Oxybis(methyl-2,1-ethanediyl) diacrylate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

- Oral: Rat: combined 28-day repeated dose oral (gavage) toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422): NOAEL general toxicity = 125 mg/kg bw/day; NOAEL reproductive toxicity at least 375 mg/kg bw/day

- Oral: Rat: EOGRTS (OECD 443; GLP); oral gavage; 10, 30, 100 mg/kg; NOAEL (reproduction) >/=100 mg/kg Read-across to tripropylene glycol diacrylate (Cas No. 42978-66-5)

Link to relevant study records

Reference

Reference 1

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

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

01 Mar 2018 - 16 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males were 10 weeks old, females were 13 weeks old
- Weight at study initiation: males weighed between 251 and 322 g, females weighed between 198 and 263 g
- Housing:
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type). Pups were housed with the dam.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: ad libitum, Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, Municipal tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22
- Humidity (%): 36 to 60
- 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:
Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. 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): Corn oil was selected as vehicle, at request of the Sponsor and based on previously performed studies.
- Concentration in vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- 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
- Once mating had occurred, the males and females were separated.

Analytical verification of doses or concentrations:

yes

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 90% and 110%). 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%).

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 50-62 days, 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 at least 14 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 40-53 days.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 16-day dose range finder with oral gavage administration of the test item in rats and in an attempt to produce graded responses to the test item.

- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

none

Parental animals: Observations and 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 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: 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.

WATER CONSUMPTION AND COMPOUND INTAKE: No data
- 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.

FUNCTIONAL TESTS
Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 9-12). These tests were performed after dosing, after completion of clinical observations and arena observations, if applicable. The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity

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 in all male parental generations:
testis weight, epididymis weight, spermatogenic profiling

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); Blood samples were collected from two of the surplus pups (if possible from one male and one female pup).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

Clinical Observations: Clinical observations were performed at least once daily for all pups. Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter.
Body Weights: Live pups were weighed individually on PND 1, 4, 7 and 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.

GROSS EXAMINATION OF DEAD PUPS:
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
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 were conducted on the following days:
Males (which sired and failed to sire): 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 or 26
Without evidence of mating: 25 days after the last day of the mating period
Females with total litter loss: Dams with no surviving pups were euthanized within 24 hours after the last pup was found dead or missing.

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

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
see table 1 and 2

The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
Selected animals: Tissues identified in Table 2 (except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina
Females with total litter loss: Mammary gland
Remaining animals: Gross lesions/masses

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: only males
- How many animals: 5/sex/group
- Parameters checked: White blood cells (WBC), Neutrophil (absolute), Lymphocyte (absolute), Monocyte (absolute), Eosinophil (absolute), Basophil (absolute), Red blood cells, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no adverse changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Animals fasted: only males
- How many animals: 5/sex/group
- Parameters checked: Alanine aminotransferase (ALAT), Glucose, Aspartate aminotransferase (ASAT), Cholesterol, Alkaline Phosphatase (ALP), Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Urea, Calcium, Creatinine, Inorganic Phosphate (Inorg. Phos)

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (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.

GROSS NECROPSY
Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup. On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.

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 was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
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 Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

see table 5

Offspring viability indices:

see table 5

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation seen after dosing among animals of all dose groups was not considered 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 a sign of systemic toxicity.
Any 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 treatment-related mortalities occurred. One female of the control group was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).

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. Body weight gain was increased in males in the second week of treatment after dosing of 125 and 375 mg/kg, and in the fifth week of treatment after dosing with 375 mg/kg. These changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and values were within the historical control range.

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. The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 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):

No toxicologically relevant changes were noted in haematological parameters. A decreased number of reticulocytes was observed in females at 125 mg/kg bw/day, which was considered unrelated to administration of the test item due to absence of a dose-related trend response. Coagulation parameters (prothrombin time and activated partial thromboplastin time) of treated rats were considered not to have been affected by treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant increases were noted for treated males (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
-increased total protein at 375 mg/kg (6%)
-increased albumin at 375 mg/kg (6%)
-increased calcium at 375 mg/kg (4%)
-increased urea at 40 and 375 mg/kg (27% and 30%, respectively)
The changes in total protein, albumin and calcium were minimal and remained within the historical control range. No dose related trend was observed for the increase in urea and values remained within the historical control range. In addition, a slight increase in sodium was observed at 125 mg/kg (1%), which occurred in the absence of a dose related trend. No treatment-related changes were noted in clinical chemistry parameters in females.

Thyroid hormone analyses:
Serum levels of T4 in F0 males were increased at 125 and 375 mg/kg (35% and 37% increase in mean values compared to concurrent control, respectively). These values remained within the historical control range and the control value was on the lower limit of this range. In addition, no effect was observed in respect to thyroid weight, therefore, this effect was considered not toxicologically relevant.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were not considered to be affected by treatment in males up to 375 mg/kg bw/day.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength was similar between control and treated animals. Motor activity was similar between treated and control groups. 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, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment were noted in the (fore)stomach of males and females and the liver and kidneys of males:
Stomach, squamous cell hyperplasia: was present in 2/5 males starting at 125 mg/kg bw/day up to marked degree and in females at 375 mg/kg bw/day up to moderate degree. This correlated with the macroscopic irregular surface.
Stomach, ulcer forestomach: was present in males starting at 125 mg/kg bw/day at minimal degree.
Stomach, inflammation forestomach: was present in males starting at 125 mg/kg bw/day up to moderate degree.
Liver, hepatocellular hypertrophy: was present in males treated at 375 mg/kg bw/day at minimal degree. This correlated with the increased liver weight.
Kidneys: an increased incidence and severity of hyaline droplet accumulation was present in males treated at 375 mg/kg bw/day up to slight degree.
The remainder 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 findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected by treatment.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was not considered to be affected by treatment. The mating indices were 90%, 100%, 90% and 100% for the control, 40, 125 and 375 mg/kg groups, respectively. Precoital time was not considered to be affected by treatment. The mean precoital times were 2.6, 2.9, 2.6 and 4.5 for the control, 40, 125 and 375 mg/kg groups, respectively. The (non-statistically) higher mean precoital time for group 375 mg/kg was considered unrelated to treatment as 8 females were mated within the first three days of mating and the median precoital time was 3 for all dose groups. Number of implantation sites was not considered to be affected by treatment. Fertility index was not considered to be affected by treatment.
The fertility indices were 100%, 90%, 78% and 90% for the control, 40, 125 and 375 mg/kg groups, respectively. A total of 4 females were not pregnant (one at dose 40 and 375 mg/kg and two females at dose 125 mg/kg). Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, as the incidences were within normal limits, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

375 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:

systemic

Effect level:

375 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:

local

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
For the pup of one female in group 375 mg/kg bw/day who was missing on Day 4, less milk in the stomach and a lean appearance were noted on Day 1. Alopecia of the whole body was noted for several pups of the control, 40 and 375 mg/kg groups. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

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 99%, 100%, 99% and 99% for the control, 40, 125 and 375 mg/kg groups, respectively. One pup of the control group (litter 48) was killed in extremis on Day 1 of lactation. One pup at 125 mg/kg and one pup at 375 mg/kg were missing on PND 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/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. Litter size was not considered affected by treatment.
Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not considered to be affected by treatment.

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

Haematological 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 not considered to be affected by treatment

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 375 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:

not examined

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.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups. Gestation index and duration of gestation were not considered to be affected by treatment. Except for one female (no. 49) of the control group, all pregnant females had live offspring. The gestation indices were 89% for the control and 100% for the 40, 125 and 375 mg/kg groups, respectively. 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 not considered to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92%, 79%, 88% and 85% for the control, 40, 125 and 375 mg/kg groups, respectively. The slightly lower post-implantation survival index for the 40 mg/kg group was considered the result of the low litter size of female no. 51 which only had one pup, but 10 implantation sites. The survival indices for all other groups were considered within normal range.Sex ratio was not considered to be affected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

375 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

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07/2020 to 04/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Jul 2016

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2000

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2016

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2000

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Oct 2008

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Jul 2000

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

CAS no: 57472-68-1

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle, Cedex, France.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 12 weeks old for males and 12 - 14 weeks old for females.
- Weight at study initiation: 250 - 350 g for males and 200 - 250 g for females
- Housing: On arrival and following the pre-test (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 (Makrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Makrolon 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 (dimensions: 48.3 x 26.7 x 20.3 cm) without cage enrichment, bedding material, food and water. Animals were separated during designated procedures/activities.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures
- Water: 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.
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY
- The feed was analysed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed, and results of these analyses are on file at the test facility.

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

IN-LIFE DATES: From: 19 Aug 2020 To: 13 Nov 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation of dosing solutions: weekly
- Preparation of dosing solutions: Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator, stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.
- Storage temperature of dosing solutions: 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.

VEHICLE
- Concentration in vehicle: 8, 25, 75 mg/mL
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurred or until 14 days had elapsed
- Proof of pregnancy: palpation / vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples for Analysis: Duplicate middle samples for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis).
- Sample Volume: Approximately 500 mg accurately weighed.
- Acceptance Criteria: For concentration, the criteria for acceptability will be mean sample concentration results within or equal to ± 10 % for solutions or ±15 % for suspensions of target concentration. For homogeneity, the criteria for acceptability will be a coefficient of variation (CV) of concentrations of ≤ 10 % for each group.

Duration of treatment / exposure:

Males for 29 days, females that delivered for 50 - 62 days, females which failed to deliver or had a total litter loss for 39 - 44 days.

Frequency of treatment:

Once a day, seven days a week.

Details on study schedule:

Females were dosed for two consecutive weeks prior to pairing (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 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 39 - 44 days.

Dose / conc.:

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

Remarks:

Group 2. Low dose.

Dose / conc.:

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

Remarks:

Group 3. Mid dose.

