Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Propylidynetrimethanol, ethoxylated, esters with acrylic acid (TMPeoTA) was tested in a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and in compliance with GLP. Wistar Han rats were treated with TMPeoTA by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day. No treatment-related changes were noted in any of the reproductive parameters investigated in this study. No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No toxicologically significant changes were noted in any of the developmental parameters investigated in this study. Based on the results of this study, the no-observed-adverse-effect levels (NOAEL) for reproduction and developmental toxicity was evaluated to be 1000 mg/kg bw/day.


 


Parental (systemic) NOAEL: 1000 mg/kg bw/day.


Parental (local) NOAEL: 100 mg/kg bw/day, based on adverse local forestomach effects.


Reproduction NOAEL: at least 1000 mg/kg bw/day.


Developmental NOAEL: at least 1000 mg/kg bw/day.


 


Based on the results of the extended one generation reproductive toxicity study (ReachCentrum, 2019C) with the read-across substance TPGDA (1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate (CAS 42978-66-5), the following no-observed-adverse-effect levels (NOAEL) were established for the read-across substance:


General toxicity (F0 and F1): at least 100 mg/kg bw/day


Reproduction: at least 100 mg/kg bw/day


Developmental: at least 100 mg/kg bw/day


 


Based on the results of the OECD 422 study with the read-across substance GPTA (CAS: 52408-84-1) (ReachCentrum, 2019A) the following NOAEL have been derived.


Parental (systemic) NOAEL: 375 mg/kg bw/day, based on treatment related mortality at 750 mg/kg bw/day.


Parental (local) NOAEL: 150 mg/kg bw/day, based on adverse local forestomach effects at 375 mg/kg bw/day (males) and 750 mg/kg bw/day (both sexes).


Reproduction NOAEL: at least 750 mg/kg bw/day.


Developmental NOAEL: at least 750 mg/kg bw/day.


 


For DNEL derivation, a NOAEL of 375 mg/kg bw/day from an OECD 408 study using GPTA (read-across substance) is used.

Link to relevant study records

Referenceopen allclose all

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:
15 Aug 2018 - 14 Nov 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD guidance document supporting OECD test guideline 443 on the extended onegeneration reproductive toxicity test, No. 151
Version / remarks:
2013
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 :
The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422) with oral exposure of the test item in rats. In this preliminary study, animals were dosed with 0, 40, 125 and 375 mg/kg/day. From dose 125 mg/kg/day, adverse findings observed were ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other test item-related findings consisted of an increase in liver weights in males and females and an increase in hyaline droplet accumulation in the male kidneys at 375 mg/kg/day. Based on the severe findings in the stomach, a parental local NOAEL was established of 40 mg/kg/day in this study. As animals are dosed for a longer time period for the Extended One-Generation Reproductive Toxicity Study (e.g. F0-males 11-13 weeks versus 4 weeks in an OECD 422 study), a maximum dose level of 100 mg/kg/day was selected.

- Inclusion/exclusion of extension of Cohort 1B : Inclusion

- Termination time for F2 : not applicable

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : Exclusion

- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : Exclusion

- Route of administration : The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6-7 week; (F1) 3 wks
- Weight at study initiation: (P) Males: 136 and 187 g; Females: 112 and 153 g; (F1) Males: mean 50 g; Females: 49 g
- Fasting period before study: During motor activity measurements, F0- female animals had no access to food for a maximum of 2 hours. During motor activity measurements, F0-female animals had no access to water for a maximum of 2 hours.
- Housing:
On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III)
During the post-mating phase, males were housed in their home cage (Macrolon type IV) with a maximum of 5 males/cage). Females were individually housed in Macrolon plastic cages (type III).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height). Pups were housed with the dam until termination or weaning (on PND 21).
During locomotor activity monitoring, F0-females were housed individually in a Hi-temp polycarbonate cage
- Diet: ad libitum, Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, Municipal tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 45 to 67
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least weekly as a solution, formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 2, 6, 20 mg/mL
- Amount of vehicle: 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (type III).
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulations. The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
see table 1
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
25 (F0) / 20 (F1)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422) with oral exposure of the test item in rats and in an attempt to produce graded responses to the test item. In this preliminary study, aninmals were dosed with 0, 40, 125 and 375 mg/kg bw/day. From dose 125 mg/kg bw/day, adverse findings observed were ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other test item-related findings consisted of an increase in liver weights in males and females and an increase in hyaline droplet
accumulation in the male kidneys at 375 mg/kg/day. As animals are dosed for a longer time period for the Extended One-Generation Reproductive Toxicity Study (e.g. F0-males 11-13 weeks versus 4 weeks in an OECD 422 study), a maximum dose level of 100 mg/kg bw/day was selected. No (severe) clinical symptoms and effects on body weight and food consumption were noted (i.e. the general health of the animals was good). The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.


Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION AND COMPOUND INTAKE: No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL TESTS – F0-Generation
Functional tests were performed on the selected 5 females during Week 10 of treatment. The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity

CLINICAL PATHOLOGY - F0-Generation and Cohort 1A animals
Sample collection:
Blood of 10 selected animals/sex/group of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy. Samples were collected from the retro-orbital sinus under anaesthesia using isoflurane in the animal facility.
The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0-animals and Cohort 1A animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.
Oestrous cyclicity (parental animals):
Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken.
Sperm parameters (parental animals):
Parameters examined in [F0, Cohort 1A] male parental generations:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing, the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:

F1-Generation until Weaning (PND 21):

Mortality/Moribundity Checks
Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.

Body Weights
Live pups were weighed individually on PND 1, 4, 7, 13 and 21. For animals of Cohort Surplus, a terminal weight was recorded on the day of scheduled necropsy.

Sex
Sex was externally determined for all pups on PND 1 and 4 (sex determination not noted for Day 13)

Anogenital Distance
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

Areola/Nipple Retention
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Clinical Pathology
On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation for determination of thyroid hormone.

F1-Generation from Weaning (PND 21) onwards:

Mortality/Moribundity Checks – Cohorts 1A, 1B and 1C
Throughout the study, animals were observed for general health/mortality and moribundity twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations– Cohorts 1A, 1B and 1C
Clinical observations were performed at least twice daily, up to the day prior to necropsy. These observations were at least conducted prior to dosing and 0-30 minutes after dosing.

Body Weights – Cohorts 1A, 1B and 1C
Animals were weekly weighed individually. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. For animals of Cohorts 1A and 1B, a terminal weight was recorded on the day of scheduled necropsy.

Food Consumption– Cohorts 1A, 1B and 1C
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.

Water Consumption – Cohorts 1A, 1B and 1C
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Vaginal Patency – Cohorts 1A, 1B and 1C
Vaginal patency (vaginal opening) was monitored daily for all females from PND 25 onwards until vaginal patency was present, by visual inspection of the vaginal area.

Balanopreputial Separation – Cohorts 1A, 1B and 1C
Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area.

Stage of Estrus Determination – Cohorts 1A, 1B and 1C
Estrous stages were determined by examining the cytology of vaginal lavage sample, taken on the day of scheduled necropsy.

Estrous Cycle Determination – Cohort 1A
Estrous stages were determined by examining the cytology of vaginal lavage samples, taken during two periods. During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.

Clinical Pathology - F1- animals of Cohort Surplus on PND 22
On PND 22, blood was collected from all Cohort Surplus animals (10/sex/group), if possible for determination of Thyroid Hormone. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.

Clinical Pathology – Cohort 1A
Blood of 10 selected animals/sex/group of F0-animals and Cohort 1A animals was collected on the day of scheduled necropsy for
- Hematology: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelets, Reticulocytes (absolute), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- Clinical Chemistry: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT),Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos)
- Thyroid hormone: Thyroxine (T4), Thyroid Stimulating Hormone (TSH)
Urinalysis: Specific gravity, White blood cells (WBC-sed.), Clarity Red blood cells (RBC-sed.), Colour Casts, pH, Epithelial cells, Blood Crystals, White blood cells (WBC), Bacteria, Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: yes
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. If possible, one pup (male or female) was selected per litter (20 litters in total). One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 μm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The following subpopulations were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy:
T-cells, T-helper cells, T-cytotoxic cells, B-cells, NK-cells, Ratio T-helper cells/ T-cytotoxic cells (Th/Tc)
The % lymphoid cells of peripheral blood mononuclear cells (PBMC) were determined using the Forward Scatter and Side Scatter.

