storage (2 to 8ºC) for up to 15 days.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Formulation rfequency: weekly
The test item formulations were ‘gritty’ consistency and the following procedure was implemented as a precautionary measure to reduce the risk of any potential irritation to the mouth and/or oesophagus during gavage administration:
-The dose was drawn up via the catheter and the external surface of the catheter wiped on paper tissue.
-The catheter surface was rinsed in a container of tap water (container 1) and wiped dry on clean paper tissue.
-Then the outside of the catheter was rinsed further in a second container of tap water (container 2) prior to intubation.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley SD rat is the species and strain of choice because there is ample experience and background data on this species and strain. It is also the species and strain used in other toxicity studies of this substance (ex. 90 days repeated dose study) which provide better results-comparison between studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number of animals: 27 litters of four male siblings and 27 litters of four female siblings; no male/female sibling relationships
- Source: Charles River (UK) Ltd. Kent, UK
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: (F0) 28-34 days, (F1) 21 days selection; 28 days allocation/formal start
- Weight at study initiation: (F0) Males: 72-132 g; Females: 52-93 g
- Fasting period before study: none
- Housing: cages compromised of polycarbonate body with stainless steel mesh lid; solid bottom cages used except during pairing when grid bottom cages were used; softwood based bark-free fiber bedding; number of animals per cage according to standards
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ad libitum SDS VRF1 Certified pelleted diet, no added antibiotic or other chemotherapeutic or prophylactic agent (restricted diet overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).
- Water (e.g. ad libitum): potable water, ad libitum (except during urine collection)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark

In-Life Dates:
16 Oct 2019 to 5 May 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 2 to 8ºC, or transferred immediately to the animal unit, and dispensed daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water):TG recomends "where possible, the use of an aqueous solution/suspension is considered first,followed by consideration of a solution/suspension in oil". Corn oil was deemed most appropriate for species selection and substance properties by the Sponsor.
- Concentration in vehicle (nominal): 0, 25, 75, 250 mg/ml (150mg/mL instead of 250 mg/mL day 21 of age to nominal day 35 of age)
- Amount of vehicle (if gavage): 4 ml/kg

Details on mating procedure:

- M/F ratio per cage: 1/1(sibling pairing was not permitted)
- Length of cohabitation: max. 2 weeks (separation at detection of mating)
- Proof of pregnancy: The day of detection of an ejected copulatory plug in situ and/or of sperm in the lavage was designated GD 0.
- After successful mating each pregnant female was caged: Mated females were transferred to individual solid bottomed cages. From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analyzed in accordance with the validated Covance Analytical Procedure (DFA/M/062/19). The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 25 μg/mL to 250 μg/mL.The formulations for Week 1 (F0 generation), Week 1 (F1 generation) and Last Week of F1 generation were sampled. For all groups, 4 × 1 mL (accurately weighed) was sampled from the middle of the formulation by Pharmacy personnel. Two samples from all groups were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. The mean concentration were within 10% of the nominal concentration, confirming the accuracy of formulation, with the exception of one formulation (Group 4 in the last week of the F1 generation) that was 10.8% of the nominal concentration. The difference from mean remained within 3%, confirming precise analysis, with the exception of Group 4, Last Week of F1 generation which was ±7.37%. The results for the Group 4 Last Week of F1 generation was not investigated, however the procedural recovery samples prepared at the same concentration as these samples was 99.9% of nominal concentration, confirming accur cy of dilution.

The original analysis for Week 1 (F0 generation) was diluted to below the quantifiable concentration range in error. The samples were rediluted from extracts to bring them within the calibration range but the system suitability failed on that occasion. Therefore, the original results are reported. The individual procedural recovery prepared at the concentration of the samples was 103.3% of nominal concentration, confirming that the calculated concentration of this group is acceptable.

Stability analyses performed previously demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

The F0 animals were dosed for 10 weeks before pairing until termination after litters are weaned. The F1 animals were dosed from PND 21 (weaning) up to at least PND 90 (Cohort 1A 13 weeks, Cohort 2A 14 weeks)

Frequency of treatment:

Daily, approx. the same time each day

Details on study schedule:

- Age at mating of the mated animals in the study (F0): 14-15 weeks (after 10 weeks dosing)
-The F0 animals were dosed for 10 weeks before pairing until termination after litters are weaned. The F1 animals were dosed from PND 21 (weaning) up to at least PND 90 (Cohort 1A 13 weeks, Cohort 2A 14 weeks)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

F1: Day 21 of age to nominal Day 35 of age - dose reduced to the maximum practicable for the dosing equipment required for weanling animals (600 mg/kg/day)

No. of animals per sex per dose:

25 males + 25 females (F0), 20 males + 20 females (F1 cohort 1A), 20 males + 20 females (F1 cohort 1B)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on results derived from a previously performed 90 day toxicity study, a combined repeated dose toxicity screening study, a deveopmental toxicity study, and a preliminary reproductive study. All dose levels of 300, 600 and 1000 mg/kg/day were generally well tolerated with minor non-adverse effects of treatment primarily limited to F0 male bodyweight, F0 female lactation body weight, F1 offspring body weight and F1 selected bodyweight, and a slight effect on F0 female food consumption prior to pairing. Low and intermediate dose levels of 100 and 300 mg/kg/day were selected to provide an approximate 3-fold dose interval. Since 600 mg/kg/day (150 mg/mL administered at 4 ml/kg) was the maximum practicable dose level for weanling animals, selected F1 animals received this reduced dose level from weaning up to approximately Day 35 of age.

- Fasting period before blood sampling for clinical biochemistry:
yes

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
Physical examination Once each week
After mating of F0 females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.
Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: :
F0 males Day that treatment commenced.
Each week.
Before necropsy.

F0 females Day that treatment commenced.
Each week until mating detected.
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 post partum.
Before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males and females Weekly, from the day that treatment commenced until paired formating.
For females after mating food consumption was performed to match the body weight recording:
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18-20 after mating
Days 1-3, 4-6, 7-13, and 14-20 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

MORTALITY:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No (ad libitum)

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
- Dry smears: For 15 days before pairing, using cotton swabs.

- Wet smears: After pairing until evidence of mating confirmed.For four days before scheduled termination (nominally Days 25 to 28 post partum). Females showing no evidence of mating - following completion of the pairing period females were separated from the male and vaginal smearing continued for up to five days or until the first estrus smear is seen. If a female shows an estrus smear during this period, she was killed as soon as practically possible and subject to macroscopic examination.

Sperm parameters (parental animals):

Motility:
A sample of sperm was expressed from the left vas deferens into prewarmed (target 37oC) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 μm depth cannula by capillary action and, where possible, at least 200 sperm per animal analysed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyser (CASA). F0 Group 2 Male 36 unable to assess 200 sperm at motility analysis.

Sperm morphology: Groups 1 and 4
A 200 L aliquot of the sperm/medium mixture (described above) was diluted with 800 L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible.

Sperm morphology: Groups 2 and 3
Fixed samples retained for possible future assessment.

Sperm count: Groups 1 and 4
The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenised for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3
Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4
After removal of the tunica, the left testis of each male was then homogenised for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenisation-resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3
Samples frozen for possible future assessment.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes , litters culled to 10 (where possible 5 males and 5 females), When the total number of pups in a litter on PND 4 was ≤ 10 pups, no litter size adjustment occurred.


PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: Clinical observations, body weight, food consumption and macropathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive system and the health (including thyroid hormone analysis), growth, development and function of the offspring. At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation, urinalysis, reproductive organ integrity and function.

The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on LD 0. The live pups were counted, sexed, weighed individually on PND 1 and examined daily for the presence of milk in the stomach and for any externally visible abnormalities daily up to and including PND 4. Additionally, on Day 1 of age, all offspring received a qualitative assessmentof body temperature, state of activity and reaction to handling and the ano-genital distance was mesured.

After being sexed dircetly before and after culling (PND 4), the pups were not sexed again until day 21 of age. From PND 5, the total live pups were counted daily and examined for ill-health, evidence of reaction to treatment again, and weighed on PND 7, 14 and 21. On day 13, male offsprings nipple/aerolae were counted.