Dose / conc.:

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

Remarks:

Group 4. High dose.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration of DPGDA in rats (Test Facility Study No. 20236861) and in an attempt to produce graded responses to the test item. 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.
- Fasting period before blood sampling for clinical biochemistry: Males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Females were not fasted.
- Culling and pups selection for blood collection (serum hormone) at necropsy:
Culling - On Day 4 post partum: to reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected.
Blood collection in pups - Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed at least once daily, up to the day prior to necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade were predefined at 1, 2, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (i.e. maximum grade 1) were scored
Once before the first administration of the test item and at weekly intervals during the treatment period, animals were observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

CLINICAL OBSERVATIONS (FUNCTIONAL OBSERVATION BATTERY TESTS)
- Time schedule: Once during the treatment period. The selected 5 males were tested once during Week 4 of treatment and the selected 5 females were tested once during the last week of lactation (i.e. Postnatal day (PND) 6 - 13). These tests were performed after clinical observations (including arena observation, if applicable).
The following tests were performed: (1) Hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent). (2) Fore- and hind-limb grip strength were recorded as the mean of three measurements, using a grip strength meter. (3) Locomotor activity (recording period: 1 hour under normal laboratory light conditions) were tested using the Kinder Scientific Motor Monitor System. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed 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 post-partum day 1, 4, 7, and 13. In order to monitor the health status animals may be weighed more often.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
Weekly, except for males and females which are housed together for mating and for females without evidence of mating. Food consumption of mated females were measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post-partum day 1, 4, 7, and 13. Food consumption were quantitatively measured.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Regular basis throughout the study.
Water consumption were monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood were collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus in the animal facility. Additional blood samples may be obtained (e.g. due to clotting of non-serum samples) in both the animal facility and in the necropsy room if permissible sampling frequency and blood volume are not exceeded. After collection, samples were transferred to the appropriate laboratory for processing.
- Anaesthetic used for blood collection: Yes, using isoflurane.
- Animals fasted: Males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before blood sampling, but water were available. Females will not be fasted overnight.
- How many animals: All parental animals (except for animals which were sacrificed in extremis or found dead and females with total litter loss.
- Parameters examined: white blood cell count (WBC), reticulocytes (absolute), neutrophils (absolute), red blood cell distribution width (RDW), lymphocytes (absolute), haemoglobin, monocytes (absolute), haematocrit, eosinophils (absolute), mean corpuscular volume (mcv), basophils (absolute), mean corpuscular haemoglobin (MCH), large unstained cells (LUC) (absolute), red blood, cell count mean corpuscular, haemoglobin concentration (MCHC) and platelets.
For Haematology: Target volume: 0.5 mL; anticoagulant used: K3-EDTA (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). A blood smear were prepared from each haematology sample. Blood smears were labelled, stained, and stored. Blood smears were evaluated when required to confirm analyser results
For Coagulation: Target Volume: 0.45 mL and anticoagulant used: Citrate (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). Coagulation Parameters: Prothrombin time (PT and Activated partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see Haematology.
- Animals fasted: see Haematology.
- How many animals: see Haematology.
- Parameters examined: Alanine aminotransferase (ALT) Creatinine, Aspartate aminotransferase (AST) Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin Chloride, Bile Acids, Calcium, Urea and Inorganic Phosphate (Inorg. Phos).
For Clinical Chemistry: Target volume: 0.5 mL; anticoagulant: Li-Heparin (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). Processing: to plasma

SERUM HORMONES: Yes
- Time of blood sample collection: see Haematology.
- Animals fasted: see Haematology.
- How many animals: see Haematology. Measurement of total T4 was conducted for parental males.
- Parameters examined: Thyroid Hormone Parameters Thyroxine (T4) and Thyroid-Stimulating Hormone (TSH; only if required)
For Thyroid Hormones: Target Volume: 1.0 mL; anticoagulant: not applicable for serum (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). After clotting and centrifugation, serum has been used.
For males, 150 μL serum for measurement of total T4 and the remaining. For females, the serum will be used for possible future measurement of total T4 and/or thyroid-stimulating hormone TSH.

Oestrous cyclicity (parental animals):

VAGINAL LAVAGE AND OESTROUS CYCLE
Oestrous 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 (pre-test 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 oestrous. This was done for all females, except for females that had to be euthanised in extremis

Sperm parameters (parental animals):

Parameters examined in male parental generation:
Testes were evaluated to assess the progression of stages of the spermatogenic cycle, cell associations, and proportions expected to be present during spermatogenesis along with assessment of interstitial and supporting cell types (Leydig cells, macrophages, vasculature, and rete testis). Any cell- or stage-specificity of testicular findings were noted.
For the testes of all selected males of Groups 1 and 4, and all males that fail to sire or died before mating detailed qualitative examination will be made, taking into account the tubular stages of the spermatogenic cycle. The examination will be conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.

Litter observations:

PUPS IDENTIFICATION, WEIGHT AND OBSERVATIONS
- Pups were identified on postnatal Day (PND) 1. Postnatal Day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Pups were randomised per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.
- Pups were observed daily for general health/mortality. 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 were performed at least once daily for all pups.
- Live pups were weighed individually on PND 1, 4, 7 and 13. Sex was externally determined for all pups on PND 1 and 4.

ANOGENITAL DISTANCE (AGD)
- Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalised 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.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
EUTHANASIA
Scheduled deaths: animals surviving until scheduled euthanasia were weighed, and deeply anesthetised using isoflurane and subsequently exsanguinated and subjected to a full post-mortem examination.
Unscheduled deaths: if necessary for humane reasons, animals were euthanised as per Test Facility SOPs. These animals were deeply anesthetised using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained but not weighed.

NECROPSY
Scheduled necropsies are summarised below:
- Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
- Females which delivered: post-partum day 14 - 16.
- Females which failed to deliver: With evidence of mating: Post-coitum Days 26 - 27
- Females with total litter loss: Dams with no surviving pups were euthanised within 24 hours after the last pup is found dead or missing.
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.

ORGAN WEIGHTS ANIMALS
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanised in poor condition or 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.
- Organs weighed at necropsy for all selected animals: cervix*; epididymis#; gland adrenal#; gland coagulation#$; gland parathyroid@; gland prostate; gland seminal vesicle#; gland thyroid#; heart; kidney#; liver; ovaries#; spleen; testes#; thymus and uterus, where:
* = weighed together with the uterus; # = paired organ weight; $ = weighed together with the seminal vesicles; @ = weighed together with the thyroid.
- Organs weighed at necropsy for all remaining animals (incl. Males that Failed to Sire, Females that Failed to Deliver and Females with Total Litter Loss) were as follows: epididymis*; gland coagulation*#; gland parathyroid$; gland prostate; gland seminal vesicle*; gland thyroid*; testes*, where:
* = Paired organ weight; # = Weighed together with the seminal vesicles; $ = Weighed together with the thyroid.

TISSUES FIXED AND PRESERVED
Representative samples of the tissues identified in the table below 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.
- Tissue Collection and Preservation for all Selected Animals, all Animals that were Sacrificed in extremis and Females with Total Litter Loss: Animal identification: Artery; aorta; body cavity; nasopharynx; bone marrow; bone, femur; bone, sternum; brain (eight levels); cervix; epididymides*; oesophagus; eye*; gland, adrenal; gland, coagulation; gland, harderian* #; gland, lacrimal; gland, mammary; gland, parathyroid$; gland, pituitary; gland, prostate; gland, salivary; gland, seminal vesicle; gland, thyroid; gross lesions/masses; gut-associated lymphoid tissue; heart; kidney; large intestine, cecum; large intestine, colon; large intestine, rectum; larynx; liver; lung; lymph node (mandibular and mesenteric site); muscle, skeletal; nerve, optic#; nerve, sciatic; ovaries; pancreas; skin; small intestine, duodenum; small intestine, ileum; small intestine, jejunum; spinal cord; spleen; stomach; testes*; thymus; tongue; trachea; urinary bladder; uterus and vagina; where:
* = Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours; # = Only collected if present in the routine section of the eye. Part of the optic nerve attached to the eye was Fixed in Modified Davidsons’s fixative. The remaining part of the optic nerve was placed in formalin; $ = Only collected if present in the routine section of the thyroid.
- Tissue Collection and Preservation for all Remaining Animals (incl. Males that Failed to Sire and females that failed to deliver): animal identification
cervix; epididymis*; gland, coagulation; gland, mammary; gland, parathyroid#; gland, pituitary; gland, prostate; gland, seminal vesicle; gland, thyroid; gross lesions/masses; ovaries; testes*; uterus and vagina, where:
* = Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours; # = Only collected if present in the routine section of the thyroid.

HISTOPATHOLOGICAL EXAMINATION
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin:
- Selected animals, and unscheduled deaths (sacrificed in extremis): see Tissues identified in above for “Tissue Collection and Preservation for all Selected Animals, all Animals that were Sacrificed in extremis and Females with Total Litter Loss” (except animal identification, aorta; nasopharynx; oesophagus; harderian gland; lacrimal gland; salivary gland; larynx; optic nerve; pancreas; skin and tongue).
- Males that failed to sire, females that failed to deliver pups and females with total litter loss: cervix; epididymis; coagulation gland; prostate gland; seminal vesicles; ovaries; testes; uterus and vagina.
- Females with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.

Postmortem examinations (offspring):

- Sacrafice: Pups, younger than 7 days were euthanised by decapitation. All remaining pups (PND 14 - 16), except for the two pups per litter selected for blood collection were euthanised by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20 %). The pups selected for blood collection on PND 14 - 16 were anesthetised using isoflurane followed by exsanguination.
- Scheduled euthanasia: On PND 4, the surplus pups were euthanised by decapitation. From two surplus pups per litter, blood was collected, if possible for exceptions. All remaining pups were euthanised 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.
- Unscheduled Deaths: Recognisable foetuses of females that were euthanised in extremis were examined externally and sexed (both externally and internally). Pups that died or were euthanised before scheduled termination were examined externally and sexed (both externally and internally). Pups found dead during the weekend were fixed in identified containers containing 70 % ethanol as they were not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
- Blood collection and thyroid hormone determination (T3, T4 and TSH): blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup). The pups selected for blood sampling were the same pups as selected for thyroid preservation.
Target volume: for PND 4 pups: 0.5 mL in total (pooled), and PND 14 - 16 pups: 1.0 mL per pup. After clotting and centrifugation serum was used as follows: for PND 4 Pups the pooled serum was used for possible future measurement of total
T4 (added by study plan amendment if applicable). And fFor PND 14 - 16 Pups 150 μL serum for measurement of total T4, and the remaining volume of serum for possible future measurement of TSH (added by study plan amendment if applicable).

