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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
developmental neurotoxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2020 - May 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 426 (Developmental Neurotoxicity Study)
Version / remarks:
October 2007
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene dibenzoate
EC Number:
202-338-6
EC Name:
Ethylene dibenzoate
Cas Number:
94-49-5
Molecular formula:
C16H14O4
IUPAC Name:
2-(benzoyloxy)ethyl benzoate
Test material form:
solid: particulate/powder
Details on test material:
Appearance: white to off white powder or flakes
Storage conditions: at room temperature protected from light
Specific details on test material used for the study:
Stability in polyethylene glycol 400: Stability for at least 5 hours at room temperature under normal laboratory conditions and 8 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL.

Test animals

Species:
rat
Strain:
Wistar
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 developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Females: time-mated, arrived on day 0 (day of successful mating) or day 1 post-coitum
- Age at study initiation: 10-15 wks
- Weight at initiation of dosing: 188-257 g
- Fasting period before study: no
- Housing: On arrival, females were individually housed in Makrolon plastic cages (MIII type, height 18 cm) until necropsy.
Pups were housed with the dam until weaning (PND 21; Subsets B-D), necropsy (Subset A, PND 21-22), or until necropsy of the dam (surplus pups). Subsets are described below at 'Details on study design'.
Subset B-D animals were housed in groups of maximum 5 animals/sex/cage in Makrolon cages (MIV type, height 18 cm).
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
- 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 food and water for a maximum of 2 hours. Analyses of food and water were available. It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 43-79
- Air changes (per hr): at least 10 (100% fresh air, no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 22 Aug 2020 To: 16 Nov 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol 400
Details on exposure:
RATIONALE FOR ROUTE OF EXPOSURE
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

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 weekly as a suspension and were heated to a maximum temperature of 60±5°C for at least 30 minutes to obtain visual homogeneity. Formulations were filled out in daily portions. If dosed on the day of formulation, formulations were released for dosing when they obtained a temperature of 40°C or lower. If formulations were not dosed on the day of formulation, they were stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 5 hours after removal from the refrigerator.

ADMINISTRATION
The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during dose administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial preparations
- Concentration in vehicle: 20, 60 or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
Time-mated females were received at the test facility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Trial formulations (not used for dosing) were prepared at target concentration levels of 20 mg/mL (low) and 200 mg/mL (high) and analyzed according to a validated analytical procedure. Analysis of accuracy of preparation and homogeneity was performed at the top, middle and bottom of the containers.
The concentration (all groups) and homogeneity of the dose formulations (low and high dose) were analysed in week 1 of treatment (duplicate sets of samples).
The accuracy of preparation was considered acceptable if the mean analyzed concentrations were 90-110% of the target concentrations. Homogeneity is demonstrated if the coefficient of variation is ≤ 10%.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 516416) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
from post-coitum day 6 up to and including lactation day 20
Frequency of treatment:
once daily, 7 days per week
Details on study schedule:
- Time-mated females were used.
- F1-animals were not dosed directly, since in a Lactational Transfer Study (Test Facility Study No. 20218705) it was demonstrated that test item was excreted into maternal milk from lactating dams exposed to 1000 mg/kg bw/day from Day 6 post-coitum to Day 13 of lactation, inclusive.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
F0: 25 females
F1: 80 animals/dose/sex divided into 4 subsets (A, B, C and D)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on previous studies conducted with Ethylene glycol dibenzoate in rats including a Combined 90-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422, Test Facility Study No. 516410), a Lactational Transfer Study (Test Facility Study No. 20218705) and a Prenatal Developmental Toxicity Study (OECD 414, Test Facility Study No. 20218707).
In the combined 90-Day study (Test Facility Study No. 516410), Wistar Han rats received 0, 100, 300 or 1000 mg/kg bw/day by daily oral gavage. A parental NOAEL of 300 mg/kg bw/day was established based on decreased total T4 levels (on average 57% and 19% for males and females, respectively; for females in combination with increased incidence of (minimal or slight) follicular cell hypertrophy in the thyroid) at 1000 mg/kg bw/day. A developmental NOAEL of 300 mg/kg bw/day was established based on a skewed sex ratio towards females (66% female pups versus 43% in the control group), a reduced live birth index (91% vs. 100% in the control group), increased postnatal loss (viability index of 95% vs. 100% in the control group) and decreased body weights on PND 1 at 1000 mg/kg bw/day (11% lower than the control group). No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day). In this study, males and females were treated for 8 weeks prior to mating, whereas females in a developmental neurotoxicity study will be treated from Day 6 post-coitum up to and including Day 20 of lactation.
In the Lactational Transfer Study (Test Facility Study No. 20218705), no maternal toxicity was recorded for Wistar Han rats receiving 1000 mg/kg bw/day by daily oral gavage from Day 6 post-coitum to Day 13 of lactation.
In the Reproduction/Developmental Toxicity Screening Test Wistar Han rats received 0, 100, 300 or 1000 mg/kg bw/day by daily oral gavage. A Maternal NOAEL of at least 1000 mg/kg bw/day was established, and a Developmental NOAEL of 300 mg/kg/day was established based on lower fetal body weights observed at 1000 mg/kg bw/day (mean combined fetal body weights showed a relative difference to concurrent controls of -8%).
Overall, these data indicated that a high dose level of 1000 mg/kg bw/day was expected to be tolerated in a developmental neurotoxicity study. The high-dose level should produce some maternal and/or developmental toxic effects, but not death nor obvious suffering. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- F1 animal assignment: On PND 4, eight pups from each litter of equal sex distribution (the first four males and first four females per litter, if possible) were selected to reduce variability among the litters. The non-selected pups were culled on PND 4.
Allocation of F1-pups for behavioural tests and neurohistopathological examination:
On PND 1, or shortly thereafter, one pup/sex/litter (if possible) was assigned to Subsets A, B, C and D, to have a total of 20 pups selected per sex per group per subset. A total of 20 females with 8 live pups/litter were selected (if possible).