- Selection and weaning of F1 animals for Cohort 1A and 1B
Two males and two females were selected from each selected litter and one male and one female from each litter, where available, were allocated to each of the two cohorts. If more were required, up to three males and three females were selected from each selected litter. Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on a nominal Day 28 of age (28 +/-2 days of age for selected F1 animals). Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

- Assessment of F1 Sexual Maturation (Cohorts 1A and 1B)
Commencing at PND 28, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, along with the body weight on that day. For Cohort 1A: a wet smear was taken daily from the day of vaginal opening until first estrus was detected. Commencing at PND 38, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, along with the body weight on that day.

In-life procedures. observations and measurements F1 (Cohorts 1A and 1B)
- Mortality/Moribundity Checks
Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment. A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

- Detailed Clinical Observations
Animals were subjected to detailed clinical observations weekly from weaning on PND 21, starting on a suitable day within one week of weaning of the majority of litters. The animals were removed from the cage for examination. The examinations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were also examined. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as
appropriate.

- Pre and Postdose Observations
Detailed observations were performed to establish and confirm a pattern of signs in
association with dosing according to the following schedule:
Week 1 - Daily.
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onward - once each week.
Detailed observations were recorded at the following times in relation to dose administration:
- Prior to dosing
- One to two hours after completion of dosing
- As late as possible in the working day

- Body weights
Recorded on Days 1, 4, 7, 14 and 21 of age; Selected F1 generation: Days 23, 25, 27* and 29* of age. (* Only applicable before formal commencement of the F1 generation at nominal 4 Weeks of age (Day 28 of age ± 2 days)). Plus before necropsy.


- Food consumption
From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.

- Oestrus Cycle Monitoring
Sexual maturation was assessed by daily examination from Day 28 of age until vaginal opening occurred. Body weight was recorded on the day of vaginal opening.
For Cohort 1A: a wet smear was taken daily from the day of vaginal opening until first estrus was detected.

- Haematology (Cohort 1A; 10 rats/sex/group):
At day of necropsy
Analysed parameters: Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute), Activated partial thromboplastin time, Fibrinogen and prothrombin time

- Clinical Chemistry (Cohort 1A; 10 rats/sex/group):
At day of necropsy
Analysed parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine kinase, Total bilirubin, Urea, Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides
Sodium, Potassium, Chloride, Sample quality

- Urinalysis (Cohort 1A; 10 rats/sex/group):
Last week of dosing
Analysed parameters: Colour, Appearance/Clarity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Blood, sodum, potassium, and chloride. microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
-Epithelial cells (Epi)
-Leucocytes (WBC)
- Erythrocytes (RBC)
- Casts
- Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals


- Thyroid stimulating hormone, TSH and T4 (Conditions, blood sample site, and anaesthetic caried based on age)
F1 - culled offspring Ten litters per group - pooled litter sample on Day 4 of age.* Plus ten male and ten female animals per group on Day 22 of age from as many litters as possible)
F1 adults- Cohort 1A - Ten male and 14# female animals per group at termination

Postmortem examinations (parental animals):

SACRIFICE
F0 animals:
- Male animals: After weaning of the F1 animals, after conifmration that no further mating was required
- Maternal animals: Day 28 post partum
- Females failing to mate: Following completion of pairing females terminated after showing an estrus smear
- Method: Carbon dioxide asphyxiation with subsequent exsanguination

GROSS NECROPSY
- F0
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues

ORGAN WEIGHTS
F0:
adrenals, brain, L+R epididymides, heart (including auricular and ventricular regions), kidneys, liver (section from two lobes), optic nerves, pituitary, prostate- dorsolateral and ventral combined, seminal vesciles (with coagulating gland), spleen, L+ R testes, thymus, thyroid with parathyroids (weighed after partial fixation), uterus with cervix and oviducts. Paired organs were weighed together. Organ to body weight percentages (using the terminal body weight) were calculated.

MICROSCOPIC EVALUATION
F0:
abnormalities, adrenals, brain (cerebellum, cerebrum, midbrain), cecum, colon, duodenum, epididymides, esophagus, eyes, femurs, heart, ileum, jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), optic nerves, ovarie, pancreas, pituitary, prostate (dorsolateral and ventral combined), rectum, scieatic nerve, seminal vesicles, skeletal muscles, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervial thoracic and lumbar levels), spleen, sternum, stomach, testes, thymus, throid with parathyroids, trachea, urinary bladder, uterus with cervix and oviducts, vagina, and vas deferens

Postmortem examinations (offspring):

SACRIFICE
F1 animals:
Unselected offspring On Day 4 and Day 22 of age.
Cohort 1A animals At approximately 13 weeks of age.
Cohort 1B animals At approximately 14 weeks of age.
- Method:
F1 adults: Carbon dioxide asphyxiation with subsequent exsanguination.
F1 offspring 14 days and older: Carbon dioxide asphyxiation with subsequent exsanguination
F1 offspring less than 14 days of age: Subcutaneous injection of sodium pentobarbitone.

GROSS NECROPSY
F1 animals:
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues

ORGAN WEIGHTS
- F1 Cohort 1A
adrenals, brain, L+R epididymides, heart (including auricular and ventricular regions), kidneys, liver (section from two lobes), lymph nodes (mesenteric, left axillary), oovaries (L+R), pituitary, prostate- dorsolateral and ventral combined, seminal vesciles (with coagulating gland), spleen, L+ R testes, thymus, thyroid with parathyroids (weighed after partial fixation), uterus with cervix and oviducts. Paired organs were weighed together. Organ to body weight percentages (using the terminal body weight) were calculated.
- F1 Cohort 1B
epididymides, ovaries (L+R), pituitary, prostate (dorsolateral and ventral combined), seminal vesciles (with coagulation gland), testes, uterus with cervix and oviducts
- Unselected F1 offspring on Day 22 of age
brain (cerebellum, cerebrum, midbrain), spleen, thymus


MICROSCOPIC EVALUATION
- F1 Cohort 1A
abnormalities, adrenals, brain (cerebellum, cerebrum, midbrain), cecum, colon, duodenum, epididymides, esophagus, eyes, femurs, heart, ileum, jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), optic nerves, ovarie, pancreas, pituitary, prostate (dorsolateral and ventral combined), rectum, scieatic nerve, seminal vesicles, skeletal muscles, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervial thoracic and lumbar levels), spleen, sternum, stomach, testes, thymus, throid with parathyroids, trachea, urinary bladder, uterus with cervix and oviducts, vagina, and vas deferens

- F1 Cohort 1B
Abnormalities

Statistics:

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population. The following data types were analyzed at each timepoint separately:
-Body weight, using absolute weights and gains over appropriate study periods
-Food consumption, over appropriate study periods
-Hematology
-Blood chemistry
-Urinalysis
-Estrous cycles
-Pre-coital interval
-Gestation length and gestation index
-Stage of estrous cycle at termination
-Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index), for before cull study periods
-Ano-genital distance, adjusted for Day 1 offspring body weight
-Sexual maturation, age and body weight at completion
-Organ weights, absolute and relative to body weight
-Corpora lutea and ovarian primordial follicle count
The following comparisons were performed:
-Group 1 vs 2, 3 and 4
-Group 1 vs 4 for ovarian follicle counts and corpora lutea data

Significant differences between the groups compared were expressed at the 5% (p<0.05) or1% (p<0.01) level.

Parametric/Non-Parametric
Tests include Bartlett's test, Willams' test, t-tests, Dunnett's tests, Kruskal-Wallis' tests, Wilcoxon rank sum tests, Cochran-Armitage tests, Chi-square tests, one- and two-tailed linear-by-linear tests

Reproductive indices:

Post implantation survival, live birth, viability, and lactation indices were calculate for F0.

Post implantation survival index (%) = (Total number of offspring born)/(Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.