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 analysed 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 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
- 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). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
-Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
- 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 Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

For each group, the following calculations were performed. Group mean values of precoitally time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:
- Mating index (%) = 100 x (Number of females mated / Number of females paired)
- Pre-coital time = Number of days between initiation of cohabitation and confirmation of mating.
- Fertility index (%) = 100 x (Number of pregnant females / Number of females mated)
- Gestation index (%)= 100 x (Number of females with living pups on Day 1 /Number of pregnant females)
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition.
- Post-implantation survival index (%) = 100 x (Total number of offspring born / Total number of uterine implantation sites)

Offspring viability indices:

- Live birth index (%) = 100 x (Number of live offspring on Day 1 after littering/ Total number of offspring born)
- Percentage live males at First Litter Check (%) = 100 x (Number of live male pups at First Litter Check / Number of live pups at First Litter Check)
- Percentage live females at First Litter Check (%)= 100 x (Number of live female pups at First Litter Check / Number of live pups at First Litter Check)
- Viability index (%)= 100 x (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering)
- Lactation index (%)= 100 x (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling))

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Two females at 375 mg/kg/day, showed piloerection on respectively Days 8 - 9 post-coitum and Days 3 - 4 of lactation. Another female also had a slightly pale and/or lean appearance between Days 2 and 5 of lactation, accompanied by a body weight loss up to 12 %. As clinical signs were transient and occurred in one or two animals only, these were considered incidental and not toxicologically relevant.
Salivation seen after dosing among males and/or females of all treatment groups was considered to be a local test item effect and likely resulting from irritating properties of the test item. Any other clinical signs noted during the treatment period (i.e. alopecia, scabs, rales and swelling of the vagina) 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/or did not show any apparent dose-related trend. At the incidence observed, these were considered not to be signs of toxicological relevance. No findings were noted during the weekly arena observations in this study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period 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 weight gain in males at 375 mg/kg/day was decreased compared with control on Day 8 of treatment, which recovered up to similar levels as control at the end of the mating period. Absolute body weights in males at 375 mg/kg/day were lower compared with control from start of treatment onwards (0.97x of control on Day 1 of treatment down to 0.95x of control on Day 15 of the mating period). Body weights and body weight gain in females were considered to have been unaffected by treatment with the test item. Considering the magnitude of change and as Group 4 males just happened to be the smallest rats in this study, no toxicological relevance was attached to this finding.

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 was considered unaffected by treatment with the test item up to 375 mg/kg/day. A trend towards lower food consumption was recorded in males and females at 375 mg/kg/day during the first week of treatment, with subsequent complete recovery. No toxicological relevance was attached to this finding.

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):

At 375 mg/kg/day, the following changes in haematology parameters were recorded (statistically significant unless stated otherwise): (1) Increased neutrophil counts in males and females (1.83x and 1.20x of control, respectively; statistically significant in males only). This was considered test item-related. (2) Decreased mean corpuscular haemoglobin concentration (MCHC) in females (0.95x of control). In the absence of changes in correlating parameters, this was considered not related to treatment with the test item. The decreased platelet counts in males at 40 mg/kg/day were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend. Coagulation parameters of treated rats were considered not to have been affected by treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following changes in clinical biochemistry parameters were recorded in females at 375 mg/kg/day: (1) Increased cholesterol levels in females at 375 mg/kg/day (1.38x of control). This was considered test item-related. (2) Increased calcium levels in females at 375 mg/kg/day (1.07x of control). This was considered test item-related. The increased mean glucose concentration recorded in females at 40 mg/kg/day was considered to be unrelated to treatment with the test item as it occurred in the absence of a dose-related trend. No toxicologically relevant changes were noted in clinical biochemistry parameters in males.

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormone analyses: Serum levels of T4 in parental males were considered unaffected by treatment with the test item up to 375 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals up to 375 mg/kg/day. Motor activity was considered to be unaffected by treatment with the test item. The higher mean values of total movements and ambulations in males at 125 mg/kg/day (not statistically significant) were caused by two animals only. Increased motor activity in females at 125 mg/kg/day was mainly apparent in intervals 5 - 7, after which the habituation profile recovered to being comparable with controls. In the absence of a dose-related trend, this was considered not related to treatment with the test item. All groups (except Group 3 females) showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with DPGDA were noted in the stomach of both sexes and liver of females. A combination of findings was recorded for the forestomach and consisted of mixed cell inflammation (up to slight), squamous cell hyperplasia (up to marked) and/or hyperkeratosis (up to moderate) in most animals at 375 mg/kg/day. Low degrees of squamous cell hyperplasia and hyperkeratosis was recorded for the forestomach in a single male at 125 mg/kg/day. In addition, a forestomach ulcer at minimal degree was recorded in a single male at 125 and at 375 mg/kg/day and in a single female at 375 mg/kg/day.
In two females at 375 mg/kg/day, a minimal degree of increased mitotic figures was recorded for the liver. The remainder 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 findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the oestrous cycle were considered not to have been affected by treatment with the test item. Most females had regular cycles of 4 days. During the pre-mating period, oestrous cycle regularity could not be determined for one female at 125 mg/kg/day as for this female only one oestrous cycle was covered due to the presence of di-oestrous during the first 7 days. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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.

Reproductive performance:

no effects observed

Description (incidence and severity):

The mating index was unaffected by treatment with the test item. All females showed evidence of mating. Pre-coital time was considered not to be affected by treatment with the test item. All paired females showed evidence of mating within 4 days, except for two females (40 and 375 mg/kg/day) for which mating took 13 and 12 days, respectively. Given the single occurrence and in the absence of a dose-related trend, this longer mating period was considered not related to treatment with the test item.

The number of implantation sites was considered not to be affected by treatment with the test item. Mean number of implantation sites were 12.6, 12.4, 12.2 and 12.2 for the control, 40, 125 and 375 mg/kg/day groups, respectively.
The fertility index was not affected by treatment with the test item. The fertility indices were 90 % for the control and 375 mg/kg/day groups, and 100 % for the 40 and 125 mg/kg/day groups.
There were 1/10 couples of the control group (one male and female) and 1/10 couples at 375 mg/kg/day (one of each sex) with no offspring. Total litter loss was observed in 1/10 females of the control group (one female) and in 1/10 females at 375 mg/kg/day (another female). No abnormalities were seen in the reproductive organs of these animals, which could account for their lack of offspring. For a female individual, no findings were seen in the reproductive organs, and the difficulty to deliver in this single female at 40 mg/kg/day was regarded unrelated to the 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.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

gross pathology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 375 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item. The pup of one female (40 mg/kg/day) that was missing on PND 3, was cold to touch, and had a small and dehydrated appearance on PND 2. For the pup of another female (control group) that was missing on PND 6, a lean appearance was noted on PND 4 and 5. 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.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Two pups of the control group, three pups at 40 mg/kg/day, and three pups at 375 mg/kg/day were found dead at first litter check; there were no dead pups in the 125 mg/kg/day group on PND 1. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups at 375 mg/kg/day were slightly reduced on Day 1 (0.97x of control for both males and females; not statistically significant), progressing up to Day 13 (0.89x and 0.90x of control for males and females, respectively; statistically significant for males only).

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

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalised for body weight) in male and female pups was considered not to be affected by treatment with the test item

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment with the test item up to 375 mg/kg/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:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment with the test item. For two pups of one litter (375 mg/kg/day) reduced size of the left testis and epididymis and/or agenesis of the left testis were recorded. Since these abnormalities were limited to two pups from the same litter, they were considered of hereditary origin rather than to be related to treatment with the test item. For the single pup of one female (40 mg/kg/day) that was found dead on PND 2 and the two pups of a different female (375 mg/kg/day) that were found dead at first litter check, no milk was observed in the stomach. The nature and incidence of these and other macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

SERUM T4 LEVELS
Serum T4 levels in male and female PND 14 - 16 pups were considered not to be affected by treatment with the test item. Any not statistically significant changes remained within the normal ranges of biological variation and were therefore regarded as not toxicologically relevant.

GESTATION INDEX AND DURATION
Gestation index (females with living pups on Lactation Day 1 compared to the number of pregnant females) and duration of gestation was not affected by treatment with the test item. Except for one female at 40 mg/kg/day with an incomplete delivery (Female No. 53), all pregnant females had live offspring. This resulted in a gestation index of 90% for the 40 mg/kg/day group, compared to 100% for the control, 125 and 375 mg/kg/day groups.

PARTURITION/MATERNAL CARE
One female in the 40 mg/kg/day group showed difficulties with parturition. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

POST-IMPLANTATION SURVIVAL INDEX
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 88, 85, 83 and 88 % for the control, 40, 125 and 375 mg/kg/day groups, respectively.

LITTER SIZE
Litter size was considered not affected by treatment with the test item. Live litter sizes were 10.8, 11.4, 10.1 and 10.4 living pups/litter for the control, 40, 125 and 375 mg/kg/day groups, respectively.

LIVE BIRTH INDEX
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 with the test item. The live birth indices were 98, 97, 100 and 97 % for the control, 40, 125 and 375 mg/kg/day groups, respectively.

VIABILITY INDEX
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 with the test item. Viability indices were 91, 97, 100 and 89 % for the control, 40, 125 and 375 mg/kg/day groups, respectively. Note that by mistake culling of one Litter (40 mg/kg/day) was performed after first litter check on PND 1 instead of PND 4. These five culled pups are included in the viability index for the 40 mg/kg/day group. Nine pups (all from the same Litter) in the control group and three pups at 40 mg/kg/day two Litters) were found dead or missing on PND 2 - 4. One female (375 mg/kg/day) had a total litter loss on PND 2. Eight pups were missing and two pups were found dead (severely cannibalized). Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend.

LACTATION INDEX
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item. The lactation indices were 98 % for the control group and 100% for each the 40, 125 and 375 mg/kg/day groups. One pup of the control group (One female, with a total litter loss) was found missing on PND 6, after it was noted with a lean appearance on PND 4 and 5. Most likely it was cannibalised. No toxicological relevance was attributed to this finding since it occurred in the control group.