Postmortem examinations (parental animals):
SACRIFICE- F0 Generation
Animals surviving until scheduled euthanasia were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies:
Males (which sired and failed to sire): After successful mating and a minimum of 10 weeks of treatment.
Females which delivered: LD 23-25.
Females which failed to deliver:
With evidence of mating: Post-coitum Days 25-27 (102,105, 108, 111, 122, 144, 179, 194, 200).
Without evidence of mating: 24 days after the last day of the mating period (Nos. 112, 168)
Females with total litter loss (No. 126): Within 24 hours after the last pup was found dead or missing.

Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy. Water was available.

GROSS NECROPSY- F0 Generation
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no
macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS - F0 Generation
The organs identified in Table 5 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios were calculated. Representative samples of the tissues identified in Table 5 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.
Postmortem examinations (offspring):
SACRIFICE -F1 Generation until weaning
Unscheduled Deaths– F1-Generation
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible). Descriptions of all external abnormalities were recorded. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. The
stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Culled Pups (PND 4) – F1-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation.

GROSS NECROPSY
See table 3 for details

Cohort 1A
Scheduled necropsy of Cohort 1A was conducted on PND 89-95. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

Cohort 1B
Scheduled necropsy of Cohort 1B was conducted on ≥ PND 97. Cohort 1B animals were not deprived of food overnight before necropsy. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort 1C
Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy and no terminal body weight was recorded. All
animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

Cohort Surplus
Scheduled necropsy of Cohort Surplus was conducted on PND 22. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGTHS
Cohort 1A
In addition to the procedures described above, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A animals of Group 1 and 4 for quantitative evaluation of follicles (primordial and small growing follicles counted together), as well as corpora lutea.
Cohort 1B
In addition to the procedures described above, the reproductive organs of all Cohort 1B animals were processed to block stage.
Statistics:
Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The motor activity data set (at least 3 groups) was compared using an overall Kruskal-Wallis.
Incidence:
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
see table 4
Offspring viability indices:
see table 4
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test-item related clinical signs were noted up to treatment with 30 mg/kg bw/day. No findings were noted during the weekly arena observations in this study.
Salivation seen after dosing among animals of the 100 mg/kg bw/day dose group during the first 8 weeks of the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than being a sign of systemic toxicity. Other clinical signs noted during the treatment period occurred within the range of
background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No test item-related mortality occurred during the study period. One female of the 10 mg/kg bw/day group was euthanized on Lactation Day 1, as she had a total litter loss.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered to have been unaffected by treatment up to 100 mg/kg bw/day. A trend towards decreased body weights and body weight gain may appeared in males at 100 mg/kg bw/day up to Week 1 of the mating period. A statistical significant decrease in bodyweight was observed at 100 mg/kg bw/day in Week 8 of treatment. The statistical significant increase in body weight gain at 30 mg/kg bw/day in Week 3 of mating was considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight in animals treated up to 100 mg/kg bw/day was similar to the control level over the treatment period. Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration 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):
No toxicologically relevant changes were noted in haematological parameters. Statistically significant lower white blood cell count (WBC) was noted in females at 10 and 100 mg/kg bw/day, which was likely the result of lower lymphocytes levels observed in these animals. In absence of a dose response and as values remained within normal range, these changes were considered unrelated to treatment with the test item. Any other statistically significant changes in haematology parameters achieving a level of statistical significance when compared to controls were considered unrelated to administration of the test item due to the minimal magnitude of the change and/or absence of a dose response. Coagulation parameters of treated rats were considered not to have been affected by treatment up to 100 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were observed in treated males at 30 and/or 100 mg/kg bw/day:
Increase in urea at 100 mg/kg/day (1.16x, 4.3 mmol/L).
• Increase in sodium at 30 and 100 mg/kg bw/day (1.01x (143.3 mmol/L) and 1.02x (143.8 mmol/L), respectively).
• Increase in potassium at 100 mg/kg bw/day (1.07x, 4.02 mmol/L)
Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment with the test item. Serum levels of TSH and T4 in F0-males and -females were not affected by treatment up to 100 mg/kg bw/kg.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis was not affected by treatment with the test item up to 100 mg/kg bw/day.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. Motor activity (total movements and ambulations) was (non-statistically) slightly increased in high dose animals. This slight increase was considered due to one female, for which a level of total movements and ambulations was recorded.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic alterations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Histopathological examination of reproductive tissues revealed no evidence of a treatment-related effect on reproduction.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were unaffected by treatment with the test item. All females had regular cycles of 4 to 5 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm motility, concentration and morphology parameters were unaffected by treatment up to 100 mg/kg bw/day. Statistically significant changes noted were considered unrelated to treatment as a dose-related response was absent, and since the opposite effect would be expected in case of toxicity.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating index was considered not to be affected by treatment. With the exception of one control and one 30 mg/kg bw/day female, all females showed evidence of mating. Precoital time was considered not to be affected by treatment. With the exception of one female (mated at Day 14), all females showed evidence of mating within the first 4 days. Number of implantation sites was considered not to be affected by treatment up to 100 mg/kg bw/day. Fertility index was considered not to be affected by treatment. The fertility indices were 79%, 96%, 100% and 88% for the control, 10, 30 and 100 mg/kg bw/day groups, respectively. A total of 5 control females, one female at 10 mg/kg bw/day and 3 females at 100 mg/kg bw/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: A dose level of 100 mg/kg bw/day was selected as the NOAEL of 40 mg/kg bw/day was established in a dose range finder. Here, adverse findings consisted of ulcers and inflammation of the forestomach and hyperplasia squamous cells from 125 mg/kg bw/day on.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest doses tesed
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. For the pup of one Female (10 mg/kg bw/day) which was missing on Day 2, absence of milk in the stomach was noted on Day 1 and for the pup of Female No. 154 which was missing on Day 2, a pale appearance was noted on Day 1. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant. No test item-related clinical signs were noted during daily detailed clinical observations in Cohort 1A,1B and 1C or during weekly arena observations.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No mortality occurred during the study period in Cohort 1A, 1B and 1C from weaning onwards that was considered to be related to treatment with the test item.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment.
Body weights and body weight gains of Cohort 1A, 1B and 1C were considered unaffected by treatment up to 100 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before and after correction for body weight was considered unaffected by treatment up to 100 mg/kg bw/day.
Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment as changes were only minimal and/or no trend was apparent regarding dose and duration of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: The following statistically significant changes were noted in male animals at 100 mg/kg bw/day.
• Increased neutrophil levels (1.3x)
• Increased lymphocyte levels (1.16x)
The statistically significant change in white blood cell count (WBC) in males at 10 mg/kg bw/day was considered not to be related to treatment with the test item as it occurred in the absence of a dose-related trend. Coagulation parameters of treated rats were considered not affected by treatment up to 100 mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female pups, culled at PND 4 and male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment. Serum TSH levels in male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment.
Cohort 1A: Clinical chemistry parameters of treated rats were considered not affected by treatment up to 100 mg/kg bw/day. Serum levels of thyroid stimulating hormone (TSH) and Total T4 were considered not to be affected by treatment up to 100 mg/kg bw/day.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Cohort 1A: Urinary parameters were unaffected by treatment.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
Balanopreputial separation and vaginal patency were considered unaffected by treatment up to 100 mg/kg bw/day. At 30 mg/kg bw/day, an increase in the age of reaching balanopreputial separation was observed (1.03x compared with control). This was likely related to Male No. 355 that had a very low body weight and had reached balanopreputial separation at age PND 54 (versus average age of reaching balanopreputial separation on PND 41.5 for control animals).
At 10 mg/kg/day, an increase in age of reaching first estrus (1.04x compared with controls) was noted in females. In absence of a dose response, this slight increase was considered unrelated to treatment.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 100 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related organ weight changes in the Cohort surplus-animals.