Live birth index (%) = (Numer of offspring on Day 1 after littering)/(Total number of offspring born) x 100

Offspring viability indices:

Viability index (%) = (Number of live offspring on Day 4 before culling)/(Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering)/(Number of live offspring on Day 4 (after culling)) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

The general condition of F0 adult animals was unaffected by treatment. There were no signs at either routine physical examination or in association with dose administration that could be attributed to Di-Trimethylolpropane

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A total of ten animals were either found dead or were killed for reasons of animal welfare. Of these, five animals had macroscopic findings that were consistent with dosing trauma and as such these deaths were unrelated to administration of Di-Trimethylolpropane (Group 3 male no.53, Group 2 females no. 228 and 229, Group 3 female no. 271 and Group 4 female no. 276). Three Control animals also died prematurely. The remaining two decedent animals (Group 2 male nos 29 and 41) either died or were killed prematurely following convulsions/seizures. No significant macroscopic or microscopic finding were observed that explained the cause of death in these two animals.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Overall the bodyweight gain for males during treatment and for females before pairing and during gestation was unaffected by administration of Di-Trimethylolpropane. On Day 1 of lactation the mean body weight for females receiving 1000 mg/kg/day was slightly but statistically significantly low at approximately 95 % of Controls (p<0.05); however the overall body weight gain for females at this dose level was significantly high when compared with Controls (p<0.01). Body weight gain during lactation for females receiving 100 or 300 mg/kg/day was similar to Controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

From Week 6 of treatment up to pairing males receiving 1000 mg/kg/day showed food consumption values that were slightly but significantly high when compared with Controls (p<0.05 - Week 6; p<0.01 - Weeks 7 to 10); food consumption for males receiving 100 or 300 mg/kg/day was similar to Controls throughout the pre-pairing treatment period. Food consumption for females before pairing, during gestation and lactation was unaffected by administration of Di-Trimethylolpropane.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological examination of males and females at scheduled termination did not reveal any changes that could be attributed to Di-Trimethylolpropane; differences were either minor, lacked a dose response or were inconsistent between the sexes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Females at 1000 mg/kg/day had high plasma phosphorous levels when compared with Controls (p<0.01). Other differences in the blood chemistry at scheduled termination were minor, lacked a dose response or were inconsistent between the sexes.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

The individual TSH concentrations in each group, at all-time points and within sexes, showed high variation. There were no observed treatment related effects on serum TSH concentrations in F0 adult animals or in the F1 offspring. There was a suggestion of low TSH levels for the F1 adult animals that received Di-Trimethylolpropane; however, as stated above, due to the high variation it cannot be stated that this was due to the test article or biological variation.

Mean T4 concentrations for F0 adult animals, F1 offspring and selected F1 adult animals were similar to Controls, showing no effects of treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological examination of F0 animals at scheduled termination did not reveal any findings that could be related to administration of Di-Trimethylolpropane up to and including 1000 mg/kg/day. Ten control females, five females treated with Di-Trimethylolpropane at 100 mg/kg/day, two females treated with Di-Trimethylolpropane at 300 mg/kg/day and eight females treated with Di-Trimethylolpropane at 1000 mg/kg/day exhibited minimal to marked mineralization in the aorta, other blood vessels at various locations, kidneys and/or stomach. Based on its anatomical distribution and the absence of necrosis, these histopathological findings were consistent with metastatic mineralization/calcification, likely due to pseudohyperparathyroidism related with pregnancy and lactation. Presence in both females from control and treated groups, metastatic mineralization is considered very unlikely to have compromised evaluation or interpretation of any test item related effects.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Pre-coital interval: The majority of animals mated within four days of pairing and the distribution of outliers between the groups showed no relationship to treatment.

Gestation Length and Gustation Index: The duration of gestation was unaffected by treatment with animals littering within the expected range of 22 to 23.5 days. The gestation index was slightly low at 1000 mg/kg/day but this was solely due to a single female (no.276) that was killed for welfare reasons following dosing trauma during gestation and as such the gestation index is deemed unaffected by treatment.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no changes in the estrous cycles that could be attributed to Di-Trimethylolpropane.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no adverse effects on sperm motility, testicular spermatid numbers, cauda epididymal sperm numbers or morphology following treatment with Di-Trimethylolpropane.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility were unaffected by administration of Di-Trimethylolpropane.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The general condition of selected F1 animals was unaffected by treatment. There were no signs at routine physical examination that could be attributed to treatment and no signs were observed in association with dose administration.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Four animals from Cohort 1A died prior to scheduled termination. For the two Controls animals (nos. 404 and 601) macroscopic examination revealed dosing trauma as the cause of death. Two males receiving 300 mg/kg/day (nos. 456 and 458) were found dead on Days 19 and 30 respectively. Terminal signs were limited to no. 456 which was observed with abnormal breathing prior to death however macroscopic examination did not reveal any abnormality. The lungs for male No. 456 showed slight multifocal lymphocytic and mononuclear cell infiltrate in a perivascular and peribronchial distribution; the lung lesions were considered to be the major factor contributing to death. For male No. 458 there were no significant microscopic findings to explain the cause of death. Most tissues examined were considered autolytic and readable. The major factor contributing to death was undetermined.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight of selected F1 animals on Day 21 of age and at formal start of the F1 generation on nominal Day 28±2 of age were unaffected by administration of Di-Trimethylolpropane and subsequent gain up to scheduled termination showed no adverse effects of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for both male and female animals did not show any adverse effects of treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological examination of males and females at scheduled termination did not reveal any changes that could be attributed to Di-Trimethylolpropane; differences were either minor, lacked a dose response or were inconsistent between the sexes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A:
Females receiving Di-Trimethylolpropane had high plasma potassium levels when compared with Controls (p<0.01); a dose response was not apparent. Other differences in the blood chemistry at scheduled termination were minor, lacked a dose response or were inconsistent between the sexes.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:
Males and females receiving 1000 mg/kg/day and females receiving 300 mg/kg/day showed low urinary pH and high specific gravity when compared with Controls; specific gravity for females at 100 mg/kg/day was also high. No other differences were apparent.

Sexual maturation:

no effects observed

Description (incidence and severity):

The age and body weight of females on completion of vaginal opening and males on completion of balano-preputial separation were unaffected by administration

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

On Day 1 of age the ano-genital distance for both male and female offspring from the treated groups were similar to Controls and showed no adverse effect of treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

On Day 13 of age the distribution of litters with male offspring that showed nipples did not show any relationship to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females that received 300 or 1000 mg/kg/day had slightly but significantly high absolute and high body weight relative thyroid weight (p<005); this was not evident in the male animals. Females at 1000 mg/kg/day also showed low absolute and body weight relative mesenteric lymph node weight (p<0.05); this was not evident in the male animals. No other differences were apparent.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination performed after at least 10 weeks of treatment revealed no test item related lesions.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related findings following treatment with Di-Trimethylolpropane from weaning to approximately 13 weeks of age (F1A cohort) or from weaning to approximately 14 weeks of age (F1B). All histological changes were considered to be unrelated to treatment and to be consistent with the pathology background observed in Sprague Dawley rats in these laboratories.

Other effects:

no effects observed

Description (incidence and severity):

Ovarian Follicle Cuonts and Corpora Lutea (Cohort 1A):
Ovarian follicle and corporalutea counts were unaffected by administration of Di-Trimethylolpropane.

Sperm Assessment (Cohort 1A):
There were no adverse effects on sperm motility, testicular spermatid numbers, cauda epididymal sperm numbers or morphology following treatment with Di-Trimethylolpropane.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Development of the F1 offspring up to weaning were unaffected by treatment at all dose levels.

Developmental immunotoxicity:

effects observed, non-treatment-related

Description (incidence and severity):

Minor differences were observed in the immunophenotyping parameters between groups for both males and females. These differences were not related to the oral gavage administration of Di-Trimethylolpropane as they were within the ranges observed in Control animals.

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment related effects were seen.

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1B)

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment related effects were seen.

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

no

Conclusions:

Oral administration of Di-Trimethylolpropane at dose levels up to and including 1000 mg/kg/day was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters, spleen cell immunophenotyping or pathology on either the F0 or F1 generation. In addition the reproductive performance and fertility of the F0 animals and the survival/development of the F1 offspring up to weaning were unaffected by treatment at all dose levels. Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg/day.