SEX RATIO
Sex ratio was considered not to be affected by treatment with the test item.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 375 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1 Summary of all investigated reproductive indices

	Group 1	Group 2	Group 3	Group 4
	Control	40 mg/kg/day	125 mg/kg/day	375 mg/kg/day
Mating index (%)	100	100	100	100
(Females mated / Females paired) * 100
Fertility index (%)	90	100	100	90
(Pregnant females / Females mated) * 100
Gestation index (%)	100	90	100	100
(Females with living pups on Day 1 / Pregnant females) * 100
Post-implantation survival index (%)	88	85	83	88
(Total number of offspring born/Total number of uterine implantation sites) * 100
Live birth index (%)	98	97	100	97
(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100
Viability index (%)	91	97	100	89
(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100
Lactation index (%)	98	100	100	100

Table 2 Organ weights summary females

		Group 1 Control	Group 2 40 mg/kg/day	Group 3 125 mg/kg/day	Group 4 375 mg/kg/day
End Of Treatment
Body W. (Gram)	Mean	277	283	289	276
	St.Dev	15	21	28	26
	N	10	9	10	10
Brain (Gram)	Mean	1.86	1.91	1.89	1.86
	St.Dev	0.04	0.09	0.02	0.03
	N	5	5	5	5
Heart (Gram)	Mean	0.799	0.807	0.851	0.828
	St.Dev	0.037	0.072	0.074	0.054
	N	5	5	5	5
Liver (Gram)	Mean	11.41	10.89	11.93	11.89
	St.Dev	0.56	1.21	0.54	0.79
	N	5	5	5	5
Thyroids (Gram)	Mean	0.0155	0.0146	0.0157	0.0144
	St.Dev	0.0031	0.0025	0.0025	0.0023
	N	10	9	10	10
Thymus (Gram)	Mean	0.191	0.195	0.218	0.216
	St.Dev	0.031	0.018	0.025	0.024
	N	5	5	5	5
Kidneys (Gram)	Mean	1.87	1.81	1.93	1.82
	St.Dev	0.08	0.14	0.13	0.10
	N	5	5	5	5
Adrenals (Gram)	Mean	0.081	0.068	0.077	0.064 *
	St.Dev	0.007	0.009	0.010	0.006
	N	5	5	5	5
Spleen (Gram)	Mean	0.507	0.475	0.502	0.479
	St.Dev	0.035	0.097	0.014	0.037
	N	5	5	5	5
Ovaries (Gram)	Mean	0.108	0.104	0.108	0.105
	St.Dev	0.013	0.010	0.008	0.010
	N	5	5	5	5
Uterus (Gram)	Mean	0.366	0.331	0.343	0.321
	St.Dev	0.066	0.036	0.017	0.025
	N	5	5	5	5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3 Reproduction data summary

	Group 1	Group 2	Group 3	Group 4
	Control	40 mg/kg/day	125 mg/kg/day	375 mg/kg/day
Females paired	10	10	10	10
Females mated	10	10	10	10
Pregnant females	9	10	10	9
Females with incomplete delivery	0	1	0	0
Females with total litter loss on Day 2	0	0	0	1
Females with total litter loss on Day 6	1	0	0	0
Females with living pups on Day 1	9	9	10	9

Conclusions:

In a combined 28-Day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422), based on the occurrence of macroscopic and microscopic stomach findings and increased neutrophil counts of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 125 mg/kg bw/day for males and females. No effects on reproduction were observed. The NOAEL for reproductive toxicity was considered to be at least 375 mg/kg bw/day both for males and females.

Executive summary:

The objectives of this OECD TG 422 study performed in compliance with CLP was to determine the potential toxic effects and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception of DPGDA. In addition, parental and reproduction (up to and including implantation) No Observed Adverse Effect Levels (NOAELs) were evaluated .Wistar Han rats were administered the test substance dose levels of 0, 40, 125, 375 mg/kg/day, based on the results of the Dose Range Finder. With the exception of the control group, all animals (10 animals/sex/group) received the test substance orally via gavage for a minimum period of 28 days using corn oil as vehicle at a dose volume of 5 mL/kg body weight. Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, measurement of thyroid hormone T4 (parental males), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction parameters were determined: mating and fertility indices, pre-coital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14 - 16 pups)).

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. Parental toxicity was observed at the highest dose level tested (375 mg/kg/day). One female at 40 mg/kg/day was sacrificed in extremis on post-coitum Day 23 because of delivery difficulties. This female delivered 3 dead pups and showed hunched posture, piloerection and pale appearance. At necropsy, 8 dead foetuses were present in the uterus. Microscopically the uterus showed characteristics of pregnancy. Findings regarded secondary to the presence of dead foetuses in the uterus were moderate multifocal hepatocellular necrosis of the centrilobular area of the liver and multifocal thrombi in the lungs. These findings in liver and lungs were attributed to the poor condition of this female. This death was regarded to be related to delivery difficulties and unrelated to the treatment with the test item. Significant body weight loss in one female at 375 mg/kg/day during the first days of lactation coincided with piloerection and a slightly pale and lean appearance. At the incidence observed, this was considered not toxicologically relevant. Body weight gain in males at 375 mg/kg/day was decreased compared with control on Day 8 of treatment, which recovered up to similar levels as control at the end of the mating period. Absolute body weights in males at 375 mg/kg/day were decreased compared with control from Day 1 of treatment up to the day of scheduled necropsy. Considering the magnitude of change and as Group 4 males just happened to be the smallest rats in this study, no toxicological relevance was attached to this finding. Relationships were suspected between gross necropsy (irregular surface of the forestomach), clinical pathology (increased neutrophil concentrations) and histopathology observations. The combination of microscopic stomach findings observed in most males and females treated at a dose of 375 mg/kg/day consisted of slight to marked squamous cell hyperplasia with slight to moderate hyperkeratosis and minimal to slight mixed cell inflammation. In addition, a forestomach ulcer at minimal degree was recorded in a single male at 125 and at 375 mg/kg/day and in a single female at 375 mg/kg/day. This combination of findings is considered adverse at the recorded incidences and severities. Mixed cell inflammation was recorded in all selected males, compared to 3/5 selected females and likely correlated to the increased neutrophils recorded for males at 375 mg/kg/day. The forestomach findings and salivation were considered to be local test item effects and likely resulting from irritating properties of the test item. The low severities of stomach findings in a single male at 125 mg/kg/day only were considered to be non-adverse. Increased mitotic figures were recorded in the liver of two females at 375 mg/kg/day and a relation to treatment with the test item could not be excluded. This alteration was considered to be non-adverse at the recorded minimal severity.

Non-adverse changes were recorded in clinical biochemistry parameters: increased cholesterol levels and increased calcium levels in females at 375 mg/kg/day. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, body weight, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), food consumption, clotting parameters, male T4 thyroid hormone levels, and organ weights). No reproductive toxicity was observed up to 375 mg/kg/day.

No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, pre-coital time, number of implantations, oestrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). Adverse changes in pup body weights were recorded at 375 mg/kg/day. Pup body weights were slightly reduced on PND 1 progressing up to approximately 10 % reduction on PND 13.

In conclusion, based on the results of this combined 28-Day repeated dose toxicity study the established No Observed Adverse Effect Level (NOAEL) of DPGDA (CAS 57472-68-1) was 125 mg/kg/day based on macroscopic and microscopic stomach findings and increased neutrophil counts). The NOAEL for reproductive toxicity was considered to be at least 375 mg/kg bw/day both for males and females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

375 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant OECD TG 422 study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Oral:

The objectives of this OECD TG 422 study performed in compliance with CLP was to determine the potential toxic effects and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception of DPGDA. In addition, parental and reproduction (up to and including implantation) No Observed Adverse Effect Levels (NOAELs) were evaluated .Wistar Han rats were administered the test substance dose levels of 0, 40, 125, 375 mg/kg/day, based on the results of the Dose Range Finder. With the exception of the control group, all animals (10 animals/sex/group) received the test substance orally via gavage for a minimum period of 28 days using corn oil as vehicle at a dose volume of 5 mL/kg body weight. Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, measurement of thyroid hormone T4 (parental males), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction parameters were determined: mating and fertility indices, pre-coital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14 - 16 pups)).

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. Parental toxicity was observed at the highest dose level tested (375 mg/kg/day). One female at 40 mg/kg/day was sacrificed in extremis on post-coitum Day 23 because of delivery difficulties. This female delivered 3 dead pups and showed hunched posture, piloerection and pale appearance. At necropsy, 8 dead foetuses were present in the uterus. Microscopically the uterus showed characteristics of pregnancy. Findings regarded secondary to the presence of dead foetuses in the uterus were moderate multifocal hepatocellular necrosis of the centrilobular area of the liver and multifocal thrombi in the lungs. These findings in liver and lungs were attributed to the poor condition of this female. This death was regarded to be related to delivery difficulties and unrelated to the treatment with the test item. Significant body weight loss in one female at 375 mg/kg/day during the first days of lactation coincided with piloerection and a slightly pale and lean appearance. At the incidence observed, this was considered not toxicologically relevant. Body weight gain in males at 375 mg/kg/day was decreased compared with control on Day 8 of treatment, which recovered up to similar levels as control at the end of the mating period. Absolute body weights in males at 375 mg/kg/day were decreased compared with control from Day 1 of treatment up to the day of scheduled necropsy. Considering the magnitude of change and as Group 4 males just happened to be the smallest rats in this study, no toxicological relevance was attached to this finding. Relationships were suspected between gross necropsy (irregular surface of the forestomach), clinical pathology (increased neutrophil concentrations) and histopathology observations. The combination of microscopic stomach findings observed in most males and females treated at a dose of 375 mg/kg/day consisted of slight to marked squamous cell hyperplasia with slight to moderate hyperkeratosis and minimal to slight mixed cell inflammation. In addition, a forestomach ulcer at minimal degree was recorded in a single male at 125 and at 375 mg/kg/day and in a single female at 375 mg/kg/day. This combination of findings is considered adverse at the recorded incidences and severities. Mixed cell inflammation was recorded in all selected males, compared to 3/5 selected females and likely correlated to the increased neutrophils recorded for males at 375 mg/kg/day. The forestomach findings and salivation were considered to be local test item effects and likely resulting from irritating properties of the test item. The low severities of stomach findings in a single male at 125 mg/kg/day only were considered to be non-adverse. Increased mitotic figures were recorded in the liver of two females at 375 mg/kg/day and a relation to treatment with the test item could not be excluded. This alteration was considered to be non-adverse at the recorded minimal severity.