Subset A:
• Neurohistopathological and morphometric evaluation of brain (Groups 1 and 4) and brain weights (PND 21-22)

Subset B:
• Behavioral ontogeny (righting reflex; beginning on PND 2)
• Locomotor activity (PND 13, 17, 25± 2, and 60±2)
• Neurohistopathological evaluation of central nervous system (CNS) and peripheral nervous system (PNS), morphometric evaluation of brain (Groups 1 and 4) and brain weights (PND 70-73)

Subset C:
• Sexual maturation: vaginal patency (from PND 25 onwards) and balanopreputial separation (from PND 35 onwards)
• Acoustic startle (PND 25±2 and 60±2)
• Biel Maze (PND 23-27)

Subset D:
• Detailed clinical (arena) observations (PND 25±2, 35± 2, 45± 2 and 60± 2)
• Biel Maze (PND 65-70)
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
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. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at least once daily, from day 6 post-coitum onwards up to the day prior to necropsy

BODY WEIGHT:
- Time schedule for examinations: Days 6, 9, 12, 15, 18 and 21 post-coitum, and on PND 1, 4, 7, 11, 14, 17 and 21 (weaning)

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, over Days 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum, and over PND 1-4, 4-7, 7-11, 11-14, 14-17 and 17-21

WATER CONSUMPTION: monitored on regular basis throughout the study by visual inspection of the water bottles

OTHER:
Detailed Arena Observations were conducted on post-coitum Days 8 and 17 and lactation Days 7 and 14 for 10 dams/group (the first 2 or 3 sequential animal Nos. per mating date). These observations were conducted prior to dosing, approximately the same time each day and with a maximum of 3 hours difference between the earliest and latest observation.
For the detailed functional observations, testing of animals was counterbalanced across dose groups to the extent possible, based on the number of animals available for each delivery date. These observations were conducted by technicians not involved in dosing or regular observations of these animals.
Animals were transferred to the separate room equipped for these purposes on the day preceding the day on which detailed functional observations were conducted:
1. Detailed clinical observations consisted of a number of tests conducted in- and outside the home cage. To the extent possible, testing of animals was counterbalanced across dose groups. The clinical observations took place in a quiet room.
2. Rectal temperature was measured immediately after the detailed clinical observations.
3. Hearing ability (Score 0 = normal/present, score 1 = abnormal/absent).
4. Pupillary reflex (both eyes; Score 0 = normal/present, score 1 = abnormal/absent).