Executive summary:

In an extended one-generation reproductive toxicity study (OECD 443) in rats, including Cohorts 1A and 1B, Di-Trimethylolpropane (Di-TMP) was administered to groups of 25 animals per sex and dose level by gavage at dose levels of 100, 300, or 1000 mg/kg/day (F0) at a volume dose of 4 mL/kg/day. Additionally, Di-TMP was administered to 40 animals per sex and dose level by gavage at dose levels 100, 300, or 1000mg/kg/day (F1, Cohorts 1A and 1B) at a volume dose of 4 mL/kg/day, except from Day 21 of age to nominal Day 35 of age when the high dose was reduced to 600 mg/kg/day which was maximum practicable for the dosing equipment required for weaning animals. A similarly constituted Control group received the vehicle (F0 & F1), corn oil, at the same volume dose as the treated groups throughout the same periods.

 

Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. All F1 animals were then dosed directly from postnatal day (PND) 21 to at least PND 90. At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation, urinalysis, reproductive organ integrity and function (Cohorts 1A and 1B). 

 

Throughout the study, 10 animals were either found dead or were killed for reasons of animal welfare, none were deemed treatment-related. Various effects were seen throughout the study, but differences and/or effects were concluded to be non-treatment-related since they were either minor, lacked a dose response or were inconsistent between the sexes.

 

Overall, administration of Di-Trimethylolpropane in F0 was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters or pathology. Estrous cycles, mating performance, pre coital interval, fertility, gestation length and index were unaffected by treatment. Regarding F1 litter responses, the general condition of offspring, litter size, offspring survival, offspring development, thyroid hormones and macropathology were unaffected by treatment. Cohorts 1A and 1B showed that the administration of Di-Trimethylolpropane was well tolerated with no treatment-related mortality and no adverse effects on general condition, body weight, food consumption, sexual maturation, estrous cycles, thyroid hormones, clinical pathology, immunophenotyping parameters, organ weights, ovarian follicle counts, sperm parameters or pathology.

 

Ultimately, oral administration of Di-Trimethylolpropane at dose levels up to and including 1000 mg/kg/day was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters, spleen cell immunophenotyping or pathology on either the F0 or F1 generation. In addition, the reproductive performance and fertility of the F0 animals and the survival/development of the F1 offspring up to weaning were unaffected by treatment at all dose levels.

 

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg/day.

 

This study is acceptable and satisfies the guideline requirements for an extended one-generation reproductive study (OECD 443) in the rat.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Extended one-generation reproductive toxicity study (EOGRTS)

No evidence of an effect on the reproductive organs was seen in a 28-day and a 90-day repeated dose toxicity study at dose levels of up to and including 1000 mg/kg bw/d. The absence of histopathological changes in reproductive organs in repeated dose toxicity study indicated that the substance would not have any adverse effects on fertility in the EOGRT. This conclusion was supported in the EOGRTS when oral administration of Di-Trimethylolpropane at dose levels up to and including 1000 mg/kg/day were well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters, spleen cell immunophenotyping or pathology on either the F0 or F1 generation. In addition the reproductive performance and fertility of the F0 animals and the survival/development of the F1 offspring up to weaning were unaffected by treatment at all dose levels. Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg/day.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity studies in rats as well as rabbits (both performed according to OECD test guideline 414) are available for Di-TMP.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July to 06 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guideline compliant study with no deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, UK
- Age at study initiation: 9 weeks old
- Weight at study initiation: 177-269 g on arrival; 197-307 g at the initiation of dosing
- Housing: 2 per cage in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings, and enrichment (devices for hiding in and an object for chewing)
- Diet (e.g. ad libitum): SDS VRF-1 breeder diet, ad libitum
- Water (e.g. ad libitum): water from the public supply, ad libitum
- Acclimation period: 3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21°C
- Humidity (%): 42-62%
- Air changes (per hr): ten or more with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hour cycle

IN-LIFE DATES: From: 27 July 2015 To: 12 August 2015

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose volume was 4 mL/kg, and the concentration of the test substance in the vehicle was 0, 25, 75 and 250 mg/mL. Dose volumes were based on the most recent body weight recorded for each animal. The control item, corn oil, was aliquoted weekly, stored in a refrigerator set to maintain 4°C and dispensed daily. The prepared control item was removed from the refrigerator and stirred for at least 30 minutes prior to dosing, and continuously during dosing. Prior to formulation, the test item was ground in a mortar, since it was supplied as flakes. The required amount of ground test item was weighed into a pre-labelled container according to instructions from the formulation computerised system (Dispense 8). The appropriate amount of vehicle was then added to the container and the formulation was magnetically stirred until visibly homogenous. The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and were stirred for at least 30 minutes before dosing and continuously during dosing.

Stability analyses performed previously in conjunction with Charles River Study No. 434348 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study for at least 8 days at 2-8°C, in the dark.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by Gas Chromatography using a validated analytical procedure. Duplicate top, middle and bottom samples (middle only for control) of 0.1 mL for each sampling time point were collected and sent to the analytical laboratory for analysis. Triplicate sets of top, middle and bottom samples (middle only for control) of the same volume were also collected and retained as backup samples. Concentration results were considered acceptable if mean sample concentration results were within 10% of theoretical concentration and individual sample concentrations were within 15% of theoretical concentration. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤10%.

Details on mating procedure:

Not applicable - time mated females were obtained direct from the supplier.

Duration of treatment / exposure:

From Days 6-19 of gestation

Frequency of treatment:

Once daily

Duration of test:

Day 0 (mating) to Day 20 of gestation (scheduled termination)

No. of animals per sex per dose:

24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen by the Sponsor following review of data from a 28 day repeated dose study of DiTMP in rats. On that study, tubular basophilia and inflammatory cell infiltration in the kidney were noted in males at all dose levels (40, 200 or 1000 mg/kg/day); however, these findings did not follow a clear dose-related pattern, were of little toxicological significance and were not noted in females. The NOAEL was therefore considered to be 1000 mg/kg bw/d.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
All animals were observed twice daily, once in the morning and once towards the end of the working day throughout the study for general health/mortality and moribundity. Animals were observed prior to dosing and regularly throughout the day for reaction to treatment on each day of dosing. The onset, intensity and duration of these any signs were recorded, with particular attention paid to the first hour after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
Animals were given a detailed examination once pretreatment and daily from Day 6 of gestation.

BODY WEIGHT: Yes
Individual body weights were recorded on Day 4 of gestation and daily from Days 6 to 20 of gestation.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured daily throughout the study, beginning on Day 4 of gestation (first measured quantity given on Day 3 of gestation).

WATER CONSUMPTION: Yes
Water consumption was monitored by visual inspection of the water bottles only. As no intergroup differences were noted, consumption was not measured by weight.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All adult animals were given an external examination, and then the contents of the cranial, thoracic and abdominal cavities were examined macroscopically, with representative samples of abnormal tissues being fixed in neutral buffered 10% formalin. The reproductive tract was dissected out and examined

Ovaries and uterine content:

The reproductive tract was dissected from the abdominal cavity. The gravid uterus weight was recorded. The ovaries and uterus were examined for number and distribution of: Corpora lutea; Implantation sites; Placentae (size, colour or shape) – only abnormalities were recorded; Live and Dead Fetuses; Early and Late Resorptions

Fetal examinations:

External abnormalities - Each fetus was examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities to the extent possible. Each implant was classified as being live, or a dead fetus (dead full term fetus that showed no signs of maceration), or a late embryonic death (macerated tissue identifiable as an embryofetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may have been of varying size).

Visceral examinations and sex - Half of the viable foetuses from each uterus were fixed in methylated ethyl alcohol, examined internally for sex and eviscerated following fixation, the viscera were not examined from fetuses prior to disposal. The remaining half of viable fetuses from each uterus were fixed in Bouins’ fluid. The foetuses fixed in Bouins’ fluid were examined for soft tissue abnormalities and sex by a freehand sectioning technique derived from Wilson.

Skeletal examination - The eviscerated carcasses were macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification.