Non-adverse changes were recorded in clinical biochemistry parameters: increased cholesterol levels and increased calcium levels in females at 375 mg/kg/day. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, body weight, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), food consumption, clotting parameters, male T4 thyroid hormone levels, and organ weights). No reproductive toxicity was observed up to 375 mg/kg/day.

No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, pre-coital time, number of implantations, oestrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). Adverse changes in pup body weights were recorded at 375 mg/kg/day. Pup body weights were slightly reduced on PND 1 progressing up to approximately 10 % reduction on PND 13.

In conclusion, based on the results of this combined 28-Day repeated dose toxicity study the established No Observed Adverse Effect Level (NOAEL) of DPGDA (CAS 57472-68-1) was 125 mg/kg/day based on macroscopic and microscopic stomach findings and increased neutrophil counts). The NOAEL for reproductive toxicity was considered to be at least 375 mg/kg bw/day both for males and females.

In addition to the OECD TG 422 study with DPGDA, the data obtained from the EOGRTS study with TPGDA can be used as source information for DPGDA. TPGDA was investigated of possible pre- and postnatal effects on development in an Extended One Generation Reproductive Toxicity Study (EOGRTS) performed according to OECD TG 443 study under GLP (Reachcentrum 2019a, Kl1). In addition, a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats was performed (OECD 443, 2019).

The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, with oral exposure of the test substance in rats. These dose levels were 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 substance 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.

No parental toxicity was observed up to the highest dose level tested. 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). 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, pre-coital time, number of implantations, oestrous cycle, sperm analysis, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg bw/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). 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 corroborative 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 oestrous, time between vaginal opening and first oestrous length and regularity of the oestrous 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).

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1), the no-observed-adverse-effect levels (NOAEL) of the test substance for general toxicity (F0 and F1), reproduction, and development was observed to be at least 100 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

 - Oral: Rat: combined 28-day repeated dose oral (gavage) toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422): NOAEL general toxicity = 125 mg/kg bw/day; NOAEL developmental toxicity 125 mg/kg bw/day

- OECD 414; GLP; New Zealand White rabbits; oral gavage; 50, 150, 450 mg/kg; NOAEL (development) 450 mg/kg bw/day:

Read across to CAS No. 42978-66-5

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07/2020 to 04/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Jul 2016

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2000

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2016

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2000

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

May 2008

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Oct 2008

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Jul 2000

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

CAS no: 57472-68-1

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle, Cedex, France.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 12 - 14 weeks old (females)
- Weight at study initiation: 200 - 250 g (females)
- Housing: On arrival and following the pre-test and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, females were individually housed in Makrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (dimensions: 48.3 x 26.7 x 20.3 cm) without cage enrichment, bedding material, food and water. Animals were separated during designated procedures/activities.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures
- Water: 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.
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY
- The feed was analysed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed, and results of these analyses are on file at the test facility.

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

IN-LIFE DATES: From: 19 Aug 2020 To: 13 Nov 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation of dosing solutions: weekly
- Preparation of dosing solutions: Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator, stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.
- Storage temperature of dosing solutions: 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.

VEHICLE
- Concentration in vehicle: 8, 25, 75 mg/mL
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples for Analysis: Duplicate middle samples for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis).
- Sample Volume: Approximately 500 mg accurately weighed.
- Acceptance Criteria: For concentration, the criteria for acceptability were mean sample concentration results within or equal to ± 10 % for solutions or ± 15 % for suspensions of target concentration. For homogeneity, the criteria for acceptability were a coefficient of variation (CV) of concentrations of ≤ 10 % for each group.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: animals were cohabitated within the same treatment group, avoiding sibling mating
- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurred or until 14 days had elapsed
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: palpation / vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Females that delivered for 50 - 62 days, females which failed to deliver or had a total litter loss for 39 - 44 days.

Frequency of treatment:

Once a day, seven days a week.

Dose / conc.:

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

Remarks:

Group 2. Low dose.

Dose / conc.:

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

Remarks:

Group 3. Mid dose.

Dose / conc.:

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

Remarks:

Group 4. High dose.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration of DPGDA in rats (Test Facility Study No. 20236861) and in an attempt to produce graded responses to the test item. 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.
- Fasting period before blood sampling for (rat) dam thyroid hormones: parental females were not fasted.
- Time of day for (rat) dam blood sampling: between 7.00 and 10.30 am

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed at least once daily, up to the day prior to necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade were predefined at 1, 2, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (i.e. maximum grade 1) were scored.
Once before the first administration of the test item and at weekly intervals during the treatment period, animals were observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

CLINICAL OBSERVATIONS (FUNCTIONAL OBSERVATION BATTERY TESTS)
- Time schedule: Once during the treatment period. The selected 5 females were tested once during the last week of lactation (i.e. Postnatal day (PND) 6 - 13). These tests were performed after clinical observations (including arena observation, if applicable).
The following tests were performed: (1) Hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent). (2) Fore- and hind-limb grip strength were recorded as the mean of three measurements, using a grip strength meter. (3) Locomotor activity (recording period: 1 hour under normal laboratory light conditions) were tested using the Kinder Scientific Motor Monitor System. Total movements and ambulations were reported. Ambulations represent movements characterised by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed 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 post-partum day 1, 4, 7, and 13. In order to monitor the health status animals may be weighed more often.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
Weekly, except for females which are housed together for mating and for females without evidence of mating. Food consumption of mated females were measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post-partum day 1, 4, 7, and 13. Food consumption were quantitatively measured.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Regular basis throughout the study.
Water consumption were monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Females which delivered on post-partum day 14 - 16; females which failed to deliver with evidence of mating on post-coitum Days 26 - 27 and females with total litter loss and Dams with no surviving pups were euthanised within 24 hours after the last pup is found dead or missing. 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.
- Organs examined: Yes
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanised in poor condition or 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.
- Organs weighed at necropsy for all selected animals: cervix*; epididymis; gland adrenal; gland coagulation; gland parathyroid@; gland thyroid; heart; kidney; liver; ovaries; spleen; thymus and uterus, where:
* = weighed together with the uterus; # = paired organ weight; @ = weighed together with the thyroid.
- Organs weighed at necropsy for all remaining animals (incl., Females that Failed to Deliver and Females with Total Litter Loss) were as follows: epididymis*; gland coagulation*; gland parathyroid$; gland thyroid*; where:
* = Paired organ weight; $ = Weighed together with the thyroid.

TISSUES FIXED AND PRESERVED
Representative samples of the tissues identified in the table below 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.
- Tissue Collection and Preservation for all Selected Animals, all Animals that were Sacrificed in extremis and Females with Total Litter Loss: Animal identification: Artery; aorta; body cavity; nasopharynx; bone marrow; bone, femur; bone, sternum; brain (eight levels); cervix; epididymides*; oesophagus; eye*; gland, adrenal; gland, coagulation; gland, harderian* #; gland, lacrimal; gland, mammary; gland, parathyroid$; gland, pituitary; gland, salivary; gland, thyroid; gross lesions/masses; gut-associated lymphoid tissue; heart; kidney; large intestine, cecum; large intestine, colon; large intestine, rectum; larynx; liver; lung; lymph node (mandibular and mesenteric site); muscle, skeletal; nerve, optic#; nerve, sciatic; ovaries; pancreas; skin; small intestine, duodenum; small intestine, ileum; small intestine, jejunum; spinal cord; spleen; stomach; thymus; tongue; trachea; urinary bladder; uterus and vagina; where:
* = Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours; # = Only collected if present in the routine section of the eye. Part of the optic nerve attached to the eye was Fixed in Modified Davidsons’s fixative. The remaining part of the optic nerve was placed in formalin; $ = Only collected if present in the routine section of the thyroid.
- Tissue Collection and Preservation for all Remaining Animals (incl. females that failed to deliver): animal identification
cervix; epididymis*; gland, coagulation; gland, mammary; gland, parathyroid#; gland, pituitary; gland, gland, thyroid; gross lesions/masses; ovaries; uterus and vagina, where:
* = Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours; # = Only collected if present in the routine section of the thyroid.

HISTOPATHOLOGICAL EXAMINATION
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin:
- Selected animals, and unscheduled deaths (sacrificed in extremis): see Tissues identified in above for “Tissue Collection and Preservation for all Selected Animals, all Animals that were Sacrificed in extremis and Females with Total Litter Loss” (except animal identification, aorta; nasopharynx; oesophagus; harderian gland; lacrimal gland; salivary gland; larynx; optic nerve; pancreas; skin and tongue).
- Females that failed to deliver pups and females with total litter loss: cervix; epididymis; coagulation gland; ovaries; uterus and vagina.
- Females with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Blood sampling:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood were collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus in the animal facility. Additional blood samples may be obtained (e.g. due to clotting of non-serum samples) in both the animal facility and in the necropsy room if permissible sampling frequency and blood volume are not exceeded. After collection, samples were transferred to the appropriate laboratory for processing.
- Anaesthetic used for blood collection: Yes, using isoflurane.
- Animals fasted: Females will not be fasted overnight.
- How many animals: All parental animals (except for animals which were sacrificed in extremis or found dead and females with total litter loss.
- Parameters examined: white blood cell count (WBC), reticulocytes (absolute), neutrophils (absolute), red blood cell distribution width (RDW), lymphocytes (absolute), haemoglobin, monocytes (absolute), haematocrit, eosinophils (absolute), mean corpuscular volume (mcv), basophils (absolute), mean corpuscular haemoglobin (MCH), large unstained cells (LUC) (absolute), red blood, cell count mean corpuscular, haemoglobin concentration (MCHC) and platelets.
For Haematology: Target volume: 0.5 mL; anticoagulant used: K3-EDTA (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). A blood smear were prepared from each haematology sample. Blood smears were labelled, stained, and stored. Blood smears were evaluated when required to confirm analyser results
For Coagulation: Target Volume: 0.45 mL and anticoagulant used: Citrate (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). Coagulation Parameters: Prothrombin time (PT and Activated partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Parameters examined: Alanine aminotransferase (ALT) Creatinine, Aspartate aminotransferase (AST) Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin Chloride, Bile Acids, Calcium, Urea and Inorganic Phosphate (Inorg. Phos).
For Clinical Chemistry: Target volume: 0.5 mL; anticoagulant: Li-Heparin (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). Processing: to plasma

SERUM HORMONES: Yes
- Parameters examined: for females, the serum will be used for possible future measurement of total T4 and/or thyroid-stimulating hormone TSH.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: yes, measured for all live pups on post partum 1. The Anogenital distance (AGD) was normalised to the cube root of body weight.