Cohort 1A and 1B:There were no test item-related organ weight changes observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment.
Cohort 1A and 1B: There were no test item-related macroscopic alterations in the F1-generation. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in several females in all dose groups in Cohort 1A and 1B, is related to a stage in the estrous cycle and is a normal finding.
Histopathological findings:
no effects observed
Description (incidence and severity):
Cohort 1A: There were no test item-related microscopic alterations in Cohort 1A of the F1-generation.. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Spermatogenesis-staging (Cohort 1A)
Stage-dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Ovarian Follicle Counts - Cohort 1A
There were no test item-related effects on the ovarian follicle counts in the F1-females (Cohort 1A) at 100 mg/kg/day when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous Cycle – F1-Generation (Cohort 1A):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had regular cycles of 4 to 5 days. Female No. 636 (30 mg/kg bw/day) had two cycles with a duration of 2 days and one of 5 days and was therefore classified as having an irregular cycle. For Female Nos. 495 (control) and 555 (10 mg/kg bw/day) estrous cycle regularity/length could not be determined. Considering the low incidence and in absence of a dose response, this finding was considered unrelated to treatment with the test item.
Sperm Analysis – F1-Generation (Cohort 1A):
At 100 mg/kg bw/day, an increase in spermcount of the epididymides was observed of 1.23x compared with controls. This was considered unrelated to treatment as a dose-related response was absent, and since the opposite effect would be expected in case of toxicity.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Cohort 1A: There were no test item-related effects on splenic lymphocyte subpopulations observed up to 100 mg/kg bw/day.
Higher T-cell and T-cytotoxic cell splenic subpopulations and lower B-cell splenic subpopulations observed for females reached statistical significance when compared with controls at 10 mg/kg bw/day. However, these shifts were slight of nature and occurred in the absence of a dose-related response and in absence of any test item-related microscopic findings in the spleen. As such, these shifts were considered to represent biological variability and were considered not to be related to treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
A study according OECD TG 443 with the read-across substance TPGDA (CAS 42978-66-5) (ReachCentrum, 2019C) is available which was conducted to provide an evaluation of possible pre- and postnatal effects on development. In addition, a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats was performed. Based on the results of the extended one generation reproductive toxicity study (ReachCentrum, 2019C) with the read-across substance TPGDA (1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] diacrylate (CAS 42978-66-5), the following no-observed-adverse-effect levels (NOAEL) were established for the read-across substance:
General toxicity (F0 and F1): at least 100 mg/kg bw/day
Reproduction: at least 100 mg/kg bw/day
Developmental: at least 100 mg/kg bw/day
Executive summary:

An oral (gavage) study according OECD TG 443 with the read-across substance TPGDA (CAS 42978-66-5) (ReachCentrum, 2019C) is available which was conducted to provide an evaluation of possible pre- and postnatal effects on development. In addition, a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats was performed. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of the substance on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

This Extended One Generation Reproductive Toxicity Study included Cohort 1 for assessment of reproductive/developmental toxicity. Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 10, 30 and 100 mg/kg bw/day. The animals of the control group received the vehicle, Corn Oil, alone. F0-males were treated for 11-13 weeks, including 10 weeks prior to mating and during the mating and post-mating period, up to and including the day before scheduled necropsy. F0-females were treated for a minimum of 14-18 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 14 weeks. From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B and 1C, were dosed up to and including the day before scheduled necropsy (i.e. for a minimum of 3 weeks). The F1-animals of Cohort Surplus and Spares (not assigned to one of the cohorts) were not dosed. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

F0-Generation: No parental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). At 30 and 100 mg/kg bw/day, a minor increase in sodium was noted in males and additionally, at 100 mg/kg bw/day, urea and potassium levels were slightly increased. As values were only slightly above the range considered normal (sodium) or remained within normal range (urea and potassium) and no corroborative microscopic findings were found, these changes were considered non-adverse. No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (females), body weight, food consumption, haematology, coagulation, thyroid hormone analysis, urinalysis, macroscopic examination, organ weights and microscopic examination).

Reproductive results – F0-generation

No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). No test item-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, sperm analysis, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Developmental results – F0-generation / F1-generation (pre-weaning)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day) during the pre-weaning phase. No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, duration of gestation, viability and weaning indices, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), and macroscopic examination).

Developmental results – F1 generation (post-weaning)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day) during the post-weaning phase. At 100 mg/kg bw/day, increases in neutrophil and lymphocyte levels were found in males. As values remained within normal range and no correlative microscopic findings were found, these changes were considered non-adverse. No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. mortality, clinical signs, body weight, food consumption, coagulation, clinical chemistry, thyroid hormone analysis, urinalysis, splenic lymphocyte subpopulation, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrous, time between vaginal opening and first estrous length and regularity of the estrous cycle, sperm analysis, macroscopic examination, organ weights, ovarian follicle and corpora lutea counts, and microscopic examination, including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Higher doses could not be tested as these were not tolerated (according to the Dose-Range Finder Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test).

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1), the following no-observed-adverse-effect levels (NOAEL) of the read across substance were established:

General toxicity (F0 and F1): at least 100 mg/kg bw/day

Reproduction: at least 100 mg/kg bw/day

Developmental: at least 100 mg/kg bw/day

A maximum dose level of 100 mg/kg bw/day was selected as a NOAEL of 40 mg/kg bw/day was established in a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test with oral exposure of the read across substance in rats. In this preliminary study, adverse findings consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females from 125 mg/kg bw/day.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For Read Across Justification please refer to Section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: A dose level of 100 mg/kg bw/day was selected as the NOAEL of 40 mg/kg bw/day was established in a dose range finder. Here, adverse findings consisted of ulcers and inflammation of the forestomach and hyperplasia squamous cells from 125 mg/kg bw/day on.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Executive summary:

No data available on TMPeoTA (CAS 28961-43-5) in relation to an extended One Generation Reproductive Toxicity Study. Data available on the read-across substance TGPDA (CAS 42978-66-5).

An oral (gavage) study according OECD TG 443 with the read-across substance TPGDA (CAS 42978-66-5) (ReachCentrum, 2019C) is available which was conducted to provide an evaluation of possible pre- and postnatal effects on development. In addition, a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats was performed. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of the substance on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

This Extended One Generation Reproductive Toxicity Study included Cohort 1 for assessment of reproductive/developmental toxicity. Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 10, 30 and 100 mg/kg bw/day. The animals of the control group received the vehicle, Corn Oil, alone. F0-males were treated for 11-13 weeks, including 10 weeks prior to mating and during the mating and post-mating period, up to and including the day before scheduled necropsy. F0-females were treated for a minimum of 14-18 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 14 weeks. From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B and 1C, were dosed up to and including the day before scheduled necropsy (i.e. for a minimum of 3 weeks). The F1-animals of Cohort Surplus and Spares (not assigned to one of the cohorts) were not dosed. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

F0-Generation: No parental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). At 30 and 100 mg/kg bw/day, a minor increase in sodium was noted in males and additionally, at 100 mg/kg bw/day, urea and potassium levels were slightly increased. As values were only slightly above the range considered normal (sodium) or remained within normal range (urea and potassium) and no corroborative microscopic findings were found, these changes were considered non-adverse. No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (females), body weight, food consumption, haematology, coagulation, thyroid hormone analysis, urinalysis, macroscopic examination, organ weights and microscopic examination).

Reproductive results – F0-generation

No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). No test item-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, sperm analysis, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Developmental results – F0-generation / F1-generation (pre-weaning)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day) during the pre-weaning phase. No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, duration of gestation, viability and weaning indices, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), and macroscopic examination).

Developmental results – F1 generation (post-weaning)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day) during the post-weaning phase. At 100 mg/kg bw/day, increases in neutrophil and lymphocyte levels were found in males. As values remained within normal range and no correlative microscopic findings were found, these changes were considered non-adverse. No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. mortality, clinical signs, body weight, food consumption, coagulation, clinical chemistry, thyroid hormone analysis, urinalysis, splenic lymphocyte subpopulation, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrous, time between vaginal opening and first estrous length and regularity of the estrous cycle, sperm analysis, macroscopic examination, organ weights, ovarian follicle and corpora lutea counts, and microscopic examination, including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Higher doses could not be tested as these were not tolerated (according to the Dose-Range Finder Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test).

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1), the following no-observed-adverse-effect levels (NOAEL) of the read across substance were established:

General toxicity (F0 and F1): at least 100 mg/kg bw/day

Reproduction: at least 100 mg/kg bw/day

Developmental: at least 100 mg/kg bw/day

A maximum dose level of 100 mg/kg bw/day was selected as a NOAEL of 40 mg/kg bw/day was established in a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test with oral exposure of the read across substance in rats. In this preliminary study, adverse findings consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females from 125 mg/kg bw/day.