Statistics:

Means and standard deviations were calculated for body weight, food consumption and selected pregnancy data. Numerical data collected on scheduled occasions for the listed variables were analysed as indicated according to relevant classification variables (e.g., sex, occasion). Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) are reported whenever possible. Inferential statistics were performed for body weight, food consumption and body weight change when possible, but excluded semi-quantitative data and any group with less than 3 observations.

ANOVA - Levene’s test was used to assess the homogeneity of group variance parametric assumption at the 5% significance level. Datasets with at least three groups was compared using an overall one-way ANOVA F-test or Kruskal-Wallis test (if parametric assumptions were not met) at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Dunnett’s or Dunn’s test, respectively, if the overall test is significant. Datasets with two groups were compared using a two-sided t-test or Wilcoxon Rank-Sum test, respectively. All significant pairwise comparisons were reported at the 0.1, 1 and 5% significance levels.

Indices:

Not applicable

Historical control data:

Available at the test facility

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no mortalities during the study. An increased incidence of ploughing behaviour (animal burrowing through bedding with its head) was noted in all treated groups; this observation was absent in Controls. This finding was transient, being noted immediately post dosing, and was no longer evident 1 hour after dosing, and was generally noted on 2-3 occasions between Days 14-20 of gestation in affected animals. All other clinical observations were considered to be incidental background findings commonly observed in this species and unrelated to treatment with DiTMP.
There were no effects on group mean body weights, group mean food consumption, and there were no abnormal findings detected at necropsy.

Pregnancy performance parameters and fetal weights were similar between all groups. Slight intergroup differences in embryonic deaths and fetal weights were noted between groups; however, these were considered to be incidental as they were within normal variation and were too small to be attributed to treatment with DiTMP.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: In all treated groups, dose-related increased incidences of misshapen scapula(e) were noted when compared with Controls

Details on embryotoxic / teratogenic effects:
In all treated groups, dose-related increased incidences of misshapen scapula(e) were noted when compared with Controls (14 fetuses in 10 litters at 100 mg/kg/day, 17 fetuses in 9 litters at 300 mg/kg/day and 23 fetuses in 10 litters at 1000 mg/kg/day, compared with 6 fetuses in 4 Control litters).
At 1000 mg/kg/day, increased incidences of unossified hyoid bone and 5th metacarpal(s) were noted when compared to Controls. Decreased incidences of ossified anterior atlas, cervical vertebral centra, phalangeal elements and sacrocaudal vertebrae with connections between centrum and arches were also observed in this group, when compared with Controls.
Renal pelvic dilation, which was noted in a small number of fetuses and litters at 1000 mg/kg/day, was considered unlikely to be related to treatment due to the low incidence and since the number of fetuses and litters affected is within background control ranges at the Test Facility.
The type and distribution of major fetal abnormalities and all other minor fetal abnormalities and variants and skeletal ossification parameters did not indicate any association with treatment. Slight intergroup differences were considered to be incidental and unrelated to treatment with DiTMP.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

skeletal malformations

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

skeletal malformations

Abnormalities:

not specified

Developmental effects observed:

not specified

Dose formulation analyses: Analysed Group 2, 3 and 4 formulations from Study Weeks 1 and 2 were found to be within the concentration acceptance criteria of ±10% of theoretical concentration (actual mean concentration range -7% to +1.7% of theoretical concentration). A low relative standard deviations of ≤4.5% also indicated that all formulations were homogenous. The absence of DiTMP from analysed control formulations was also confirmed.

Summary of Group Incidences of Treatment Related Clinical Observations

	Dose Group/Dose Level (mg/kg bw/d)
Observation	1 (0)	2 (100)	3 (300)	4 (1000)
Ploughing behaviour	0/24	11/24	14/24	24/24

Group Incidences of Minor Fetal Abnormalities and Variants

Abnormality/Variant	Group/Dose Level (mg/kg bw/d)
	1 (0)	2 (100)	3 (300)	4 (1000)
	Incidence of Fetuses (Litters)
Skeletal
Basisphenoid incompletely ossified, bilateral jugal misshapen	0	0	1(1)	0
Basioccipital misshapen	0	0	0	1(1)
Cranial bone(s) discrete unossified/incompletely ossified area(s)	3(2)	0	1(1)	2(1)
Cervical vertebral arch(es) increased ossification	11(7)	5(4)	12(8)	8(7)
Scapula(e) misshapen	6(4)	14(10)	17(9)	23(10)
Bilateral humerus incompletely ossified	0	0	0	1(1)
Sternebra bifurcated, xiphoid cartilage incomplete	0	1(1)	0	0
Rib(s) minimally kinked	0	1(1)	1(1)	2(2)
Rib(s) incompletely ossified	0	0	0	1(1)
Additional ossified area arising from sternebra	2(2)	0	1(1)	1(1)
Rib(s) costal cartilages asymmetrically aligned	7(7)	7(6)	6(6)	1(1)
Rib(s) costal cartilage(s) not attached to sternum	4(3)	2(1)	2(2)	1(1)
Pelvic girdle cranial displacement: Unilateral	1(1)	0	0	0
Pelvic girdle caudal displacement
Unilateral:	0	0	1(1)	0
Bilateral:	0	1(1)	0	0
Number with minor abnormality/variant	31(18)	29(15)	37(19)	36(16)
Total number examined skeletally	164(24)	150(24)	157(24)	161(24)
Number of ribs
13th vestigial rib(s)	4(3)	2(2)	0	1(1)
13th reduced rib(s)	2(2)	0	1(1)	3(1)
13 complete rib(s)	149(24)	142(24)	152(24)	155(24)
Vestigial supernumerary rib(s) on 1st lumbar vertebra	9(6)	6(5)	3(3)	2(2)
Reduced supernumerary rib(s) on 1st lumbar vertebra	0	0	1(1)	0

Group Incidences of Skeletal Ossification Parameters

Parameter	Group/Dose Level (mg/kg bw/d)
	1 (0)	2 (100)	3 (300)	4 (1000)
	Incidence of Fetuses (Litters)
Incomplete ossification affecting:
≥4 skull bones	6(4)	2(2)	5(3)	7(5)
≤3 skull bones	26(14)	14(7)	16(9)	21(12)
Cervical vertebral arch(es)	1(1)	1(1)	1(1)	0
Thoracic vertebral centrum(a)	9(7)	12(9)	8(6)	8(7)
Lumbar vertebral arch(es)	0	0	1(1)	0
Lumbar vertebral centrum(a)	0	0	1(1)	0
Pubis/es	5(3)	5(4)	1(1)	2(2)
Ischium/a	1(1)	0	3(2)	0
Sacral vertebral arch(es)	18(3)	6(5)	6(3)	8(6)
2nd and/or 4th metacarpal(s)	5(4)	1(1)	3(3)	1(1)
Unossified:
Hyoid	13(9)	4(4)	8(4)	20(12)
5th metacarpal(s)	29(13)	20(10)	29(14)	41(13)
5th metatarsal(s)	0	1(1)	1(1)	0
Ossified:
Anterior arch of atlas	51(17)	51(20)	41(13)	36(17)
>2 cervical vertebral centra	15(11)	13(8)	17(8)	10(8)
One or more sacrocaudal vertebrae with connection between centrum and arch(es)	46(19)	46(21)	53(17)	35(16)
Phalangeal elements	23(9)	33(12)	26(11)	13(8)
Mean number of caudal vertebral centra	4.1	4.0	4.1	3.8
Number of sternebrae retarded:
0	69(24)	71(22)	85(21)	60(19)
1	75(22)	58(21)	59(21)	70(22)
2	18(11)	12(11)	11(9)	27(14)
>2	2(2)	9(4)	2(2)	4(4)
Total number examined skeletally	164(24)	150(24)	157(24)	161(24)

Conclusions:

The NOAEL for maternal toxicity was considered to be 1000 mg/kg bw/d, however a developmental NOAEL could not be established.