- Pups were identified on postnatal Day (PND) 1: post-partum day 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Pups were randomised per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.
- Pups were observed daily for general health/mortality. The number of live and dead pups was determined on post-partum day 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Clinical observations were performed at least once daily for all pups.
- Live pups were weighed individually on post-partum day 1, 4, 7 and 13. Sex was externally determined for all pups on post-partum day 1 and 4.
- Areola/nipple retention: All male pups in each litter were examined for the number of areola/nipples on post-partum day 13.
- Blood collection and thyroid hormone determination (T3, T4 and TSH): blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup). The pups selected for blood sampling were the same pups as selected for thyroid preservation. Target volume: for post-partum day 4 pups: 0.5 mL in total (pooled), and post-partum day 14 - 16 pups: 1.0 mL per pup. After clotting and centrifugation serum was used as follows: for PND 4 Pups the pooled serum was used for possible future measurement of total T4 (added by study plan amendment if applicable). And for post-partum day 14 - 16 Pups 150 μL serum for measurement of total T4, and the remaining volume of serum for possible future measurement of TSH (added by study plan amendment if applicable).
- In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, pre-coital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early post-natal pup development (mortality, clinical signs, body weights, sex (ratio), and macroscopy.

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 analysed 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 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
- 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). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
-Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
- 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 Fisher’s exact test whenever the overall test is significant.

Indices:

For each group, the following calculations were performed. Group mean values of precoitally time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:
- Mating index (%) = 100 x (Number of females mated / Number of females paired
- Pre-coital time = Number of days between initiation of cohabitation and confirmation of mating.
- Fertility index (%) = 100 x (Number of pregnant females / Number of females mated)
- Gestation index (%)= 100 x (Number of females with living pups on Day 1 /Number of pregnant females)
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition.
- Post-implantation survival index (%) = 100 x (Total number of offspring born / Total number of uterine implantation sites)

- Live birth index (%) = 100 x (Number of live offspring on Day 1 after littering/ Total number of offspring born)
- Percentage live males at First Litter Check (%) = 100 x (Number of live male pups at First Litter Check / Number of live pups at First Litter Check)
- Percentage live females at First Litter Check (%)= 100 x (Number of live female pups at First Litter Check / Number of live pups at First Litter Check)
- Viability index (%)= 100 x (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering)
- Lactation index (%)= 100 x (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling))

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Two females at 375 mg/kg/day, showed piloerection on respectively Days 8 - 9 post-coitum and Days 3 - 4 of lactation. Another female also had a slightly pale and/or lean appearance between Days 2 and 5 of lactation, accompanied by a body weight loss up to 12 %. As clinical signs were transient and occurred in one or two animals only, these were considered incidental and not toxicologically relevant.
Salivation seen after dosing among females of all treatment groups was considered to be a local test item effect and likely resulting from irritating properties of the test item. Any other clinical signs noted during the treatment period (i.e. alopecia, scabs, rales and swelling of the vagina) 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/or did not show any apparent dose-related trend. At the incidence observed, these were considered not to be signs of toxicological relevance. No findings were noted during the weekly arena observations in this study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period 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 and body weight gain in females were considered to have been unaffected by treatment with the test item.

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 was considered unaffected by treatment with the test item up to 375 mg/kg/day. A trend towards lower food consumption was recorded in females at 375 mg/kg/day during the first week of treatment, with subsequent complete recovery. No toxicological relevance was attached to this finding.

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):

At 375 mg/kg/day, the following changes in haematology parameters were recorded (statistically significant unless stated otherwise): (1) Increased neutrophil counts in females (1.20x of control; not statistically significant). This was considered test item-related. (2) Decreased mean corpuscular haemoglobin concentration (MCHC) in females (0.95x of control). In the absence of changes in correlating parameters, this was considered not related to treatment with the test item. The decreased platelet counts in males at 40 mg/kg/day were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend. Coagulation parameters of treated rats were considered not to have been affected by treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following changes in clinical biochemistry parameters were recorded in females at 375 mg/kg/day: (1) Increased cholesterol levels in females at 375 mg/kg/day (1.38x of control). This was considered test item-related. (2) Increased calcium levels in females at 375 mg/kg/day (1.07x of control). This was considered test item-related. The increased mean glucose concentration recorded in females at 40 mg/kg/day was considered to be unrelated to treatment with the test item as it occurred in the absence of a dose-related trend.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals up to 375 mg/kg/day. Motor activity was considered to be unaffected by treatment with the test item. Increased motor activity in females at 125 mg/kg/day was mainly apparent in intervals 5 - 7, after which the habituation profile recovered to being comparable with controls. In the absence of a dose-related trend, this was considered not related to treatment with the test item. All groups (except Group 3 females) showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights and organ: body weight ratios were considered unaffected by treatment with the test item. Lower adrenal gland weights (absolute and relative to body weight) were recorded for females at 375 mg/kg/day. In the absence of a histopathological correlate, this weight change was regarded unrelated to the treatment with the test item. Remaining changes which reached statistical significance (lower absolute epididymis weight at 40 mg/kg/day) were considered not to be test item-related due to the lack of a dose-related pattern and/or general overlap and variability in individual values.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with DPGDA were noted in the stomach and liver of females. A combination of findings was recorded for the forestomach and consisted of mixed cell inflammation (up to slight), squamous cell hyperplasia (up to marked) and/or hyperkeratosis (up to moderate) in most animals at 375 mg/kg/day. In addition, a forestomach ulcer at minimal degree was recorded in a single female at 375 mg/kg/day. In two females at 375 mg/kg/day, a minimal degree of increased mitotic figures was recorded for the liver. The remainder 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 findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 88, 85, 83 and 88 % for the control, 40, 125 and 375 mg/kg/day groups, respectively

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Litter size was considered not affected by treatment with the test item. Live litter sizes were 10.8, 11.4, 10.1 and 10.4 living pups/litter for the control, 40, 125 and 375 mg/kg/day groups, respectively.

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

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 with the test item. The live birth indices were 98, 97, 100 and 97 % for the control, 40, 125 and 375 mg/kg/day groups, respectively. Two pups (different litters) of the control group, three pups (from two different litters) at 40 mg/kg/day, and three pups (from two litters) at 375 mg/kg/day were found dead at first litter check; there were no dead pups in the 125 mg/kg/day group on PND 1. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation index (females with living pups on Lactation Day 1 compared to the number of pregnant females) and duration of gestation was not affected by treatment with the test item. Except for one female at 40 mg/kg/day with an incomplete delivery, all pregnant females had live offspring. This resulted in a gestation index of 90% for the 40 mg/kg/day group, compared to 100% for the control, 125 and 375 mg/kg/day groups.

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

One female in the 40 mg/kg/day group showed difficulties with parturition. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

gross pathology

haematology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 375 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups at 375 mg/kg/day were slightly reduced on Day 1 (0.97x of control for both males and females; not statistically significant), progressing up to Day 13 (0.89x and 0.90x of control for males and females, respectively; statistically significant for males only).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment with the test item.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was considered not affected by treatment with the test item. Live litter sizes were 10.8, 11.4, 10.1 and 10.4 living pups/litter for the control, 40, 125 and 375 mg/kg/day groups, respectively.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalised for body weight) in male and female pups was considered not to be affected by treatment with the test item.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment with the test item. For two pups of a single litter (375 mg/kg/day) reduced size of the left testis and epididymis and/or agenesis of the left testis were recorded. Since these abnormalities were limited to two pups from the same litter, they were considered of hereditary origin rather than to be related to treatment with the test item. For the single pup of a female (40 mg/kg/day) that was found dead on PND 2 and the two pups of another female (375 mg/kg/day) that were found dead at first litter check, no milk was observed in the stomach. The nature and incidence of these and other macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Other effects:

no effects observed

Description (incidence and severity):

- No clinical signs occurred among pups that were considered to be related to treatment with the test item. The pup of one female (40 mg/kg/day) that was missing on PND 3, was cold to touch, and had a small and dehydrated appearance on PND 2. For the pup of another female (control group) that was missing on PND 6, a lean appearance was noted on PND 4 and 5. 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.
- Post-Implantation Survival: The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 88, 85, 83 and 88 % for the control, 40, 125 and 375 mg/kg/day groups, respectively.
- Serum T4 levels in male and female PND 14 - 16 pups were considered not to be affected by treatment with the test item. Any not statistically significant changes remained within the normal ranges of biological variation and were therefore regarded as not toxicologically relevant.
- Viability Index: 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 with the test item. Viability indices were 91, 97, 100 and 89 % for the control, 40, 125 and 375 mg/kg/day groups, respectively. Note that by mistake culling of one litter (40 mg/kg/day) was performed after first litter check on PND 1 instead of PND 4. These five culled pups are included in the viability index for the 40 mg/kg/day group. Nine pups (all from the same litter) in the control group and three pups at 40 mg/kg/day (two litters) were found dead or missing on PND 2-4. One female (375 mg/kg/day) had a total litter loss on PND 2. Eight pups were missing and two pups were found dead (severely cannibalised). Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend.
- Lactation Index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item. The lactation indices were 98 % for the control group and 100% for each the 40, 125 and 375 mg/kg/day groups. One pup of the control group (one female, with a total litter loss) was found missing on PND 6, after it was noted with a lean appearance on PND 4 and 5. Most likely it was cannibalised. No toxicological relevance was attributed to this finding since it occurred in the control group.
- Areola/Nipple Retention: Treatment with the test item up to 375 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1 Summary of all investigated reproductive indices