GENERAL REPRODUCTION DATA
For each female, confirmation of pregnancy and delivery day were recorded.
Females were allowed to litter normally. 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). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Litter observations:
STANDARDISATION OF LITTERS: see under details of study design

The following parameters were examined in the F1 generation:
MORTALITY/MORIBUNDITY:
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.
Pups showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

CLINICAL OBSERVATIONS:
Clinical observations were performed at least once daily (home cage). For all Subset D animals, a hand-held observation was additionally conducted on PND 7 and 14.

BODY WEIGHT:
Live pups were weighed individually on PND 1, 4, 7, 11, 14, 17 and 21 (all animals).
From PND 21 onwards, all animals were weighed weekly, from weaning onwards.
Animals of each subset were also weighed on the day of acquisition of righting reflex, balanopreputial separation and vaginal opening, i.e. for Subset B animals on the day of acquiring righting reflex, and for Subset C animals for the first animal per litter for which vaginal opening and balanopreputial separation was reached.

FOOD CONSUMPTION:
Food consumption was quantitatively measured weekly, starting from within one week after all animals were weaned.

WATER CONSUMPTION:
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles. No quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL AND BEHAVIORAL TESTS:
Detailed Arena Observations:
Detailed Arena Observations were conducted on PND 25±2, 35±2, 45±2 and 60±2, for all Subset D animals. Observations/measurements were conducted in separate room(s) specially equipped for these purposes or in the study room (detailed recorded in the study raw data). When conducted in a separate room, animals were transferred to this room on the day preceding the day on which detailed functional observations were conducted. These observations were conducted approximately the same time each day and with a maximum of 3 hours difference between the earliest and latest observation.
1. Detailed clinical observations consisted of a number of tests conducted in- and outside the home cage. To the extent possible, testing of animals was counterbalanced across dose groups. The clinical observations took place in a quiet room.
2. Rectal temperature was measured immediately after the detailed clinical observations.
3. Hearing ability (Score 0 = normal/present, score 1 = abnormal/absent).
4. Pupillary reflex (both eyes; Score 0 = normal/present, score 1 = abnormal/absent).

Righthing reflex:
Acquisition of the righting reflex was observed daily, beginning on PND 2 for all Subset B animals (20/sex/group) and continued for the respective pups until positive.

Acoustic startle response:
Acoustic startle response (habituation) was assessed using the StartleMonitor System (Kinder Scientific, Poway, USA). This was performed on PND 25± 2 and 60± 2, for all Subset C animals (20 animals/sex/group) in a sound-attenuated room.
To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose group and sex. The animals were tested in sets of up to 3. The test sessions consisted of a five-minute acclimation period with a 65 ± 5-dB broadband background white noise. The startle stimulus for each trial was a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 milliseconds in duration). Responses were recorded during the first 250 milliseconds following onset of the startle stimulus for each trial. The test session consisted of 50 trials with an eight-second intertrial interval. Average response amplitude (AveN), average maximum response amplitude (MaxN) and average latency to achieving the maximum response amplitude (Tmax) were analyzed in five blocks of 10 trials each.

Locomotor activity:
Locomotor activity was tested on PND 13, 17, 25±2, and 60± 2, for all Subset B animals (20 pups/sex/group) using the Kinder Scientific Motor Monitor System. Recording period was one hour under normal laboratory light conditions.
To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose groups.
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.

Learning and memory test (Biel Maze):
Cognitive function (learning and memory retention) was conducted for all Subset C animals (20 animals/sex/group) between PND 23-27 and for all Subset D pups (20 pups/sex/group) between PND 65-70; “Day 1” as specified below occurred within these respective periods.
A water-filled Biel Maze was used. A total of 4 trials to verify swimming ability and adapt the animals to the water maze conditions, 5 trials for learning and 1 for memory assessment was performed.
The time to escape and number of errors (animal deviates from the correct channel with all four feet) were recorded. Animals were allowed three minutes to escape (except for the straight channel swimming test for which the animals were allowed two minutes) after which they were removed.
Each animal was tested as follows:
Day 1: Four consecutive trials in the straight channel.
Day 2: Five trials in Path A (minimum inter-trial interval of one hour)
Day 9: One trial in Path A.

SEX AND SEXUAL MATURATION:
Sex was externally determined for all pups on PND 1 and 4.
Vaginal patency (vaginal opening) was monitored daily for all females from Subset C (20 rats/group) from PND 25 onwards. until vaginal patency was present, by visual inspection of the vaginal area.
Balanopreputial separation (prepuce opening) was monitored daily for all males from Subset C (20 rats/group) from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area.
Body weight was recorded on the day of acquisition of vaginal patency/balanopreputial separation.