Executive summary:

The potential for DiTMP to cause prenatal developmental toxicity was investigated in pregnant Sprague-Dawley rats, according to OECD 414. DiTMP was administered to groups of 24 time-mated rats by gavage at doses of 0, 100, 300 or 1000 mg/kg bw/d in corn oil. Animals were dosed over Days 6-19, inclusive, of gestation (where the day of detection of mating was Day 0 of gestation) and regularly checked for clinical signs of toxicity, body weight and food consumption performance and were killed on Day 20 of gestation for examination of pregnancies and embryofetal development.

Dosing with DiTMP was not associated with any maternal body weight or food consumption effects or gross necropsy findings. A dose-related increased incidence of ploughing behaviour was noted in all treated groups when compared to controls; however, as this findings was transient, noted only on a few occasions in each individual and is thought to be a reaction to unpleasant taste of dosing formulations, it was considered not to be adverse at any level.

Pregnancy performance and fetal weights in all groups were similar, and there were no major fetal abnormalities which were considered to be related to treatment with DiTMP. In all treated groups, a dose-related increased incidence of misshapen scapula(e) was noted when compared with controls. This finding is difficult to interpret as it had only recently been noted at the Test Facility in a small number of studies at low incidence, therefore background control data on this finding is limited, and the incidences in all treated groups on this study are above the background range. The significance of this finding could therefore not be determined. At 1000 mg/kg/day, a general trend toward delayed ossification was noted (which included increased incidences of unossified hyoid bones and 5th metacarpals and decreased incidences of ossified anterior atlas, cervical vertebral centra, phalangeal elements and sacrocaudal vertebrae with connections between centrum and arches) when compared with Controls. It was therefore concluded from the results of this study, that the maternal NOAEL was 1000 mg/kg bw/d. A developmental NOAEL cannot be established due to an increased and dose-related incidence of misshapen scapulae at all dose levels and a trend towards delayed ossification at 1000 mg/kg bw/d.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-18 to 2019-10-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 January 2001

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Di-Trimethylolpropane
- Chemical name (IUPAC): 2,2’-[Oxybis(methylene)]bis[2-ethylpropane-1,3-diol]
- Analytical purity: 99%
- Lot/batch No.: 170703932
- Expiration date of the lot/batch: 13 July 2019
- Storage condition of test material: Room temperature

Species:

rabbit

Strain:

New Zealand White

Remarks:

specific-pathogen-free

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia S.r.l.
- Age at study initiation: 13 weeks of age (females) and at least 25 weeks of age (males)
- Weight at study initiation: At least 2.5 kg body weight (females) and more than 3.5 kg body weight (males)
- Fasting period before study: not specified
- Housing: The rabbits were housed in a limited access animal facility. The animals were individually housed in grid-bottomed metal cages suspended over trays. Each cage tray held absorbent paper which was inspected and changed as necessary.
- Diet: A commercially available laboratory rabbit diet (2RB, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water: ad libitum to each cage via water bottles
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C ± 2 °C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): The rooms were lit by artificial light for 12 hours each day.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

2% CMC in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of the test item was suspended in the vehicle (2% carboxymethyl cellulose in water) during stirring and heating up to approximately 60 °C. Once the test item was dissolved, the formulations were kept between 35-40 °C in water bath on magnetic stirrer. The formulations were prepared daily. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was composed by carboxymethyl cellulose 2% in water. Administrations of corn oil at dose volume of 2 mL/kg and above are not tolerated by pregnant rabbit (EPA Archived Document OPPTS 870.3770, 1996). The solubility test of the test item in corn oil at dose volume of 1 mL/kg indicated it was not soluble and no suspension was formed. Therefore, it was decided to select another vehicle in order to avoid toxicity on pregnant rabbits and the selected vehicle of carboxymethyl cellulose 2% in water gave a good suspension with the test item.
- Concentration in vehicle: 2% carboxymethyl cellulose in water
- Amount of vehicle (if gavage): 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in this study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (5.5 hour at +40°C). The range of tested concentrations was between 20 and 200 mg/mL. Acceptance criteria for the validation was linearity R > 0.98, accuracy 85-115% and precision CV<10%.

In addition samples of the formulations prepared during the study (the first and the last week of treatment) were analysed to check the homogeneity and concentration (acceptance criteria ± 15% of the theoretical value for concentration and CV < 10% for homogeneity). Chemical analysis was carried out by the Analytical Chemistry Department at RTC. The software used for this activity was Analyst 1.6.2.

Details on mating procedure:

- Impregnation procedure: co-housed
- M/F ratio per cage: 1 male/1 female per cage (one female was placed into the home cage of the one male)
- Length of cohabitation: Each female remained with the male (in the home cage of the male) for at least 1 hour after successful mating was observed.
- Proof of pregnancy: Day of coitus referred to as day 0 of gestation

Duration of treatment / exposure:

Day 6 to day 28 post coitum

Frequency of treatment:

Once a day

Duration of test:

Females that completed the scheduled treatment period were euthanised on day 29 post coitum.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Medium dose

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a previous preliminary study on embryo-foetal development in the rabbit (RTC no. Y0220, not GLP-compliant).
The outcome of this preliminary study indicated that the treatment with Di-Trimethylolpropane at 1000 mg/kg/day caused maternal toxicity in terms of mortality and reduction in body weight. At this dose level, one female died, and the mean body weight was significantly reduced during the last week of treatment (7% reduction on Day 23 post-partum and 13% reduction on Day 26 post-partum).
No adverse changes were detected at 100 and 300 mg/kg/day. Based on the outcome of this study, the high dose level for the subsequent reproductive/developmental toxicity study should be lower than 1000 mg/kg/day.
Therefore, the dose levels of 100, 300 and 750 mg/kg/day were selected for the subsequent prenatal developmental toxicity study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once daily during treatment at the same time interval each day
- Cage side observations checked include abrasions/injuries on the body, hair loss, detection of faecal matters and staining on cage tray.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group (Day 0 post coitum) and on days 3, 6, 9, 12, 15, 18, 21, 23, 26 and 29 post coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal and group (as a mean) determined: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 (post coitum)
- Organs examined: Ovaries and uteriCAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once daily during treatment at the same time interval each day
- Cage side observations checked include abrasions/injuries on the body, hair loss, detection of faecal matters and staining on cage tray.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group (Day 0 post coitum) and on days 3, 6, 9, 12, 15, 18, 21, 23, 26 and 29 post coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal and group (as a mean) determined: Yes
- Time schedule for examinations: Food consumption was recorded on Days 3, 6, 9, 12, 15, 18, 21, 23, 26 and 29 of post coitum starting from Day 0 post coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 (post coitum)
- Organs examined: Ovaries and uteri

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
- Other: Gross evaluation of placentae

Fetal examinations:

- Examination of number, sex and weight of all living foetuses: yes
- Examination of number and sex of dead foetuses: yes
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes [all per litter]
- Head examinations: Yes: [half of all living foetuses, i.e. every other second living foetus]

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the homogeneity of data.

Statistical analyses of non-continuous variables were carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams’ test.

Indices:

Group mean values for body weight and food consumption of pregnant females, terminal body weight, gravid uterus weight, absolute weight gain (terminal body weight minus gravid uterus weight minus body weight at Day 0 post coitum), litter size, intra-uterine deaths, corpora lutea count, total implantation loss and pre- and post-implantation loss were calculated. Data from non-pregnant animals were not included in group mean calculations. Animals which aborted were included in body weight calculations up to the day of abortion.

Litter data: Pre-implantation loss was calculated as a percentage from the formula:
Pre impl. Loss% = (no. of corpora lutea – no. of implantations)/(no. of corpora lutea) x 100

Post-implantation loss was calculated as a percentage from the formula:
Post impl. Loss% = (no. of implantations – no. of live foetuses)/(no. of implantations) x 100

Total implantation loss was calculated as a percentage from the formula:
Total impl. Loss% = (no. of corpra lutea – no. no. of live fetuses)/(no. of corpora lutea) x 100

Sex ratios of the foetuses were calculated as the percentage of males.
The number of foetuses affected with structural deviations and the corresponding litter
percentage were calculated. All derived values (e.g. means, percentages, ratios) were first
calculated within the litter and the group values derived as a mean and standard deviation
of individual litter values. Foetal structural deviations were expressed as the percentage
of affected foetuses relative to all foetuses examined per group, as well as in terms of the
mean litter percentage of affected litters.