Based on read-across the same NOAEL values for general toxicity, reproduction and development from an extended one generation reproduction study should apply for TMPeoTA (CAS 28961-43-5) as well.

See justification for read-across attached in section 13.

 

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
NA
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
NA
Specific details on test material used for the study:
Identification: Propylidynetrimethanol, ethoxylated, esters with acrylic acid
Appearance: Clear Liquid
Batch: 180330317
Purity/Composition: 99.96%
Test item storage: At room temperature
Stable under storage conditions until: 29 March 2019 (expiry date)

Test Facility Test Item Number: 209397/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Yes, maximum temperature: 220°C
Chemical name (IUPAC, synonym or trade name): Propylidynetrimethanol, ethoxylated, esters with acrylic acid; Trade name: MIRAMER M3130
CAS No: 28961-43-5
EC No: 500-066-5
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar rat strain (Crl:WI(Han) rats) from Charles River Deutschland, Sulzfeld, Germany.

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
On 08 Aug 2018, female Crl: WI(Han) rats were received and on 22 Aug 2018, male Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of dosing, males were 10-11 weeks old and weighed between 281 and 331 g and
females were 13-14 weeks old and weighed between 202 and 240 g. A health inspection was performed before the initiation of dosing.

A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phasewas replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported. Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

The animals were allowed to acclimate to the Test Facility toxicology accommodation for 6 days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males).

On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.

During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment,bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.

Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.

Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.

ENVIRONMENTAL CONDITIONS
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20-23°C with an actual daily mean relative humidity of 44 to 66%. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item. The dosing formulations were stirred continuously during dose administration.


VEHICLE
- Justification for use and choice of vehicle (if other than water):
Corn oil. Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment was complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 1 to 200 mg/mL, Test Facility Study No. 20149504.

- Concentration in vehicle:
0, 20, 60, 200 mg/mL
Details on mating procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis. All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility.

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20149504).

Duplicate sets of samples (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Concentration results were considered acceptable
if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.

Duplicate sets of samples (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20149504) 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. Stability data have been retained in the study records for Test Facility Study No. 20149504.
Duration of treatment / exposure:
Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 51-55 days (most females) or 63 days (one female at 300 mg/kg/day), i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which did not mate or failed to deliver were treated for 52 days or 42 days, respectively.
Frequency of treatment:
The test item and vehicle were administered to the appropriate animals by once daily oral avage 7 days a week for a minimum of 28 days.
Details on study schedule:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Detection of mating was not confirmed in first instance for female no. 45. Evidence of mating was obtained by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the
mating in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle (corn oil)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 male/10 female per dose
Control animals:
yes, concurrent vehicle
Details on study design:
A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported. Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy. During the dosing period, these observations were performed directly after dosing based on the results of the dose range finder

BODY WEIGHT: Yes
- Time schedule for examinations:
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.



Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.
Sperm parameters (parental animals):
Parameters examined included testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology (see attached study report).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- eight pups from each litter of equal sex distribution were selected.; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals (see attachedd study report)

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):
SACRIFICE
Scheduled necropsies were conducted on the following days:
Males: Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 25 (nos. 43) and Day 27 (nos. 56, 58 and 78).
Without evidence of mating: 24 days after the last day of the mating period (no. 51).

All males were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted.

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were
stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

GROSS NECROPSY
- Full Gross necropsy was performed (See attached study report)

HISTOPATHOLOGY / ORGAN WEIGHTS
- A full histopathological evaluation was performed and organ weights collected in accordance to OECD 422 testing guideline requirements (see attached study report). Representative samples of tissues identified were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. All tissues as defined under Histology – F0-Generation (section 4.12.6) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported. For the testes of all selected males of Groups 1 and 4 and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. A peer review on the histopathology data was performed by a second pathologist.

Organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 7-16 PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

The pup of dam 71 that died before scheduled termination was examined externally and sexed (both externally and internally). The stomach of this pup was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day.

On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

GROSS NECROPSY
- Gross necropsy consisted of xternal and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
A full histopathological evaluation was perfomed and oragn weights noted in accordance to requirements in OECD 422 testing guideline (see attached study report).
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Reproductive indices:
Mating index (%); Precoital time; Fertility index (%); Gestation index (%); Duration of gestation;
Offspring viability indices:
Post-implantation survival index (%); Live birth index (%); Percentage live males/females at First Litter Check (%), Viability index (%); Lactation index (%);
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No findings were noted during the weekly arena observations in this study.

Salivation was noted in all animals at 300 and 1000 mg/kg/day. At 300 mg/kg/day, this clinical sign was observed from Day 14 of treatment onwards in both sexes. At 1000 mg/kg/day, salivation was noted in most males and females from Day 6 and 8 of treatment, respectively, onwards. At 1000 mg/kg/day, rales were observed for two males and three females. This clinical sign was noted on incidental occasions, with a maximum of 6 consecutive days.

In addition, for one female at 1000 mg/kg/day (no. 71) hunched posture, piloerection and squeaking were noted on Day 6 of treatment. Based on these symptoms, the dosing of this female was omitted for one day. On Day 7, apart from a slight wheezing, no symptoms were
observed during the morning check and therefore the dosing of this female was resumed. During the remaining premating period, this female presented with hunched posture, rales and piloerection. However, as these symptoms were no longer observed after Day 1 of the mating
period they were considered to be transient and therefore not toxicologically relevant. The other clinical signs noted (i.e. a wound and alopecia) occurred incidentally and represented normal background findings. At the incidence observed, these findings were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were two premature decedents in Group 4 females (1000 mg/kg/day).

Female no.79 was found dead on Day 16 of treatment (Day 2 of mating), showing advanced autolysis and watery-clear fluid in the thoracic cavity. During the clinical observations of this female, no symptoms were noted that were considered to be related to this death. In addition, there were no microscopic findings that indicated a cause of her moribundity. The cause of death was undetermined, and therefore a possible relation with treatment could not be excluded.

Female no.74 was euthanized for humane reasons on Day 21 of treatment (post-coitum Day 4). On the day of sacrifice, breathing abnormalities (i.e. gasping), a hunched posture, closed eyes (ptosis), cold feet and excessive salivation were observed (recorded in Study Daybook). At necropsy, an irregular surface of the forestomach was noted and the intestines were distended with gas. During microscopic evaluations, gavage-related findings in the form of luminal exudate and an acute inflammation of the trachea were observed. Therefore, considering the sudden onset of the clinical signs and the corresponding microscopic findings, the moribundity of female no. 74 was considered to be related to the gavage procedure rather than compound-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight (gain) was reduced in males at 1000 mg/kg/day from Day 8 of treatment onwards, reaching statistical significance during the mating period. This resulted in a 7% lower mean body weight at the end of the treatment period when compared with concurrent control. No remarkable changes in body weight (gain) were noted in males treated up to 300 mg/kg/day. Body weights and body weight gain of treated females remained in the same range as controls
over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded. The statistically significance indicated at the relative food consumption of females at 1000 mg/kg/day on Days 7-11 was considered to be unrelated to treatment since no trend was apparent regarding dose and duration 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):
Haematological parameters of treated rats were considered not to have been affected by treatment. While few changes were statistically significant ( i.e. MCV and MCH in females at 1000 mg/kg/day), the alterations in these hematological parameters were considered unrelated to administration of the test item due to the minimal magnitude of the change, absence of a dose response, and/or as values generally remained within historical control data. The higher mean value of reticulocytes in females at 1000 mg/kg/day, was mostly caused by one female (no. 77) with a value above the P-95 value of historical control range5. As this
concerned a single animal, the higher reticulocytes value was considered incidental and unrelated to treatment. Any statistically significant changes in haematology parameters at 100 or 300 mg/kg/day (i.e.haemoglobin (both sexes) and haematocrit (females)) were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

No toxicologically relevant changes were noted in coagulation parameters. In females at 100, 300 and 1000 mg/kg/day, an increase of 1.6, 4.2 and 6.9% in mean
Activated Partial Thromboplastin Time (APTT) was noted when compared with concurrent control. However, as no statistical significance was achieved and all values remained within historical control range, this increase was considered to be not toxocilogically relevant.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean total bilirubin concentration was increased with 14 and 24% in males and females at 1000 mg/kg/day, respectively, when compared with concurrent controls (statistically significant in females only). All values remained within historical control range. At 1000 mg/kg/day, mean bile acid concentration of males and females were 1.7 and 2.3 times higher, respectively, than concurrent control mean. No statistical significance was achieved, however for 3/5 males and 1/5 females, the individual value was above the upper limit of historical control data. In females at 300 mg/kg/day, mean bile acid concentration was statistically significantly increased when compared with concurrent control. However, as this was mainly caused by two females with extremely high values (nos. 64 and 70), and as values of remaining females at 300 mg/kg/day remained within normal limits and values of high-dose females were all at least 2 times lower, the mean increase in females at 300 mg/kg/day was considered unrelated to treatment.