CLINICAL PATHOLOGY
Blood of 10 F1-animals/sex/group (2-3 animals/sex/group per mating date, if possible) from Subset A (non-perfused animals; PND 21-22) and Subset B (PND 70-73) were collected on the day of scheduled necropsy. Animals were not fasted overnight.
Samples were collected using the sequence Group 1-2-3-4 on necropsy Day 1, Group 2-3-4-1 on necropsy Days 2, Group 3-4-1-2 on necropsy Day 3 etc. (if practically and/or logistically feasible). Samples were collected between 7.00 and 10:30 a.m., from the aorta under isoflurane anesthesia as part of the necropsy procedure from Surplus animals at necropsy (PND 24-25) since blood samples collected at necropsy from Subset A animals were not processed into serum and therefore not subjected to thyroid hormone analysis. It should be noted that the F0 animals to which these spare animals belonged were dosed up to and including PND 20.
Blood samples at a target volume of 1.0 mL (Subset A (non-perfused), Surplus (non-perfused) and Subset B animals) were collected into tubes without anticoagulant. Blood samples were processed for serum.
Serum samples retained for possible future analysis of T3, T4 and TSH were maintained by the Test Facility in the freezer (≤-75°C). Under these storage conditions, samples are stable for 6 months.
Postmortem examinations (parental animals):
SACRIFICE AND GROSS NECROPSY
Scheduled necropsy on LD 21-25 for females that delivered, post-coitum day 26 for females which failed to deliver, within 24 hrs after the last pup was found dead or missing for a female with total litter loss. Animals were fasted overnight before necropsy.
All animals were subjected to an external, thoracic and abdominal examination. All macroscopic abnormalities were recorded.
No terminal body weight was recorded and no organs were weighed
Postmortem examinations (offspring):
TERMINAL PROCEDURES
Unscheduled deaths:
Pups that were sacrificed in extremis, younger than 7 days, were euthanized by decapitation.
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.
Descriptions of all external abnormalities were recorded. 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):
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation.
Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

Surplus pups:
Animals were not fasted overnight before necropsy.
Surplus pups were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%), and were examined externally and sexed (both externally and internally).
From several Surplus pups (as defined in the study records), a blood sample was collected from the aorta since from a few Subset A animals the collected blood sample was stored at room temperature too long to allow a meaningful analysis of these samples. These pups were deeply anaesthetized using isoflurane prior to blood sampling and were exsanguinated after blood sampling.

Subset A and B:
Animals were not fasted overnight before necropsy.
Subset A and B animals selected for neuropathology were euthanized by whole body (in situ) fixation perfusion.
Subset A and B animals not selected for whole body perfusion were deeply anaesthetized using isoflurane and subsequently exsanguinated.
Necropsy procedures for Subsets A and B animals were counterbalanced across dose groups as much as practically feasible.
Animals selected for neuropathology:
On PND 21-22 (Subset A) or PND 70-73 (Subset B), all animals had a terminal body weight taken. A total of 10 animals/sex/group (first 2-3 animals/sex/group per necropsy date, if possible) per Subset A and B were first anesthetized with isoflurane. The animals were subsequently sacrificed by whole body (in situ) fixation perfusion.
After perfusion, the cranium was removed, exposing the brain. The skull including the brain was placed in 10% buffered formalin and allowed to fix for at least 7 days prior to removal from the skull. The fixed brains were removed and weighed, and the length and maximum width of the brain was measured for all animals selected for neuropathology. Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues.
After sacrifice all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to tissues of the central and peripheral nervous system. Descriptions of all abnormalities were recorded and fixed in 10% buffered formalin. Sex was determined both externally and internally.
Animals selected for unfixed brain weights:
On PND 21-22 (Subset A) or PND 70-73 (Subset B), the remaining animals of Subsets A and B had a terminal body weight taken and were deeply anaesthetized using isoflurane.
A blood sample was subsequently collected from the aorta from all these animals.
Animals were subsequently exsanguinated and subjected to a gross necropsy. The brains were immediately removed, weighed and immersion fixed in 10% buffered formalin.
After sacrifice all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to tissues of the central and peripheral nervous system. Descriptions of all abnormalities were recorded. Abnormalities were fixed in 10% buffered formalin. Sex was determined both externally and internally

Subset C and D:
Animals were not fasted overnight before necropsy.
On PND 63-70 (Subset C) or PND 78-81 (Subset D), all surviving animals were euthanized by an oxygen/carbon dioxide procedure. After sacrifice all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to tissues of the central and peripheral nervous system. Descriptions of all abnormalities were recorded. Abnormalities were fixed in 10% buffered formalin. Sex was determined both externally and internally.