Historical control data:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hair loss was recorded in a few animals (no more than 2 per dose group) in all dose groups. Reduced and soft faeces were seen in medium- and high-dose groups with a similar incidence (up to 2 animals). Red staining and foetuses in cage tray were recorded in the 2 females that were humanely sacrificed before end of study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Unscheduled deaths occurred during the study. Two females, one in the medium dose group and one in the high dose group, were humanely sacrificed for abortion on days 25 and 23 post coitum, respectively.
Fate: The total number of females at term was 20 in the control group, 18 in the low and high dose groups, and 19 in the mid-dose group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease of terminal body weight (5% decrease) recorded at the end of the study was observed in the high-dose females receiving 750 mg/kg/day (4016.11 g) when compared to controls (4240.50 g). No significant changes were detected in body weight between control and the low- or medium-dose females.

Statistically significant reduction in absolute body weight gain was recorded in the high-dose females receiving 750 mg/kg/day starting from Day 12 post coitum up to the end of the study. At the end of the study (i.e. day 29 post coitum), there was a 39.9% decrease in the mean absolute body weight gain of the high-dose females (318.89 g) compared to that of the control females (530.50 g).

See Tables 1 and 2 in box „Any other information on results incl. tables”.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a consistent trend of reduced food consumption in the high dose females receiving 750 mg/kg/day starting from Day 9 post coitum. On day 29 post coitum, there was a 15.6% decrease (with statistical significance) in the mean food consumption of the high-dose females (141.38 g) compared to that of the control females (167.51 g).

See Table 3 in box "Any other information on results incl. tables" for results.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

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:

effects observed, non-treatment-related

Description (incidence and severity):

There was slight decrease in uterus weight in all treated groups compared to controls. However, no dose-response relationship or statistical significance in the decrease was observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted during the macroscopic observation of the females. The sporadic changes such as hair loss in two high-dose females or abrasion in one high-dose female were not attributable to treatment.

Of the two females that were humanely sacrificed before end of study, no macroscopic findings were seen in the high-dose female, whereas moderate hair loss of the abdominal region was seen in the medium-dose female.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

Slight maternal toxicity was present in the high dose females receiving 750 mg/kg/day as demonstrated by a statistically significant reduction of absolute body weight gain, food consumption and terminal body weight. No significant differences in these parameters were detected in the low- and medium-dose females when compared to controls.

At necropsy all animals at term did not show abnormalities that could be considered related to treatment.

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Two females (one in the medium-dose group and one in the high-dose group) were humanely sacrificed on days 25 and 23 post coitum, respectively, due to abortions. This is considered not to be treatment-related since this low incidence of abortion is within the range of the historical control data.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No significant difference observed between treated and control animals. See Table 4 in box "Any other information on results incl. tables" for results.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No significant difference observed between treated and control animals. See Table 4 in box "Any other information on results incl. tables" for results.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No significant difference observed between treated and control animals. See Table 4 in box "Any other information on results incl. tables" for results.

Dead fetuses:

no effects observed

Description (incidence and severity):

No significant difference observed between treated and control animals. See Table 4 in box "Any other information on results incl. tables" for results.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Two low dose females and one high dose female were found not pregnant at final sacrifice.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no difference in the mean foetal weight of both sexes, but there was a significant decrease in male foetal weight at the highest dose.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No significant difference observed between treated and control animals.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant difference observed between treated and control animals.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No significant difference observed between treated and control animals.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No abnormalities were detected in all groups (treated and control).

Skeletal malformations:

no effects observed

Description (incidence and severity):

No treatment-related findings were recorded.

Malformation recorded in one foetus of the control group (lumbar hemivertebrae) and in two different foetuses of the low-dose group (articulation point absent in the pelvic girdle and thoracic hemivertebrae) were considered spontaneous in origin.

Visceral malformations:

no effects observed

Description (incidence and severity):

No abnormalities were detected in all groups (treated and control).

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

The uterine examinations did not reveal any effects on foetuses on term. The mean foetal and litter weights of all treated groups were comparable to controls. At the external and internal examination of foetuses no treatment-related abnormalities were described. The examination of foetal fixed head and skeleton did not reveal any treatment-related findings.

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

No treatment-related developmental effects were observed.

Key result

Developmental effects observed:

no

Treatment related:

no

Table 1: Summary of maternal terminal body weight group data

Group		Terminal body weight [g]
Control	N	20
	Mean [g]	4240.50
	SD	229.04
Low dose	N	18
	Mean [g]	4080.00
	SD	202.22
Medium dose	N	19
	Mean [g]	4145.79
	SD	259.51
High dose	N	18
	Mean [g]	4016.11*
	SD	254.40

N = number of animals; SD = standard deviation; * = mean value of group is significantly different from control (Statistical analysis: Kruskall Wallis test and William’s test if group differences are different from control at p < 0.05)

Table 2: Summary of the maternal absolute body weight gain group data

		Post coitum
Group		Day 3	Day 6	Day 9	Day 12	Day 15	Day 18	Day 21	Day 23	Day 26	Day 29
Control	N	20	20	20	20	20	20	20	20	20	20
	Mean [g]	0.00	83.00	139.00	195.50	310.50	322.50	347.50	380.00	450.00	530.50
	SD	0.000	39.484	42.785	67.938	121.373	111.868	122.426	125.865	131.869	134.926
Low dose	N	18	18	18	18	18	18	18	18	18	18
	Mean [g]	0.00	75.56	117.22	167.78	233.33*C	265.56	289.44	304.44	347.78	383.33*D
	SD	0.000	49.967	40.410	60.348	87.515	72.048	90.064	97.088	126.749	150.646
Medium dose	N	20	20	20	20	20	20	20	20	19	19
	Mean [g]	0.00	67.00	109.50	151.50	222.00	234.50	255.00	278.00	355.79	416.84
	SD	0.000	60.966	69.546	75.413	160.348	198.321	237.985	241.739	142.568	140.121
High dose	N	19	19	19	19	19	19	19	19	18	18
	Mean [g]	0.00	73.68	68.42+C	82.11+C	127.37+C	164.74*C	162.11*C	162.11+C	267.22+D	318.89+D
	SD	0.000	34.835	66.855	144.936	225.606	251.868	264.902	272.918	133.320	201.111

N = number of animals; SD = standard deviation; *D Dunnett LSD Test Significant at the 0.05 level; +D Dunnett LSD Test Significant at the 0.01 level; *C Cochran and Cox Test Significant at the 0.05 level; #C Cochran and Cox Test Significant at the 0.001 level

Table 3: Summary of food consumption group data

		Post coitum
Group		Day 3	Day 6	Day 9	Day 12	Day 15	Day 18	Day 21	Day 23	Day 26	Day 29
Control	N	20	20	20	20	20	20	20	20	20	20
	Mean [g]	186.84	205.52	212.62	204.35	186.08	209.81	206.60	201.95	172.37	167.51
	SD	25.954	15.581	18.251	21.461	31.237	25.565	23.504	36.350	29.793	31.199
Low dose	N	18	18	18	18	18	18	18	18	18	18
	Mean [g]	185.54	216.10	212.42	202.87	184.09	210.55	218.25	227.77	177.28	191.37
	SD	18.290	28.044	16.907	17.372	30.567	31.961	31.958	52.702	38.200	34.313
Medium dose	N	20	20	20	20	20	20	20	20	19	19
	Mean [g]	191.47	215.08	201.93	193.47	174.00	187.02	192.96	179.37	153.29	154.17
	SD	26.979	25.751	29.414	26.430	43.247	43.034	45.757	67.851	31.852	33.764
High dose	N	19	19	19	19	19	19	19	19	18	18
	Mean [g]	187.52	206.44	180.14#C	168.43+C	136.54+D	166.51+D	175.91*C	166.73	148.28	141.38*D
	SD	20.933	18.452	29.868	41.955	35.970	45.617	54.445	59.560	27.136	32.587