In females at 1000 mg/kg/day, mean urea concentration was decreased with 14% and mean inorganic phosphate concentration was decreased with 54% when compared with concurrent controls. No statistical significance was achieved and as all values remained within historical control data9, these changes were considered not to be toxicologically relevant.

No other treatment-related changes were noted in clinical biochemistry parameters. Mean cholesterol values of treated males were statistically significanty increased when compared with concurrent controls. As the mean concurrent control value was relatively low and all values of treated males remained within the historical control range10 this change was considered unrelated to treatment. The statistically significant change in mean potassium concentration of males at 100 mg/kg/day was considered to be unrelated to treatment in the absence of a dose-related trend.

Thyroid hormone analyses:
Serum levels of T4 in F0 males were considered not to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength were unaffected by treatment.
At end of treatment, the mean grip strength of the hind legs of the males and the fore legs of the females at 1000 mg/kg/day were both decreased with 14% when compared with concurrent control. These differences were not attributed to treatment since they showed no clear dose-related response and statistical significance was not achieved. Moreover, all values in treated rats remained within the historical control ranges.

A treatment-related lower motor activity was noted in males at 1000 mg/kg/day. When compared with concurrent control, the overall mean total movements and ambulations were decreased with 46% and 43%, respectively (statistically significant for total movements only). Mean values were at the lower limit of historical control ranges. During the first interval of motor activity testing, mean counts of total movements and ambulations in treated males were
decreased when compared with concurrent control. In males at 100 and 300 mg/kg/day, mean counts recovered to similar values as concurrent control during subsequent intervals. The decrease noted in males at 100 and 300 mg/kg/day during the first interval was therefore considered not to be toxicologically relevant. In males at 1000 mg/kg/day, mean values generally remained lower than concurrent control throughout the motor activity testing. All groups showed a habituation profile with a decreasing trend in activity over the duration of the test period.

In females, motor activity was similar between treated and control females. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. All females had regular cycles of 4 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There was 1/10 control treated couple (male 03 and female 43), 3/10 couples treated at 100 mg/kg/day (male 11 & female 51, male 16 & female 56, male 18 & female 58) and 3/10 couples treated at 1000 mg/kg/day (male 34 & female 74, male 38 & female 78, and male 39 & female 79) without offspring. For two males, the lack of mating or offspring was related to reduced sperm of the epididymides and hypoplasia of the testes (male no. 11) or tubular atrophy and vacuolation of the testes (male no. 38). Since there were no other 1000 mg/kg/day males affected and these findings can occasionally be found in control males, these findings were considered to be unrelated to treatment with the test item. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis (male 11 excluded).

As female no. 79 at 1000 mg/kg/day was found dead on Day 2 of the mating period, reproduction data is available of only 9 females in the high dose group.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was considered not to be affected by treatment. Except for one female at 100 mg/kg/day (no. 51), all females showed evidence of mating.
The mating indices were 100% for the control, 300 and 1000 mg/kg/day groups and 90% for the 100 mg/kg/day group. The unsuccessful mating of female no. 51 was linked to the reduced sperm of the epididymides and hypoplasia of the testes noted in male no. 11. Based on the single occurrence in a male of the low dose group and the fact that these findings occasionally can be found in control males, these findings were considered to be unrelated to the test item.

Fertility index was considered not to be affected by treatment. The fertility indices were 90, 78, 100 and 88% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. In total, one controle female, two females at 100 mg/kg/day and one female at 1000 mg/kg/day were not pregnant. For female no. 78, the non-pregnancy was related to tubular atrophy (moderate) and vacuolation (slight) of the testes noted in male no. 38 (1000 mg/kg/day). Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, this was considered unrelated to treatment.

Number of implantation sites was considered not to be affected by treatment.

Precoital time was considered not to be affected by treatment. Most females showed evidence of mating within 4 days.
For developmental effects the following was concluded:

- Gestation index and duration of gestation were considered not to be affected by treatment. All pregnant females had live offspring and thus the gestation index was 100% for all groups.

- No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

- The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 90, 92, 92 and 91% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
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:
other: No reproductive parameters affected
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The slightly lower mean pup body weights noted at 1000 mg/kg/day were mainly caused by a single litter (no. 75). All pups of this litter had a body weigth below the P5-value of historical control data on PND 1 and 4. At PND 13, the body weights of only two males and one
female were within normal limits. Pup body weights in other litters at 1000 mg/kg/day remained within historical control data. Therefore, as the lower pup body weights were observed in a single litter and in the absence of signs that could be related to lack of maternal care of dam 75 (e.g. no milk), the lower pup body weights were considered not to be toxicologically relevant.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment. Values remained within the historical control range.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Histopathological findings:
no effects observed
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Gestation index and duration of gestation were considered not to be affected by treatment. All pregnant females had live offspring and thus the gestation index was 100% for all groups.

No signs of difficult or prolonged parturition were noted among the pregnant females.Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 90, 92, 92 and 91% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Litter size was considered not affected by treatment. Live litter sizes were 11.3, 11.6, 11.9 and 12.1 living pups/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment.
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100% for the control, 100 and 300 mg/kg/day groups, respectively, and 99% for the 1000 mg/kg/day group. One pup of the 1000 mg/kg/day group (dam 71, pup no. 12) was found dead at first litter check. The sex of this pup could not be determined due to cannibalism, therefore it was sexed as female. No toxicological relevance was attributed to this dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 100% for the control, 100 and 1000 mg/kg/day groups, respectively, and 97% for the 300 mg/kg/day group. Three pups at 300 mg/kg/day (dam 62, pup no. 6; dam 63, pup nos. 6 and 12) went missing on PND 4 (most likely cannibalised). No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. The lactation indices were 97% for the control and 100% for the 100, 300 and 1000 mg/kg/day groups, respectively.
Two pups of the control group (dam 49, pup nos. 1 and 6) went missing on PND 5 and 6 (most likely cannibalised). No toxicological relevance was attributed to these missing pups since the mortality incidence remained within the range considered normal for pups of this age.




Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental parameters affected
Key result
Reproductive effects observed:
no
Treatment related:
no
Conclusions:
Wistar Han rats were treated with Propylidynetrimethanol, ethoxylated, esters with acrylic acid (TMPeoTA) by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day in accordance to OECD 422. Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the no-observed-adverse-effect levels (NOAEL) for reproduction and developmental toxicity was evaluated to be1000 mg/kg bw/day.
Executive summary:

The objectives of this OECD 422 study were to determine the potential reproductive and developmental toxicity of Propylidynetrimethanol, ethoxylated, esters with acrylic acid (TMPeoTA) when given orally by gavage for a minimum of 28 days to Wistar Han rats, evaluating male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were 0, 100, 300 and 1000 mg/kg/day, based on the results of a preliminary dose range finding study. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.

No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day). No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the no-observed-adverse-effect levels (NOAEL) for reproduction and developmental toxicity was evaluated to be 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
37
Species:
rat
Quality of whole database:
OECD 422 (study performed with the target substance TMPeoTA)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

An OECD 422 study was performed to determine the potential reproductive and developmental toxicity of Propylidynetrimethanol, ethoxylated, esters with acrylic acid (TMPeoTA) when given orally by gavage for a minimum of 28 days to Wistar Han rats, evaluating male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were 0, 100, 300 and 1000 mg/kg/day, based on the results of a preliminary dose range finding study. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day). No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse effect level (NOAEL) of the substance were established:

Parental (systemic) NOAEL: 1000 mg/kg bw/day.