HISTOLOGY
All PNS and CNS tissues (including the skull containing olfactory bulbs) collected from
10 perfusion fixed animals/sex/group of Subset A and B were investigated.
PND 21-22 CNS tissues (Subset A):
For the 10 perfusion fixed animals/sex/group, CNS tissues selected for quantitative histopathology was embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with HE.
For morphometric analysis, 3 consecutive sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections were obtained. These sections of the brain of perfusion-fixed Subset A animals of Groups 1 and 4 initially were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet.
PND 70-73 CNS and PNS tissues (Subset B):
For the 10 perfusion fixed animals/sex/group, CNS and PNS selected for quantitative histopathology were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with HE.
At the cervical and lumbar position of the spinal cord a longitudinal (1-1.5 cm) and transversal cut (approx. 3 mm thickness) were made, including the cervical and lumbar swellings. At the position of both the cervical and lumbar swellings, multiple ganglia (if possible) were dissected with the dorsal and ventral nerve roots attached.
The spinal cord and peripheral nerve sections were included both cross or transversal and longitudinal sections.
For morphometric analysis, 3 consecutive sections were taken from neocortical, hippocampal and cerebellar areas to ensure homologous sections were obtained. These sections of the brain of perfusion-fixed Subset B animals of Groups 1 and 4 initially were also stained for myelin and cell bodies using Luxol Fast Blue and Cresyl Violet.

HISTOPATHOLOGY
- Morphometric (quantitative) analyses of brain tissues was performed for Subset A and B of Groups 1 and 4.
Microscopic morphometric measurements were performed on specified sections of brain. Analyses included measurements from neocortical, hippocampal, and cerebellar areas. Slides were scanned using a Hamamatsu Nanozoomer whole slide scanner and imported into the Visiopharm software for measurements. These measurements were performed in a blinded random fashion for all dose groups.
Statistics:
Data collected/processed in ToxData:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation 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 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.
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 were compared using a Steel-test (many-to-one rank test).
For the startle data set normal distribution was determined by using the Kolmogorov-Smirnov, Cramer-von Mises and Anderson-Darling tests. As the majority of the data was not normally distributed on PND 25 and 60, the Kruskal-Wallis test was applied to determine overall statistical significance.
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.
Data from Provantis: see "Any other info on mat/method
Reproductive indices:
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on PND 4 before culling/Number live offspring on PND 1 after littering) x 100
Weaning index (%): (Number of live offspring on PND 21/Number of live offspring on PND 4) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were noted that were considered to be related to toxicity of the test item.
Salivation seen after dosing among a few animals at 300 and 1000 mg/kg bw/day on several days during 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 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.
No clinical signs were observed for females at 100 mg/kg bw/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality occurred during the study period.
One control female was sacrificed on post-coitum Day 24 because of 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 not affected by treatment with the test item.
For one female at 1000 mg/kg bw/day, a notable weight loss was recorded over lactation Days 14-17 along with a reduced food intake. This was ascribed to a dysfunctioning water bottle due to which the animal presumably had no or limited access to water for a maximum of approximately three days. On PND 17, this animal was found to have hunched posture and its pups had piloerection and ptosis as well as lower weight gain between lactation Days 14 and 17. The water bottle was replaced on PND 17 following which this animal gained weight and had normal food intake. Also, no clinical signs were observed for the pups from this day onwards and weight gain resumed to normal values. This incidental occurrence was therefore considered not to have affected the outcome of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Absolute and relative food consumption were considered not affected by treatment with the test item.
For one female at 1000 mg/kg bw/day, a reduced food intake was recorded over lactation Days 14-17. This was unrelated to treatment with the test item as outlined under body weight.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Arena observation parameters as determined on post-coitum Days 8 and 17 and lactation Days 7 and 14 were considered not affected by treatment with the test item. Incidences of observed symptoms did not show a dose-related trend, and/or had incidences/severities that were similar to the control group.
All examined animals had a normal hearing and pupillary reflex, and rectal temperature was similar across the dose groups on all measurement occasions.