	Group 1	Group 2	Group 3	Group 4
	Control	40 mg/kg/day	125 mg/kg/day	375 mg/kg/day
Mating index (%)	100	100	100	100
(Females mated / Females paired) * 100
Fertility index (%)	90	100	100	90
(Pregnant females / Females mated) * 100
Gestation index (%)	100	90	100	100
(Females with living pups on Day 1 / Pregnant females) * 100
Post-implantation survival index (%)	88	85	83	88
(Total number of offspring born/Total number of uterine implantation sites) * 100
Live birth index (%)	98	97	100	97
(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100
Viability index (%)	91	97	100	89
(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100
Lactation index (%)	98	100	100	100

Table 2 Implantation sites summary females

		Group 1 Control	Group 2 40 mg/kg/day	Group 3 125 mg/kg/day	Group 4 375 mg/kg/day
At necropsy
Implantations	Mean	12.6	12.4	12.2	12.2
	St.Dev	2.1	1.8	3.0	1.6
	N	9	10	10	9

Table 3 Developmental data F0 - generation – lactation

	Group 1	Group 2	Group 3	Group 4
	Control	40 mg/kg/day	125 mg/kg/day	375 mg/kg/day
Litters Total	9	9	10	9
Duration Of Gestation
Mean (+)	21.4	21.4	21.3	21.3
St.Dev.	0.5	0.5	0.5	0.5
N	9	0.5	0.5	9
Dead Pups At First Litter Check
Litters Affected (#)	2	2	0	2
Total	2	3	0	3
Mean (+)	0.2	0.3	0.0	0.3
St.Dev.	0.4	0.7	0.0	0.7
N	9	9	10	9
Living Pups At First Litter Check
% Of Males / Females (#)	60 / 40	51 / 49	47 / 53	53 / 47
Total	97	103	101	94
Mean (+)	10.8	11.4	10.1	10.4
St.Dev.	3.2	2.9	4.0	2.5
N	9	9	10	9
Postnatal Loss
% Of Living Pups	9.3	2.9	0.0	10.6
Litters Affected (#)	1	2	0	1
Total (#)	9	3	0 ##	10
Mean (+)	1.0	0.3	0.0	1.1
St.Dev.	3.0	0.7	0.0	3.3
N	9	9	10	9
Culled Pups
Total	26	30	29	22
Living Pups Day 4 P.P.
Total	62	70	72	62
Mean (+)	6.9	7.8	7.2	6.9
St.Dev.	2.4	0.7	1.9	2.6
N	9	9	10	9
Breeding Loss Days 5 - 13 P.P. % Of Living Pups At Day 4 P.P.	1.6	0.0	0.0	0.0
Litters Affected (#)	1	0	0	0
Total (#)	1	0	0	0
Mean (+)	0.1	0.0	0.0	0.0
St.Dev.	0.3	0.0	0.0	0.0
N	9	9	10	9
Living Pups Day 13 P.P.
% Of Males / Females (#)	54 / 46	51 / 49	44 / 56	48 / 52
Total	61	70	72	62
Mean (+)	6.8	7.8	7.2	6.9
St.Dev.	2.7	0.7	1.9	2.6
N	9	9	10	9

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 4 Developmental data

	Group 1	Group 2	Group 3	Group 4
	Control	40 mg/kg/day	125 mg/kg/day	375 mg/kg/day
Total number of offspring born	99	106	101	97
Total number of uterine implantation sites	113	124	122	110
Number of live offspring on Day 1 after littering	97	103	101	94
Number of live offspring on Day 4 (before culling)	88	100	101	84
Number of live offspring on Day 4 (after culling)	62	70	72	62
Number of live offspring on Day 13 after littering	61	70	72	62

Conclusions:

In a combined 28-Day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422), based on the occurrence of macroscopic and microscopic stomach findings and increased neutrophil counts of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 125 mg/kg bw/day for males and females. The NOAEL for reproduction was considered to be ≥ 375 mg/kg bw/day for maternal females and developmental toxicity 125 mg/kg bw/day based on decreased body weights of pups.

Executive summary:

The objectives of this OECD TG 422 study performed in compliance with GLP was to determine the potential toxic effects and to evaluate the potential to affect reproductive performance such as gonadal function, mating behaviour, conception and early postnatal developmentof DPGDA. In addition, parental and reproduction (up to and including implantation) No Observed Adverse Effect Levels (NOAELs) were evaluated. Wistar Han rats were administered the test substance dose levels of 0, 40, 125, 375 mg/kg/day, based on the results of the Dose Range Finder. With the exception of the control group, all animals (10 animals/sex/group) received the test substance orally via gavage for a minimum period of 28 days using corn oil as vehicle at a dose volume of 5 mL/kg body weight. Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, pre-coital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14 - 16 pups)).

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. Parental toxicity was observed at the highest dose level tested (375 mg/kg/day). One female at 40 mg/kg/day was sacrificed in extremis on post-coitum Day 23 because of delivery difficulties. This female delivered 3 dead pups and showed hunched posture, piloerection and pale appearance. At necropsy, 8 dead foetuses were present in the uterus. Microscopically the uterus showed characteristics of pregnancy. Findings regarded secondary to the presence of dead foetuses in the uterus were moderate multifocal hepatocellular necrosis of the centrilobular area of the liver and multifocal thrombi in the lungs. These findings in liver and lungs were attributed to the poor condition of this female. This death was regarded to be related to delivery difficulties and unrelated to the treatment with the test item. Significant body weight loss in one female at 375 mg/kg/day during the first days of lactation coincided with piloerection and a slightly pale and lean appearance. At the incidence observed, this was considered not toxicologically relevant. Relationships were suspected between gross necropsy (irregular surface of the forestomach), clinical pathology (increased neutrophil concentrations) and histopathology observations. The combination of microscopic stomach findings observed in most females treated at a dose of 375 mg/kg/day consisted of slight to marked squamous cell hyperplasia with slight to moderate hyperkeratosis and minimal to slight mixed cell inflammation. In addition, a forestomach ulcer at minimal degree was recorded in a single female at 375 mg/kg/day. This combination of findings is considered adverse at the recorded incidences and severities. Mixed cell inflammation was recorded in 3/5 of selected females and likely correlated to the increased neutrophils. The forestomach findings and salivation were considered to be local test item effects and likely resulting from irritating properties of the test item. Increased mitotic figures were recorded in the liver of two females at 375 mg/kg/day and a relation to treatment with the test item could not be excluded. This alteration was considered to be non-adverse at the recorded minimal severity.

Non-adverse changes were recorded in clinical biochemistry parameters: increased cholesterol levels and increased calcium levels in females at 375 mg/kg/day. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, body weight, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), food consumption, clotting parameters, and organ weights). No reproductive toxicity was observed up to 375 mg/kg/day.No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, pre-coital time, number of implantations, oestrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). Adverse changes in pup body weights were recorded at 375 mg/kg/day. Pup body weights were slightly reduced on PND 1 progressing up to approximately 10% reduction on PND 13. The developmental toxicity observed in this study occurred at dose levels associated with parental toxicity. This included adverse findings in the stomach/forestomach. It could not be excluded that these stomach effects were related to the observed developmental toxicity. No developmental toxicity was observed at dose levels which were non-toxic to the parents. No toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size, pup mortality, clinical signs, anogenital distance, areola/nipple retention and T4 thyroid hormone levels).

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 Effects Level (NOAEL) were established: Maternal NOAEL of 125 mg/kg/day (based on macroscopic and microscopic stomach findings and increased neutrophil counts), reproduction NOAEL of at least 375 mg/kg/day and developmental NOAEL of 125 mg/kg/day (based on decreased body weights of pups).

Reference 2

Endpoint:

developmental toxicity

Remarks:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar- Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on August 2019

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: ReachCentrum SA
- Batch number of test material: 180003P040
- Expiration date of the lot/batch: 31. Dec 2018
- Purity/Composition: 100% (UVCB)
- Appearance: clear, colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under storage conditions: until 31. Dec 2018
- Stability of the test substance in the vehicle: Stability for at least 24 hours at room temperature protected from light, stability for at least 8 days in
the refrigerator, and stability of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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 solution and dosed within 6 hours after adding the vehicle to the test item. 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.

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han), outbred, SPF-Quality

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males), 13 weeks (females)
- Weight at study initiation: males: 251- 322 g; females: 198- 263 g
- Housing: individually (females during the post-mating phase and lactation phase with the pups) and grouping (pretest, males during the post-amting phase
- Diet: ad libitum; pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum
- Acclimation period: for 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70 (daily mean relative humidity of 36 to 60%)
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 d
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): single housing

Duration of treatment / exposure:

males: 29 days; females: 50-62 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and the duration of pregnancy and at least 14 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 40-53 days.

Frequency of treatment:

daily (7d/week)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

375 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- in total 10 animals/ sex/ dose
- 5 animals /sex/ dose were selected for functional tests (males only), clinical pathology, collection of full list of organs/tissues at macroscopic examination, organ weights (full list) and histopathology (full list)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A test study was performed in male and female Wistar rats. The test substance was orally (gavage) applied at concentrations of 0, 557 and 1855 mg/kg body weight per day for 14 days to four animals per test group and sex. No treatment related alterations were observed in any of the observed parameters at 1855 mg/kg body weight (high dose) dose level. Hence, this short-term repeated dose oral toxicity study was performed at similar dose levels.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
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: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: First day of treatment (prior to dosing) and weekly thereafter (after dosing)
- Body Weight Gains: Calculated against the body weight on Day 1 (premating, mating and lactation periods) or Day 0 (postcoitum period).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Time schedule for examinations: 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.
- Relative Food Consumption Calculated against the body weight for scheduled intervals.