Parental (local) NOAEL: 100 mg/kg bw/day, based on adverse local forestomach effects.

Reproduction NOAEL: at least 1000 mg/kg bw/day.

Developmental NOAEL: at least 1000 mg/kg bw/day.

 

Furthermore, a study according to OECD TG 443 with the read-across substance TPGDA (CAS 42978-66-5) (ReachCentrum, 2019C) is available which was conducted to provide an evaluation of possible pre- and postnatal effects on development. In addition, a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats was performed. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, the study provided and/or confirmed information about the effects of the substance on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

This Extended One Generation Reproductive Toxicity Study included Cohort 1 for assessment of reproductive/developmental toxicity. Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 10, 30 and 100 mg/kg bw/day. The animals of the control group received the vehicle, Corn Oil, alone. F0-males were treated for 11-13 weeks, including 10 weeks prior to mating and during the mating and post-mating period, up to and including the day before scheduled necropsy. F0-females were treated for a minimum of 14-18 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 14 weeks. From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B and 1C, were dosed up to and including the day before scheduled necropsy (i.e. for a minimum of 3 weeks). The F1-animals of Cohort Surplus and Spares (not assigned to one of the cohorts) were not dosed. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

F0-Generation: No parental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). At 30 and 100 mg/kg bw/day, a minor increase in sodium was noted in males and additionally, at 100 mg/kg bw/day, urea and potassium levels were slightly increased. As values were only slightly above the range considered normal (sodium) or remained within normal range (urea and potassium) and no corroborative microscopic findings were found, these changes were considered non-adverse. No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (females), body weight, food consumption, haematology, coagulation, thyroid hormone analysis, urinalysis, macroscopic examination, organ weights and microscopic examination).

Reproductive results – F0-generation

No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). No test item-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, sperm analysis, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Developmental results – F0-generation / F1-generation (pre-weaning)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day) during the pre-weaning phase. No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, duration of gestation, viability and weaning indices, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), and macroscopic examination).

Developmental results – F1 generation (post-weaning)

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day) during the post-weaning phase. At 100 mg/kg bw/day, increases in neutrophil and lymphocyte levels were found in males. As values remained within normal range and no correlative microscopic findings were found, these changes were considered non-adverse. No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. mortality, clinical signs, body weight, food consumption, coagulation, clinical chemistry, thyroid hormone analysis, urinalysis, splenic lymphocyte subpopulation, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrous, time between vaginal opening and first estrous length and regularity of the estrous cycle, sperm analysis, macroscopic examination, organ weights, ovarian follicle and corpora lutea counts, and microscopic examination, including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Higher doses could not be tested as these were not tolerated (according to the Dose-Range Finder Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test). In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1), the following no-observed-adverse-effect levels (NOAEL) of the read across substance were established:

General toxicity (F0 and F1): at least 100 mg/kg bw/day

Reproduction: at least 100 mg/kg bw/day

Developmental: at least 100 mg/kg bw/day

In conclusion a maximum dose level of 100 mg/kg bw/day was selected as a NOAEL of 40 mg/kg bw/day was established in a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test with oral exposure of the read across substance in rats. In this preliminary study, adverse findings consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females from 125 mg/kg bw/day.

In order to support the read-across approach for reproductive toxicity one additional reproductive toxicity screening assay is available. A study according to OECD TG 422 was conducted to determine the potential toxic effects of the structural analogue and read-across substance TGPDA (CAS 42978-66-5, ReachCentrum, 2019B) when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. Wistar Han rats were treated with the substance by daily oral gavage at dose levels of 40, 125 and 375 mg/kg bw/day. The rats of the control group received the vehicle, Corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results:

Parental toxicity was observed in the (fore)stomach of males from 125 mg/kg and in females at 375 mg/kg. These changes consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other treatment-related but non-adverse changes were observed in the liver at microscopic examination. An absolute increase of 31% and a relative increase of 25% in liver weight was observed at dose 375 mg/kg. At microscopic examination, hepatocellular hypertrophy in the liver was observed at minimal severity and was in the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities. In the kidneys an increase in hyaline droplet accumulation was recorded in males which was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden et al., 1991). This male rat specific protein is not present in female rats nor in higher mammals, including man (Sahota et al., 2013). The increased hyaline droplet accumulation in the male kidneys at 375 mg/kg/day was not accompanied by indicators of tubular damage and therefore this was considered to be non-adverse. Functional observations were not performed for females and therefore, possible treatment related effects on the functional parameters could not be evaluated. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (males), body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), macroscopic examination and organ weights).

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (375 mg/kg bw/day).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (375 mg/kg bw/day).

In conclusion based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the NOAEL of the read-across substance were established:

Parental systemic NOAEL: at least 375 mg/kg bw/day

Parental local NOAEL: 40 mg/kg (based on findings in the (fore)stomach)

Reproduction NOAEL: at least 375 mg/kg bw/day

Developmental NOAEL: at least 375 mg/kg bw/day

 

Conclusion

Based on the effects observed in the combined repeated dose toxicity studies available for the target substance (TMPeoTA) and the source substance (TPGDA) as well as from the results of the extended one-generation study (TGPDA), it is concluded that the target substance (TMPeoTA) is not expected to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In all studies, no impairment of fertility and development of the offspring were observed up to the highest dose tested and tolerability of the substances was limited by local effects in the (fore)stomach rather than any systemic signs of toxicity.

Effects on developmental toxicity

Description of key information

A study was designed to screen for potential maternal, embryotoxic and teratogenic effect of the substance in rats. The study was conducted equivalent or similar to OECD Guideline No. 414 in compliance with Good Laboratory Practices. TMPeoTA was administered orally, admixed in corn oil, to one group of 30 bred rats at a dosage level of 1000 mg/kg bw/day, from gestation Day 6 through 15. Control group consisting of 30 bred rats received the corn oil vehicle on a comparable regimen, at 10 mL/kg. Throughout gestation, all rats were observed twice daily for signs of toxicity. Body weights were recorded at appropriate intervals. All surviving animals were sacrificed on gestation Day 20 for the scheduled caesarean section. Fetuses were sexed, weighed and examined for external, skeletal and soft tissue anomalies and developmental variations. Clinical signs of toxicity such as salivation prior to and following dosing, urogenital matting and hair loss from various body surfaces were observed. Mean body weight gains were significantly reduced over the entire treatment period.No embryotoxic effects were apparent. Developmental variations were not remarkably different between the control and test material group. No statistically significant or biologically meaningful differences occurred between the control and treatment regarding the occurrence of malformations. TMPeoTA produced substantial maternal toxicity at a dose level of 1000 mg/kg bw/day but did not induce a teratogenic or embryotoxic effect in rats, thus NOAEL was established at 1000 mg/kg bw/day.

 

A prenatal developmental toxicity study according to OECD TG 414 on rabbits is available for the structural analogue TGPDA (CAS 42978-66-5) (ReachCentrum, 2018). Since adverse effects were neither observed for maternal nor for developmental toxicity, a maternal and developmental No Observed Adverse Effect Level (NOAEL) of at least 450 mg/kg bw/day was established.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Jun 2018 - 25 Sep 2018
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:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 15-21 weeks old
- Weight at study initiation: between 3043 g and 4268 g
- Housing: housed individually in cages with perforated floors (Ebeco, Germany)
- Diet: ad libitum, Pelleted diet for rabbits (Global Diet 2030 from Harlan)
- Water: ad libitum, Municipal tap water
- Acclimation period: least 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 38 to 97
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension; protected from light. If not used on the same day of preparation, the aliquots were stored in the refrigerator set to maintain 4°C for a maximum of 8 days. On the day of use, they were removed from the refrigerator and allowed to warm to room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically
possible, the dosing formulations and vehicle were continuously stirred until and during dosing.m Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: 1% Aqueous carboxymethyl cellulose with 0.5% Tween 80
Analytical verification of doses or concentrations:
yes
Remarks:
Acquity UPLC system
Details on analytical verification of doses or concentrations:
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
The females arrived on Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
from Day 6 to Day 28 post-coitum
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of the tolerability study and dose range finder and in an attempt to produce graded responses to the test item. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity including mortality was observed at 750 mg/kg. Therefore, the dose levels selected in the current main teratology study were 0, 50, 150 and 450 mg/kg bw/day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
All animals (including female no. 75 euthanized before planned necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. In addition, the gastro-intestinal tract was examined for signs of irritation/corrosion; the oesophagus was examined internally. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed. Each ovary and uterine horn of all animals was dissected and examined as quickly as possible.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]

- Soft tissue examinations: Yes: [all per litter]

- Skeletal examinations: Yes: [all per litter]
Subsequently, the skeletal examination was done on all fetuses from all groups from Groups 1 and 4. Since no possible treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group.