Reproductive function / performance (P0)

Description (incidence and severity):
One female at 100 mg/kg bw/day and one female at 300 mg/kg bw/day did not deliver offspring and were sent for necropsy on post-coitum Day 26. One control female was sent for necropsy on post-coitum Day 24 as this female had total litter loss. This female was recorded to be delivering pups on post-coitum Day 23, but no pups were present during the first litter check on post-coitum Day 24. Therefore, it was not included in the calculation of postnatal loss, live birth index and viability index of the control group. These incidences were considered not to be related to treatment with the test item, as they occurred in the absence of a dose-related trend and may be encountered in this type of study. All other females delivered offspring.
Duration of gestation was considered not to be affected by treatment with the test item.
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.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Pre-weaning:
No clinical signs occurred among pups that were considered to be related to treatment with the test item .
The nature and incidence of recorded clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.

Post-weaning:
Post-weaning clinical observations revealed no clinical signs that were considered to be related to treatment with the test item.
Any clinical signs noted after weaning 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 with the test item.
No clinical signs were recorded for control group females.

Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pre-weaning:
Litter size was considered not affected by treatment with the test item.
Mean litter sizes were 10.6, 10.3, 10.5 and 10.6 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment with the test item. The live birth indices were 100% for the control, 300 and 1000 mg/kg bw/day group each, and 99% for the 100 mg/kg bw/day group.
Two pups at 100 mg/kg bw/day and one pup at 1000 mg/kg bw/day were found dead at first litter check. 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.
Viability index (the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1) was considered not to be affected by treatment with the test item.
Viability indices were 100% for the control group, 99% for the 100 and 300 mg/kg bw/day groups, and 98% for the 1000 mg/kg bw/day group.
Two pups each at 100 and 300 mg/kg bw/day and four pups at 1000 mg/kg bw/day were found dead or missing on PND 3 or 4. Two of these missing pups at 1000 mg/kg bw/day felt cold at first litter check. In addition, one pup at 1000 mg/kg bw/day was sacrificed for ethical reasons on PND 1 due to a wound on the right front leg. 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 and remained within the range considered normal for pups of this age.
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 21, resulting in a weaning index of 100% for all groups.

Post-weaning: no mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, pre-weaning body weights of male and female pups were lower than control means from lactation Day 4 onwards, achieving a level of statistical significance between PND 7 and 17 on most occasions for males and on PND 11 for females (see also clin. signs F0). On lactation Day 21, mean pre-weaning body weight of both sexes combined was 0.96x of control mean.
Body weights of pups at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item.

Post-weaning:
At 300 and 1000 mg/kg bw/day, mean body weight of males was lower than the control means from Day 1 of post-weaning onwards (0.95x of control for both groups), and remained lower during the first four or two weeks of post-weaning respectively (statistically significant). Mean absolute body weight at these dose levels was similar to control means at the end of the study period. This was reflected by a statistically significantly higher body weight gain of males at 1000 mg/kg bw/day from Day 22 of post-weaning onwards (1.07x of control at the end of the study period). Mean body weight gain of males of the 300 mg/kg bw/day group showed no apparent dose-related changes throughout the study period.
Body weight gain of females of the 1000 mg/kg bw/day group was higher than control means during most of the study period (1.06x of control at the end of the study period), but mean absolute body weights remained similar to control means throughout the study period. It should be noted that since body weights on Day 1 for animals of this age are relatively low, rather large changes in weight gain can occur with small increases in absolute body weight.
.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Absolute and relative food consumption were considered not affected by treatment with the test item.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum T3, T4 and TSH levels on PND 21-22 and PND 70-73 were considered not to be affected by treatment with the test item.
All variations in these parameters, including the statistically significantly higher total T3 value for males of the 300 mg/kg bw/day group on PND 70-73, occurred in the absence of a dose-related trend.
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio during lactation was not affected by treatment with the test item.
Time to achieving balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening) in females were considered not affected by treatment with the test item.
Mean body weight on the day of achieving balanopreputial separation or vaginal opening
was similar across the dose groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Fresh and fixed brain weight were considered not affected by treatment with the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Up to weaning no macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.