WATER CONSUMPTION: not quantitative
- Subjective appraisal was maintained during the study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Females which delivered on PND 14-16, females which failed to deliver post-coitum Day 25 or 26 (with evidence of mating) or 25 days after the last day of the mating period (without evidence of mating), dams with no surviving pups were euthanized within 24 hours after the last pup was found dead or missing

- Organs examined:
Selected animals: Tissues identified in table 5 (see any other information on materials and methods) with the exception of animal identification, aorta, nasopharynx, esophagus, harderian
gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue,
Females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina,
Females with total litter loss: Mammary gland,
Remaining animals: Gross lesions/masses.

OTHER: Hematology, coagulation, clinical chemistry, thyroid hormones (see any other information on materials and methods)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No (exception: if no macroscopic visible implantation sites were present)
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Indices:

Gestation index, postimplantation survival index, live birth index, viability index, lactation index (for detailed formulas see "Any other information on materials and methods"

Historical control data:

Historical control data to allow comparison with concurrent controls were provided by the test facility for the following parameters: Maternal body weight gain (period 2015- end 2017), clinical chemistry (period 2015- end 2017), thyroid hormone analyses (period 2015- end 2017), post-implantation survival index (period 2015 - May 2018)

Clinical signs:

no effects observed

Description (incidence and severity):

Salivation seen after dosing among animals of all dose groups was not considered 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 a sign of systemic toxicity

Mortality:

no mortality observed

Description (incidence):

One female of the control group (no. 49) was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A decreased number of reticulocytes was observed in females at 125 mg/kg, which was considered unrelated to administration of the test item due to absence of a dose-related trend response.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

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):

Test item-related irregular surface was observed in the (fore)stomach in 2/10 females treated at 375 mg/kg/day.
The remainder 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 one control female and 3 females treated with 125 mg/kg, is related to a stage in the estrous cycle and is a normal finding.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the (fore)stomach (2/5females). Squamous cell hyperplasia was present in females at 375 mg/kg/day up to moderate degree. This correlated with the macroscopic irregular surface.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92%, 79%, 88% and 85% for the control, 40, 125
and 375 mg/kg groups, respectively. The slightly lower post-implantation survival index for the 40 mg/kg group was considered the result of the low litter size of female no. 51 which only had one pup, but 10 implantation sites. The survival indices for all other groups were considered within normal range.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- No deficiencies in maternal care were observed.
- Litter size was not considered affected by treatment. Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

> 375 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse developmental effects observed up to and including the highest dose tested.

Fetal body weight changes:

not examined

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Dead pubs at the first litter check: 11 in the control group and 1 daed pub in the exposure group 40 mg/kg bw/d

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

One litter each was affected in the control, 125 and 375 mg/kg bw/d group (one dead pub), representing 1, 1.2 and 1% of living pubs

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- The post-implantation survival index, the live birth index, viability index and lactation index were not affected by the treatment.
- No clinical signs occurred among pups that were considered to be related to treatment. For the pup of female no. 71 (375 mg/kg) who was missing on Day 4, less milk in the
stomach and a lean appearance were noted on Day 1. Alopecia of the whole body was noted for several pups of the control, 40 and 375 mg/kg groups (not toxicological relevant as within the range considered normal)
- No macroscopic findings among the pubs

Dose descriptor:

NOAEL

Effect level:

> 375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse developmental effects observed up to and including the highest dose tested.

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1 Developmental indeces

Dose in mg/kg bw/d	0	40	125	375
Post-implantation survival index (%)	92	79	88	85
Live birth index (%)	90	99	100	100
Viability index (%)	99	100	99	99
Lactation index (%)	100	100	100	100

Table 2 Developmental data

Dose in mg/kg bw/d	0	40	125	375
Total number of offspring born	109	84	85	104
Total number of uterine implantation sites	118	106	97	123
Number of live offspring on day 1 after littering	98	83	85	104
Number of live offspring on day 4 (before culling)	97	83	84	103
Number of live offspring on day 4 (after culling)	63	65	56	70
Number of live offspring on day 13 after littering	63	65	56	70

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed no adverse signs of developmental toxicity in in maternal animals and their offspring up to and including the tested dose of 375 mg/kg bw/d.

Therefore, the NOAEL for developmental toxicity was set to be at least 375 mg/kg bw/d.

Executive summary:

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP. 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.

The following developmental parameters were determined: Number of implantation sites, gestation index and duration, parturition and maternal care, live birth index, viability index and lactation index, post-implantation survival index and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

Gestation index and duration of gestation were not considered to be affected by treatment. Except for one female of the control group, all pregnant females had live offspring. The gestation indices were 89% for the control and 100% for the 40, 125 and 375 mg/kg groups, respectively. 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 not considered to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92%, 79%, 88% and 85% for the control, 40, 125 and 375 mg/kg groups, respectively. The slightly lower post-implantation survival index for the 40 mg/kg group was considered the result of the low litter size of female no. 51 which only had one pup, but 10 implantation sites. The survival indices for all other groups were considered within normal range. Litter size was not considered affected by treatment. Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively. 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 90%, 99%, 100% and 100% for the control, 40, 125 and 375 mg/kg groups, respectively.

Eleven pups of the control group and one pup at 40 mg/kg were found dead at first litter check. Eight of these pups were of the control group female, which had a total litter loss. No toxicological relevance was attributed to these dead pups since the mortality incidence mainly occurred in the control group and did not show a dose-related trend. 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 99%, 100%, 99% and 99% for the control, 40, 125 and 375 mg/kg groups, respectively. Three dead/missing pups were present throughout the study (one pub of the control, 125 and 375 mg/kg bw/d expsoure group), however, 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 not considered to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups. The postnatal pub deveopment was unaffected by the treatment.

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no developmental toxicity was observed up to and including the highest tested dose of 375 mg/kg test item due to the abscence of respective adverse effects under the conditions of this study.

Reference 3

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant OECD TG 422 study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The objectives of this OECD TG 422 study performed in compliance with GLP was to determine the potential toxic effects and to evaluate the potential to affect reproductive performance such as gonadal function, mating behaviour, conception and early postnatal development of DPGDA. In addition, parental and reproduction (up to and including implantation) No Observed Adverse Effect Levels (NOAELs) were evaluated. Wistar Han rats were administered the test substance dose levels of 0, 40, 125, 375 mg/kg/day, based on the results of the Dose Range Finder. With the exception of the control group, all animals (10 animals/sex/group) received the test substance orally via gavage for a minimum period of 28 days using corn oil as vehicle at a dose volume of 5 mL/kg body weight. Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, pre-coital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14 - 16 pups)).

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. Parental toxicity was observed at the highest dose level tested (375 mg/kg/day). One female at 40 mg/kg/day was sacrificed in extremis on post-coitum Day 23 because of delivery difficulties. This female delivered 3 dead pups and showed hunched posture, piloerection and pale appearance. At necropsy, 8 dead foetuses were present in the uterus. Microscopically the uterus showed characteristics of pregnancy. Findings regarded secondary to the presence of dead foetuses in the uterus were moderate multifocal hepatocellular necrosis of the centrilobular area of the liver and multifocal thrombi in the lungs. These findings in liver and lungs were attributed to the poor condition of this female. This death was regarded to be related to delivery difficulties and unrelated to the treatment with the test item. Significant body weight loss in one female at 375 mg/kg/day during the first days of lactation coincided with piloerection and a slightly pale and lean appearance. At the incidence observed, this was considered not toxicologically relevant. Relationships were suspected between gross necropsy (irregular surface of the forestomach), clinical pathology (increased neutrophil concentrations) and histopathology observations. The combination of microscopic stomach findings observed in most females treated at a dose of 375 mg/kg/day consisted of slight to marked squamous cell hyperplasia with slight to moderate hyperkeratosis and minimal to slight mixed cell inflammation. In addition, a forestomach ulcer at minimal degree was recorded in a single female at 375 mg/kg/day. This combination of findings is considered adverse at the recorded incidences and severities. Mixed cell inflammation was recorded in 3/5 of selected females and likely correlated to the increased neutrophils. The forestomach findings and salivation were considered to be local test item effects and likely resulting from irritating properties of the test item. Increased mitotic figures were recorded in the liver of two females at 375 mg/kg/day and a relation to treatment with the test item could not be excluded. This alteration was considered to be non-adverse at the recorded minimal severity.

Non-adverse changes were recorded in clinical biochemistry parameters: increased cholesterol levels and increased calcium levels in females at 375 mg/kg/day. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, body weight, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), food consumption, clotting parameters, and organ weights). No reproductive toxicity was observed up to 375 mg/kg/day. No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, pre-coital time, number of implantations, oestrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). Adverse changes in pup body weights were recorded at 375 mg/kg/day. Pup body weights were slightly reduced on PND 1 progressing up to approximately 10% reduction on PND 13. The developmental toxicity observed in this study occurred at dose levels associated with parental toxicity. This included adverse findings in the stomach/forestomach. It could not be excluded that these stomach effects were related to the observed developmental toxicity. No developmental toxicity was observed at dose levels which were non-toxic to the parents. No toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size, pup mortality, clinical signs, anogenital distance, areola/nipple retention and T4 thyroid hormone levels).

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 Effects Level (NOAEL) were established: Maternal NOAEL of 125 mg/kg/day (based on macroscopic and microscopic stomach findings and increased neutrophil counts), reproduction NOAEL of at least 375 mg/kg/day and developmental NOAEL of 125 mg/kg/day (based on decreased body weights of pups).

There are valid data available for the structurally related tripropylene glycol diacrylate (TPGDA) (CAS No. 42978-66-5). These data were adopted from TPGDA, respectively, for DPGDA by read-across:

TPGDA was investigated in a teratogenicity study according to OECD 414 and in compliance with GLP (2019). The objectives of this study were to determine the potential of the test substance 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/day, based on the results of the dose range finderand 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.

The following parameters and end points were evaluated in this study for the F0-generation:

mortality/moribundity, clinical signs, body weights, food consumption, gross necropsyfindings, 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. 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 groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance 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 sacrificedin extremison Days 4 and 7 of treatment at 750 mg/kg.

Justification for classification or non-classification

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

The available screening studies are reliable and suitable for classification purposes under Regulation 1272/2008. As no substance-related adverse findings were noted up to the highest test dose, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.