- Head examinations: Yes: [half per litter]
Statistics:
Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
see table 1
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicological relevant findings up to 450 mg/kg.
Reduced faeces production (up to a moderate degree) was observed in 14, 18, 19 and 18 females in the control, 50, 150 and 450 mg/kg groups, respectively, on several days during treatment. As all groups were affected (including the vehicle control group) and in the absence of a dose-related trend, no toxicological significance was attached to this observation. Incidental findings of note were red fluid on the manure tray in one high dose female, and lean appearance and/or piloerection in another high dose female. These findings were considered not to be toxicological relevant as they were observed in the vehicle control group at the same incidence (red fluid on the manure tray, lean appearance), were transient (lean appearance and piloerection) and all gravid females in this study had a normal pregnancy.
One female in Group 3 was noted with missing toes (two missing toes on the right foreleg and 1 missing toe on the left foreleg; taken from the study daybook). This finding was considered to be a congenital abnormality and thus unrelated to treatment with the test item. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain, and/or were observed in control females only. They were therefore of no toxicologically relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatmentwith the test item.
One female from the high dose group had to be euthanized after 4 days of treatment. Immediately after dosing on post-coitum Day 9, she was observed with respiratory problems (gasping) and moribund. At necropsy, red foamy content of the trachea, several reddish foci in left caudal lobe of the lungs and grey-white discolouration of the left caudal lobe of the lungs were noted. Taken together, these findings were indicative of complications during the oral gavage procedure. Therefore, the preterm sacrifice of this female was considered unrelated to the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight, body weight gain before and after correction for (gravid) uterus weight of treated Group 2, 3 and 4 females were unaffected by the treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded up to 450 mg/kg. In the high and mid dose group each, a trend towards slightly lower absolute and relative food consumption compared to the concurrent control group was noted from Days 6-15 and Days 12-15 post-coitum, respectively. As changes were only slight (reaching no statistical significance) and transient (complete recovery was seen from Days 15-18 post-coitum onwards), it was not considered as adverse.
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:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. Moreover, the examination of the gastro-intestinal tract and the internal examination of the oesophagus did not reveal any signs of irritation and/or corrosion. The observation of missing toes in one Group 3 female was considered to be a congenital abnormality and thus unrelated to treatment with the test item. Remaining incidental findings among control and treated animals included watery-clear cysts of the oviducts, alopecia, scabs or an isolated wound. These findings are occasionally seen among rabbits used in these types of study, and in the absence of a dose relationship they were considered unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pre-implantation loss was 3.6, 3.0, 3.0 and 10.5% for the control, 50, 150 and 450 mg/kg groups, respectively. The higher pre-implantation loss observed in the 450 mg/kg group could largely be attributed to one female (no. 76) who had 11 corpora lutea, but only 3 implantation sites, resulting in a pre-implantation loss of 72.7%. After excluding this female pre-implantation loss was 7.2%. As all values remained within the available historical control range the slightly higher pre-implantation loss in the high dose group was considered unrelated to treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Overall, the number of pregnant females, corpora lutea and implantation sites, and pre-and post-implantation loss in the control and treatment groups were considered to be unaffected by treatment. Mean pre-implantation loss was 3.6, 3.0, 3.0 and 10.5% for the control, 50, 150 and 450 mg/kg groups, respectively. The higher pre-implantation loss observed in the 450 mg/kg group could largely be attributed to one female (no. 76) who had 11 corpora lutea, but only 3 implantation sites, resulting in a pre-implantation loss of 72.7%. After excluding this female pre-implantation loss was 7.2%. As all values remained within the available historical control range the slightly higher pre-implantation loss in the high dose group was considered unrelated to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 450 mg/kg.
Mean combined (male and female) fetal body weights were 37.8, 38.2, 39.1 and 38.0 gram for the control, 50, 150 and 450 mg/kg groups, respectively.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 450 mg/kg.
Mean sex ratios (males:females) were 46:54, 50:50, 44:56 and 56:44 for the control, 50, 150 and 450 mg/kg groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.4, 9.8, 10.1 and 9.3 fetuses/litter for the control, 50, 150 and 450 mg/kg groups, respectively. As all values remained within the available historical control range the slightly lower litter size in the high dose group was considered unrelated to treatment.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to 450 mg/kg/day.
Two external malformations were observed in this study. Two fetus of Groups 2 and 4, respectively, both had an open eye with relating findings of the eye/lens noted at soft tissue cephalic examination (i.e. eyelids not joined, lens attached to cornea and/or lens misshapen). Due to the single occurrence of this malformation in a low and high dose fetus, it was considered to be chance finding. A flexure of the carpal and/or tarsal was the other malformation and this occurred in the control group in one fetus (both tarsals, without apparent skeletal origin) and in late resorptions in two fetuses (one or both carpals, not skeletally examined). Because carpal and/or tarsal flexures were observed at the control level only, this was considered spontaneous in origin. External variations were not observed in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment at 450 mg/kg/day.
The only fetus with a malformation was one fetus of the high dose, which had a vertebral anomaly with associated rib anomaly. A vertebral anomaly is the most common skeletal malformation among historical control fetuses and at the single occurrence noted in this study, it is considered spontaneous in origin. A statistical significant increase in the litter incidence of fetuses with caudal shift of pelvic girdle was observed in the high dose group. This variation occurred at an incidence of 21.5%
versus 8.7% per litter in the concurrent control group. Because the litter incidence of caudal shift of pelvic girdle was well within the historical control data range (mean 16.5% per litter; P5 – P95: 2.8 - 34.5% per litter), the higher incidence of this finding in Group 4 was considered to have occurred by chance and not to be toxicologically relevant. All other variations noted were not considered treatment related as they occurred infrequently, at frequencies that were within the range of available historical control data, or were observed in control fetuses only.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 450 mg/kg/day. Visceral malformations occurred in 0 (0), 1 (1), 4 (3) and 3 (2) fetuses (litters) in the control, 50, 150 and 450 mg/kg groups, respectively.
In Group 4, the three malformed foetuses (out of two litters) had a malpositioned testis. This malformation was also noted in Group 3 fetus, but not in fetuses at the control and low dose levels. Nevertheless, because a malpositioned testis is one of the most common visceral malformations in historical control fetuses, this was considered not to be treatment related. The other malformed Group 3 fetuses all had a multiple cardiovascular malformation. As no cases occurred in Group 4 and none of
the individual malformations was uncommon among historical control fetuses these were considered not toxicologically relevant. The affected fetus in Group 2 had a diverticulum of the intestine, which at the single occurrence at the low dose was considered spontaneous in origin.
All variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only, and/or at frequencies that were within the range of available historical control data.
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
The objectives of this study OECD 414 study (ReachCentrum, 2019) were to determine the potential of the read-across substance TGPDA (CAS 42978-66-5) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive.No maternal toxicity was observed in the 50, 150 and 450 mg/kg groups. No developmental toxicity was observed in the 50, 150 and 450 mg/kg bw/d groups. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) of at least 450 mg/kg was established
Executive summary:

The objectives of this study OECD 414 study (ReachCentrum, 2019) were to determine the potential of the read-across substance TGPDA (CAS 42978-66-5) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 50, 150, 450 mg/kg bw/day, based on the results of the dose range finder and tolerability study. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity was observed at 750 mg/kg. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents, and maternal pregnancy data. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test item in 1% aqueous carboxymethyl cellulose with 0.5% Tween 80 were prepared accurately and homogenously.