Post-weaning:
There were no test item-related gross observations recorded for Subsets A, B, C and D animals.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings:
no effects observed
Description (incidence and severity):
Brain dimensions (length and width of brain) of Subset A and B animals were considered not affected by treatment with the test item.
There were no microscopic findings in the peripheral and central nervous tissues examined for the control or high dose (1000 mg/kg bw/day) group males or females of Subset A and B after treatment with the test item based on examination of the H&E and Luxol Fast Blue/Cresyl Violet stains.
Morphometric brain measurements for Subset A and B animals were comparable with control values.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Detailed clinical observations:
Arena observation parameters as determined on PND 25, 35, 45 and 60 were considered not affected by treatment with the test item. Incidences of observed symptoms did not show a dose-related trend.
One male of the 100 mg/kg bw/day group had a small right pupil on PND 60 which did not dilate due to an eye lesion, and one female of the 1000 mg/kg bw/day group had a dilated left pupil on PND 60 which did not become smaller . All other animals had a normal pupillary reflex. All animals had a normal hearing reflex and rectal temperature was considered not affected by treatment with the test item. The statistically significantly higher rectal temperature of females of the 1000 mg/kg bw/day group on PND 45 was considered not to be related to treatment with the test item since no such change was apparent on other measurement occasions.

Righting reflex:
The day on which righting reflex was acquired was considered not affected by treatment with the test item. Animals showed - on average - a positive righting reflex on PND 2 or 3.

Acoustic startle response:
Acoustic startle response on PND 25 and PND 60 was considered not affected by treatment with the test item. Means of average and maximum response amplitude and latency to maximum response amplitude remained similar to control means on all occasions.

Motor activity:
Motor activity was considered not affected by treatment with the test item.
PND 13
The lower mean motor activity of males and females in the 1000 mg/kg bw/day group, (0.75x and 0.66x of control for total movements and ambulations, respectively for males and 0.82x and 0.68x of control for total movements and ambulations, respectively for females; not statistically significant) was attributed to high values in three control group animals. When discounting these high control values in the mean calculations, the mean control values were 3238 and 870 counts for total movements and ambulations of males, and 3439 and 1043 counts for total movements and ambulations of females. These mean values were similar to or even lower than the means including these higher individual values. It was therefore considered that these variations in motor activity did not represent a change related to treatment with the test item.
In relation to the above described variations in individual control values, variations in mean motor activity levels across the dose groups did not show a dose-related trend and were therefore considered not to represent an effect of treatment with the test item.
PND 17
Motor activity was considered not affected by treatment with the test item.
PND 25
The higher mean motor activity of males in the 1000 mg/kg bw/day group (1.29x and 1.30x of control for total movements and ambulations, respectively) was ascribed to a lower motor activity of control males during intervals 6, 7 and 8. Also, a clear dose-related trend was absent; motor activity/habituation pattern was similar across all test item-treated groups. Variations of these parameters were therefore considered not to be related to treatment with the test item.
Motor activity of females was considered not affected by treatment with the test item.
All male and female groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
PND 60
Mean motor activity (total movements and ambulations) of females at 100, 300 and 1000 mg/kg bw/day appeared higher than the control means (not statistically significant). In absence of a dose-related trend, these variations were considered not to be related to treatment with the test item.
All male and female groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Learning and memory test (Biel maze):
No effect on learning and memory was observed that was considered to be related to treatment with the test item on PND 23-27 and PND 65-70.
The straight channel trial confirmed swimming ability for all rats. The statistically significantly higher mean time to reach the platform of females of the 100 and 300 mg/kg bw/day groups during the first trial of the straight channel swimming test occurred in the absence of a dose-related trend and was therefore considered not to be related to treatment with the test item. During subsequent swimming trials, the mean time to reach the platform was similar across all groups.
Mean time required to reach the platform and number of errors made during the five consecutive swimming trials in the Biel maze was similar between treated and control animals on PND 23-27 and PND 65-70 and showed an expected downward trend over these trials. Any statistically significant changes occurring during the swimming trials occurred in the absence of a dose-related trend and were therefore considered not to be related to treatment with the test item.
The memory swimming trial conducted seven days after the swimming trials also showed that the mean time required to reach the platform and number of errors made was similar between treated and control animals on PND 23-27. In the memory swimming trial conducted on PND 65-70, the mean time to reach the platform by 1000 mg/kg bw/day group male rats appeared higher than controls (not statistically significant). However, the intra-group variation was high, and on PND 23-27 mean time to reach the platform of these animals was similar to control levels. As such, this variation was considered not to be related to treatment with the test item.
On PND 23-27 and PND 65-70, the memory swimming trial showed no apparent dose-related differences compared to mean values in the last swimming trial conducted seven days earlier.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Dose formulation analysis:

Accuracy
In Group 1 (vehicle), no test item was detected.
The concentrations analyzed in the formulations of Groups 2 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
For the formulation of Group 3, the mean accuracy was below the target concentration
(i.e. 89% of target). This result was accepted as it was only 1% below the target range.
Homogeneity
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Applicant's summary and conclusion

Conclusions:
In a developmental neurotoxicity study performed in rats according to OECD 426 and GLP principles, the maternal and developmental (including neurotoxicity endpoints) NOAEL for the test item were established to be at least 1000 mg/kg bw/day.
Executive summary:

A developmental neurotoxicity study was performed in rats according to OECD 426 and GLP principles with the test item. Time-mated female rats (25/dose) were given 0, 100, 300 and 1000 mg/kg bw/day from day 6 post-coitum up to and including lactation day 20. 

For the F0-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption and gross necropsy findings.
For the F1-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, sexual maturation (vaginal patency and balanopreputial separation), functional and behavioral tests including detailed clinical observations, righting reflex,, acoustic startle response, learning and memory test (Biel Maze) and motor activity, clinical biochemistry (T3, T4, TSH), gross necropsy findings, brain weights and dimensions and histopathologic examination of central and peripheral nervous tissues (including brain morphometry).
In addition, the following reproduction/developmental parameters were determined for the F0-generation: gestation duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex and macroscopy).
Formulation analyses confirmed that formulations of test item in polyethylene glycol 400 were prepared accurately and homogenously.

F0-generation
Enlargement of the liver was recorded for two animals at 1000 mg/kg bw/day and for one animal at 300 mg/kg bw/day. This necropsy finding could not be supported by other morphological changes since liver weight and histopathology were not part of this study. Although a relationship to treatment could not be excluded, its low incidence was considered to support the non-adversity of this finding.
No treatment-related changes were noted in clinical appearance, body weight and food consumption, and no test item-related mortality occurred during the study period.

F1-generation – pre-weaning
A non-adverse lower mean pre-weaning body weight was recorded at 1000 mg/kg bw/day, for both sexes from lactation Day 4 onwards (0.96x of control mean on lactation Day 21, for both sexes combined). Body weights of pups at 100 and 300 mg/kg bw/day were considered not affected by treatment with the test item. Since this body weight variation was slight and other pre-weaning developmental parameters remained unaffected by treatment with the test item, this was considered not adverse.

No test item-related changes were noted in any of the other pre-weaning developmental parameters investigated in this study (i.e. duration of gestation, parturition, sex ratio, maternal care, litter size, live birth index, viability index, weaning index, clinical signs and macroscopy).

F1-generation - post-weaning
A non-adverse lower mean body weight and/or higher weight gain was recorded for males and/or females during the initial weeks of post-weaning in the 300 and 1000 mg/kg bw/day groups, but absolute body weights attained values similar to control levels during the study period.
No test item-related changes were noted in any of the other F1-generation
post-weaning developmental parameters investigated in this study (i.e mortality, clinical signs, food consumption, balanopreputial separation, vaginal opening, total T3, T4 and TSH levels (See attached graphs) and macroscopy).

F1-generation - neurotoxicity results
No test item-related changes were noted in any of the F1-generation/
(neuro-)developmental parameters investigated in this study (i.e. time to acquire righting reflex, arena observations, hearing and pupillary reflex, rectal temperature, learning and memory, acoustic startle response, motor activity, gross brain dimensions, brain weights, brain morphometry, and peripheral and central nervous tissue histopathology).
In conclusion, based on the results of this Developmental Neurotoxicity Study, the following No Observed Adverse Effect Levels (NOAEL) of Ethylene Glycol Dibenzoate were established:

Maternal NOAEL: at least 1000 mg/kg bw/day

Developmental (including neurotoxicity endpoints) NOAEL: at least 1000 mg/kg bw/day