N = number of animals; SD = standard deviation; *D Dunnett LSD Test Significant at the 0.05 level; +D Dunnett LSD Test Significant at the 0.01 level; *C Cochran and Cox Test Significant at the 0.05 level; #C Cochran and Cox Test Significant at the 0.001 level

Table 4: Summary of reproductive/developmental endpoint group data

Group		Corpora Lutea	Implantation	Pre-Imp. Loss	Post-Imp. Loss	Early Resorption	Late Resorption	Male Sex Percent	Dead foetuses	Live foetuses	Male foetal weight mean [g]	Female foetal weight mean [g]	Litter weight [g]
Control	Mean	9.60	9.30	0.30	4.03	0.00	0.25	50.10	0.00	9.05	45.35	43.10	390.81
	SD	2.21	2.49	0.57	8.09	0.00	0.79	15.94	0.00	2.39	6.11	5.61	81.10
	N	20	20	20	20	20	20	20	20	2039	20	20	20
Low dose	Mean	8.72	8.00	0.72	9.87	0.11	0.06	44.17	0.00	7.83	43.96	43.44	332.11
	SD	2.08	2.57	1.02	14.75	0.32	0.24	21.63	0.00	2.46	5.35	6.05	91.32
	N	18	18	18	18	18	18	18	18	18	18	18	18
Medium dose	Mean	9.89	9.68	0.21	2.86	0.11	0.26	48.16	0.00	9.32	44.03	43.11	392.62
	SD	2.11	2.26	0.42	6.50	0.32	0.93	17.11	0.00	2.40	7.91	7.16	77.63
	N	19	19	19	19	19	19	19	19	19	19	19	19
High dose	Mean	9.50	9.28	1.11	5.49	0.06	0.00	48.89	0.00	9.22	40.35*	40.28	370.84
	SD	1.86	1.27	2.32	7.24	0.24	0.00	16.33	0.00	1.35	3.37	3.63	48.22
	N	18	18	18	18	18	18	18	18	18	18	18	18

N = number of animals or litters (depending on endpoint); SD = standard deviation; * = mean value of group is significantly different from control (Statistical analysis: Kruskall Wallis test and William’s test if group differences are different from control at p < 0.05)

Conclusions:

In a prenatal developmental toxicity study performed in accordance with OECD 414, slight maternal toxicity was observed in rabbits at the highest tested dose of 750 mg/kg/day without any indication of developmental effects. Hence, 300 mg/kg/day is determined as the NOAEL for maternal toxicity and 750 mg/kg/day as the NOAEL for developmental toxicity.

Executive summary:

In a prenatal developmental toxicity study performed in accordance with OECD 414, di-trimethylolpropane (99% purity) was administered to New Zealand White pregnant rabbits (20 females per dose) in 2% carboxymethyl cellulose in water at dose levels of 0, 100, 300 or 750 mg/kg/day from day 6 to day 28 post coitum. Control animals received 2% carboxymethyl cellulose in water as vehicle at the same treatment period as the test item-treated animals. Animals were sacrificed on day 29 post coitum and subjected to post mortem examination. The females treated with 750 mg/kg/day exhibited slight toxicity as indicated by the reduction in body weight, absolute body weight gain and food consumption when compared with control females. No significant differences of the aforementioned parameters were observed in the females treated with 100 or 300 mg/kg/day. At necropsy, macroscopic examination of the females revealed no treatment-related changes.

 

The uterine examinations did not reveal any effects on the foetuses. The mean foetal and litter weights of all treated groups were comparable to controls. At the external and internal examination of foetuses no treatment-related abnormalities were described. The examination of foetal fixed head and skeleton did not reveal any treatment-related findings.

 

Based on the results from this study, 300 mg/kg/day is determined as the NOAEL (No-Observed-Adverse-Effect-Level) for maternal toxicity and 750 mg/kg/day as the NOAEL for developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two GLP- and OECD test guideline-compliant studies are available.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Prenatal developmental toxicity study with Di-TMP in rats and rabbits

In a prenatal developmental toxicity study performed in accordance with OECD 414, di-trimethylolpropane (99% purity) was administered to New Zealand White pregnant rabbits (20 females per dose) in 2% carboxymethyl cellulose in water at dose levels of 0, 100, 300 or 750 mg/kg/day from day 6 to day 28 post coitum. Control animals received 2% carboxymethyl cellulose in water as vehicle at the same treatment period as the test item-treated animals. Animals were sacrificed on day 29 post coitum and subjected to post mortem examination. The females treated with 750 mg/kg/day exhibited slight toxicity as indicated by the reduction in body weight, absolute body weight gain and food consumption when compared with control females. No significant differences of the aforementioned parameters were observed in the females treated with 100 or 300 mg/kg/day. At necropsy, macroscopic examination of the females revealed no treatment-related changes.

 

The uterine examinations did not reveal any effects on the foetuses. The mean foetal and litter weights of all treated groups were comparable to controls. At the external and internal examination of foetuses no treatment-related abnormalities were described. The examination of foetal fixed head and skeleton did not reveal any treatment-related findings. Based on the results from this study, 300 mg/kg/day is determined as the NOAEL (No-Observed-Adverse-Effect-Level) for maternal toxicity and 750 mg/kg/day as the NOAEL for developmental toxicity.

 

Justification for selection of Effect on developmental toxicity: via oral route: In the rats, a dose-related increased incidence of misshapen scapula(e) was noted when compared with controls. This finding is difficult to interpret as it had only recently been noted at the Test Facility in a small number of studies at low incidence; background control data on this finding is limited, and the incidences in all treated groups on this study are above the background range. It is therefore concluded from the results of this study that the maternal NOAEL was 1000 mg/kg bw/d and the developmental LOAEL was <100 mg/kg bw/day. Further data from the Test Facility on the background incidence of this finding from ongoing and future studies will enable the toxicological significance of the finding (variation) to be put in context.

 

On the other hand, in the rabbits, a prenatal developmental toxicity study, slight maternal toxicity was observed in rabbits at the highest tested dose of 750 mg/kg/day without any indication of developmental effects. Hence, 300 mg/kg/day is determined as the NOAEL for maternal toxicity and 750 mg/kg/day as the NOAEL for developmental toxicity.

Toxicity to reproduction: other studies

Description of key information

No other studies.

Justification for classification or non-classification

The results of the 28-day repeated dose toxicity study do not indicate the reproductive system as a target of toxicity. A 90-day repeated dose toxicity study in the rat has also been conducted where no toxicologically relevant findings were noted.

The rat developmental toxicity study shows a dose-related increase in the incidence of a single foetal skeletal finding in all treated groups in the apparent absence of maternal toxicity. Interpretation of this finding (currently classed by the Test Facility as a minor abnormality) is difficult as it had only become apparent in controls in two studies prior to the Di-TMP study. Due to the increased background incidence of the finding, it is likely to be reclassified as a variation by the Test Facility. Furthermore, the relevance of the finding can only reliably be put in context when further background data from ongoing/future studies become available.

In a prenatal developmental toxicity rabbit study of Di-TMP performed in accordance with OECD 414, slight maternal toxicity was observed in rabbits at the highest tested dose of 750 mg/kg/day without any indication of developmental effects. Hence, 300 mg/kg/day is determined as the NOAEL for maternal toxicity and 750 mg/kg/day as the NOAEL for developmental toxicity.

In an extended one generation reproductive toxicty study, 100, 300, and 1000 mg/kg/day of Di-TMP was adminitered via oral gavage. The substance well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters, spleen cell immunophenotyping or pathology on either the F0 or F1 generation. In addition the reproductive performance and fertility of the F0 animals and the survival/development of the F1 offspring up to weaning were unaffected by treatment at all dose levels. Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg/day.

Given the lack of adverse reproductive findings in the rat from repeated dose toxicity studies, the uncertainty of the data interpretation in the rat developmental toxicity study and the lack of developmental effects in the rabbits, no classification for the test item Di-TMP is therefore justified for reproductive toxicity under according to EC Regulation 1272/2008 taking a weight-of-evidence approach.