No maternal toxicity was observed in the 50, 150 and 450 mg/kg groups. No developmental toxicity was observed in the 50, 150 and 450 mg/kg bw/d groups. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) of at least 450 mg/kg was established. Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg bw/d.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For read-across justification please refer to IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg.
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Executive summary:

No data available on TMPeoTA (CAS 28961-43-5) for developmental toxicity in second species (rabbits). Data available on the read-across substance TGPDA (CAS 42978-66-5).

The objectives of this study OECD 414 study (ReachCentrum, 2019) were to determine the potential of the read-across substance TGPDA (CAS 42978-66-5) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 50, 150, 450 mg/kg bw/day, based on the results of the dose range finder and tolerability study. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity was observed at 750 mg/kg. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents, and maternal pregnancy data. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test item in 1% aqueous carboxymethyl cellulose with 0.5% Tween 80 were prepared accurately and homogenously.

No maternal toxicity was observed in the 50, 150 and 450 mg/kg groups. No developmental toxicity was observed in the 50, 150 and 450 mg/kg bw/d groups. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) of at least 450 mg/kg was established. Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg bw/d.

Based on read-across the same NOAEL values for developmental effects in second species (rabbit) should apply for TMPeoTA (CAS 28961-43-5) as well.

See justification for read-across attached in section 13.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 28, 1984 to December 11, 1984
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study is equivalent or similar to OECD Guideline 414 in compliance with Good Laboratory Practices
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague Dawley COBS CD rats
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding laboratories, Inc., Portage, Michigan
- Age at study initiation: 13 wk
- Weight at study initiation: 225-297 g
- Housing: Individually housed in wire mesh cages suspended above cage board
- Diet: Purina certified rodent chow; ad libitum
- Water: Tap water; ad libitum
- Acclimation period: 14 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 0.16 °C
- Humidity (%): 40 %
- Photoperiod (h dark / h light): 12 h dark / 12 h light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solution was prepared with corn oil using magnetic stir plate and bars


VEHICLE: CORN OIL
- Lot/batch no. (if required): May 14 85
- Purity: 100 %
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Presence of vaginal plug referred to as Day 0 of pregnancy
Duration of treatment / exposure:
10 d, from Day 6 to 15 of gestation, inclusive
Frequency of treatment:
Once daily
Duration of test:
25 d
Remarks:
Doses / Concentrations:
0 and 1,000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
30 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a range-finding study, the test dosage (1,000 mg/kg/d) was selected since it was anticipated to induce some degree of maternal toxicity but one which would not likely affect maternal survival.
- Rationale for animal assignment: Random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From Day 0 through 20 of gestation


BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 6, 9, 12, 16 and 20
- Mean body weight changes were calculated for each corresponding interval of gestation additionally for Day 6-16, 16-20
and 0-20


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20



Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
1. The fetal sex ratios were compared by the Chi-square test with Yates' correction factor.
2. The number of litters with malformations and developmental variations were compared by Fisher's Exact Test
3. The numbers of early and late resorptions, dead fetuses and postimplantation losses were compared by the Mann-Whitney U-test
4. Mean numbers of corpora lutea, total implantations, viable fetuses, mean fetal and maternal body weights, and maternal body weight gain at each interval were analyzed by a one-way ANOVA and Dunnett's test
Indices:
- Fetal sex ratios, number of litters with malformations and developmental variations, numbers of early and late resorptions, dead fetuses and postimplantation losses
- Numbers of corpora lutea, total implantations and viable fetuses
Historical control data:
Yes, WlL historical control data appended in the report
Details on maternal toxic effects:
Maternal toxic effects: yes

Details on maternal toxic effects:
- Two rats died due to an intubation error
- Slight decrease in mean body weight gain was observed during gestation Day 12-16
- Significant decrease in mean body weight gain was noticed during gestation Day 6-16
- Clinical signs of toxicity such as salivation prior to and following dosing; urogenital matting and hair loss from various body surfaces were observed
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
other: urogential matting and hair loss
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
- Two malformed fetuses occurred and are known to occur spontaneously in this strain of rat
- No statistically significant or biologically meaningful embyrotoxic/teratogenic effects occurred in the treatment group
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
no

None

Conclusions:
C-661 produced substantial maternal toxicity at a dose level of 1,000 mg/kg bw/day but did not induce a teratogenic or embryotoxic effect in rats.
Executive summary:

A study was designed to screen for potential maternal, embryotoxic and teratogenic effect of the substance in rats. The study was conducted equivalent or similar to OECD Guideline No. 414 in compliance with Good Laboratory Practices.

The test substance was administered orally, admixed in corn oil, to one group of 30 bred rats at a dosage level of 1,000 mg/kg bw/day, from gestation Day 6 through 15. Control group consisting of 30 bred rats received the corn oil vehicle on a comparable regimen, at 10 mL/kg. Throughout gestation, all rats were observed twice daily for signs of toxicity. Body weights were recorded at appropriate intervals. All surviving animals were sacrificed on gestation Day 20 for the scheduled Cesarean section. Fetuses were sexed, weighed and examined for external, skeletal and soft tissue anomalies and developmental variations.

Clinical signs of toxicity such as salivation prior to and following dosing, urogenital matting and hair loss from various body surfaces were observed. Mean body weight gains were significantly reduced over the entire treatment period. No embryotoxic effects were apparent. Developmental variations were not remarkably different between the control and test material group. No statistically significant or biologically meaningful differences occurred between the control and treatment regarding the occurrence of malformations.  

In conclusion, TMPeoTA produced substantial maternal toxicity at a dose level of 1,000 mg/kg bw/day but did not induce a teratogenic or embryotoxic effect in rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A study similal/equivalent to OECD TG 414 (Klimisch score 1) is available fo TMPeoTA. A study similal/equivalent to OECD TG 414 (Klimisch score 1) is available for the read-across substance TGPDA (CAS 42978-66-5).
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 study on Rats:

TMPeoTA was tested for potential maternal, embryotoxic and teratogenic effects in rats (Cytec, 1984). The study was conducted equivalent or similar to OECD Guideline No. 414 in compliance with Good Laboratory Practices. The test substance was administered orally, admixed in corn oil, to one group of 30 bred rats at a dosage level of 1000 mg/kg bw/day, from gestation Day 6 through 15. Control group consisting of 30 bred rats received the corn oil vehicle on a comparable regimen, at 10 mL/kg. Throughout gestation, all rats were observed twice daily for signs of toxicity. Body weights were recorded at appropriate intervals. All surviving animals were sacrificed on gestation Day 20 for the scheduled caesarean section. Fetuses were sexed, weighed and examined for external, skeletal and soft tissue anomalies and developmental variations. Clinical signs of toxicity such as salivation prior to and following dosing, urogenital matting and hair loss from various body surfaces were observed. Mean body weight gains were significantly reduced over the entire treatment period. No embryotoxic effects were apparent. Developmental variations were not remarkably different between the control and test material group. No statistically significant or biologically meaningful differences occurred between the control and treatment regarding the occurrence of malformations.  In conclusion, the substance produced substantial maternal toxicity at a dose level of 1000 mg/kg bw/day but did not induce a teratogenic or embryotoxic effect in rats.

 

Prenatal developmental study on Rabbits:

The objectives of this study (ReachCentrum, 2019) were to determine the potential of the read-across substance TGPDA (CAS 42978-66-5) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 50, 150, 450 mg/kg bw/day, based on the results of the dose range finder and tolerability study. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity was observed at 750 mg/kg. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents, and maternal pregnancy data. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test item in 1% aqueous carboxymethyl cellulose with 0.5% Tween 80 were prepared accurately and homogenously. No maternal toxicity was observed in the 50, 150 and 450 mg/kg groups. No developmental toxicity was observed in the 50, 150 and 450 mg/kg bw/d groups. In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) of at least 450 mg/kg was established. Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg bw/d.

  

Conclusion

Based on the available prenatal developmental toxicity studies with the target substance (TMPeoTA) and the source substance (TGPDA) on rats and rabbits it is concluded that the TMPeoTA is not expected to induce a teratogenic or embryotoxic effect. Impairment of prenatal development was observed neither for the target nor for the source substances up to and including dose levels inducing maternal toxicity. Moreover, in all cases the tolerability of the source and the target substances was limited by local effects in the (fore)stomach rather than any systemic signs of toxicity.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008:

The available information are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information, the target substance (TMPeoTA) is not classified for toxicity to reproduction or developmental toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.

Additional information