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Toxicological information

Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Remarks:
Prenatal Developmental Toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-25 to 2018-01-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
developmental toxicity
Remarks:
Prenatal Developmental Toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2017-06-25 to 2018-01-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Eastman Chemical Company (United States); Batch no. V988701802
- Expiration date of the lot/batch: None
- Purity test date: 2017-01-25

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (18°C to 24°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: previously shown to be stable over the range of concentrations used on this studyfor at least 10 days under refrigerated (2°C to 8°C) conditions

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid

OTHER SPECIFICS:
Purity: > 99.0%
Species:
rabbit
Strain:
New Zealand White
Remarks:
Hra:(NZW)SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc. (Denver, PA)
- Age at study initiation: approximately 7 months old at the time of mating by the supplier
- Weight at study initiation: 2949 g to 3901 g on Gestation Day 0
- Fasting period before study: No
- Housing: Upon arrival, all rabbits were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel-O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). The bedding was changed at least twice weekly. Nesting material was not required because the females were euthanized prior to the date of expected parturition. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rabbit LabDiet®5322 - The basal diet was offered in 25-g increments 3 times per dayon the dayof arrival and in increased amounts over the next few days, until the animals gradually achieved ad libitum status prior to the dose administration period; basal diet was offered ad libitum thereafter. Kale (1 leaf at each occasion) was provided to each animal daily for environmental enrichment and to aid in maintaining the animal's gastrointestinal health, beginning upon animal receipt and continuing throughout the duration of the study. Kale present in the cage at the time of providing a new leaf was discarded. The diet was supplemented with extra kale, celery sticks, and haycubes as necessary.
- Water (e.g. ad libitum): Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum
- Acclimation period: time-mated rabbits received on Gestation Day 1, 2, 3, or 4.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 61°F to 71°F (16°C to 22°C)
- Humidity (%): 30% to 70%
- Air changes (per hr): minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours) / 12-hour dark photoperiod

IN-LIFE DATES: From: 2017-06-30 To: 2017-07-28
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
medium viscosity, 400–800 cps, USP
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle and test substance formulations were administered orally by gavage, via appropriately sized rubber catheters, once daily during Gestation Days 7–28. The dose volume for all groups was 5 mL/kg. Following administration of each dose, the catheter was flushed with 5 mL of deionized water to ensure delivery of the entire dose.

VEHICLE
- Justification for use and choice of vehicle (if other than water): carboxymethylcellulose (CMC); reason for choice not specified
- Concentration in vehicle: 0, 20, 50, or 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required): Lot Nos. 2GA0044 and 1GE0858, retest dates: 31 Jul 2017 and 16 Apr 2018, respectively
- Purity: 0.5% carboxymethylcellulose in deionized water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A validated HPLC/UV method was used and extended for the determination of DPGDB concentration in suspension formulations. Method specificity/selectivity, calibration reproducibility, precision, and accuracy and were assessed and validated, satisfying SOP specified criteria. In addition, the results of the assessment of test substance homogeneity and, following 10 days of refrigerated storage, resuspension homogeneity in formulations prepared at target concentrations of 20 and 100 mg DPGDB/mL met the applicable protocol-specified acceptance criteria.
Details on mating procedure:
One hundred six time-mated female New Zealand White rabbits were received in good health from Covance Research Products, Inc., Denver, PA, on 30 Jun 2017. The time-mated rabbits were received on Gestation Day 1, 2, 3, or 4.
Duration of treatment / exposure:
The vehicle and test substance formulations were administered orally by gavage, via appropriately sized rubber catheters, once daily during Gestation Days 7–28. The dose volume for all groups was 5 mL/kg. Dosage levels of 100, 250, and 500 mg/kg/day were selected for the current study.
Frequency of treatment:
once daily during Gestation Days 7–28
Duration of test:
during Gestation Days 7–28.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle Control (Group 1)
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
500 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
The experimental design consisted of 3 test substance-treated groups and 1 control group, composed of 24 rabbits/group.


Group Number Treatment Dosage/Dose Level Number of
Females
(mg/kg/day)

1 Vehicle Control 0 24
2 DPGDB 100 24
3 DPGDB 250 24
4 DPGDB 500 24







Control animals:
yes
Details on study design:
- Dose selection rationale:
Dosage levels were selected based on results of a previous dose range-finding prenatal developmental toxicity study in rabbits with DPGDB, and were provided by the Sponsor
Representative after consultation with the Charles River Study Director. In the previous study, time-mated rabbits were dosed (via oral gavage) with 100, 250, 500, and 1000 mg/kg/day of DPGDB during Gestation Days 7–28. Significant toxicity was noted at the 1000 mg/kg/day dosage level as evidenced by abortions, body weight losses, and reduced food consumption, resulting in early termination of this dosage group. At the 500 mg/kg/day dosage level, excreta-related findings were noted without significant effects on body weights or food consumption. Dosage levels of 100 and 250 mg/kg/day were well tolerated. Therefore, dosage levels of 100, 250, and 500 mg/kg/day were selected for the current study to provide the evaluation of potential dose-response and determination of a no-observed-adverse-effect level (NOAEL).

The selected route of administration for this studywas oral (gavage) because this is a potential route of exposure for humans.

- Rationale for animal assignment (if not random): The bred females were assigned to groups using a WTDMS™ computer program, which randomized the animals based on stratification of the Gestation Day 0 body weights in a block design.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations were recorded daily from the dayof receipt through Gestation Day 29 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1, 2, and 4 hours following dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on Gestation Days 0 (by supplier under conditions that were not compliant with GLPs, but in accordance with the supplier’s SOPs), 5, and 7–29. Group mean body weights were calculated for each of these days. Mean bodyweight changes were calculated for each corresponding interval and also for Gestation Days 7–10, 10–13, 13–20, 20–29, and 7–29. Gravid uterine weight was collected and net bodyweight (the Gestation Day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Individual food consumption was recorded on Gestation Days 5–29. Food intake was reported as g/animal/dayand g/kg/dayfor the corresponding bodyweight change intervals.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Gestation Day 29 Laparohysterectomy: The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovarywas recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss. Maternal tissues with gross lesions were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. In addition, the adrenal glands, stomach, heart, and kidneys were preserved in 10% neutral-buffered formalin for microscopic examination.

Unscheduled Deaths: For females that were found dead, euthanized in extremis, or aborted during the course of the study, maternal tissues with gross lesions were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Inaddition, the adrenal glands, stomach, heart, and kidneys were preserved in 10% neutral-buffered formalin for microscopic examination.The number and location of implantation sites, corpora lutea, and viable fetuses were recorded.

OTHER:

CLINICAL PATHOLOGY:
Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 females/group from all groups at necropsy (Gestation Day29), and from females that were euthanized in extremis or exhibited signs of toxicity during the study. For females euthanized in extremis, blood was collected prior to euthanasia, and animals were euthanized immediatelyfollowing blood collection by an intravenous injection of sodium pentobarbital via a marginal ear vein. The animals were not fasted prior to blood collection. Blood samples were collected from a marginal ear vein (or other site as deemed necessary). Blood was collected in tubes containing K2EDTA (hematology) or no anticoagulant (serum chemistry).

Parameters listed in Table 2. and Table 3. were evaluated for Haematology and Clinical Chemistry, respectively.
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

The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. Selected tissues were examined microscopically from all females found dead and euthanized in extremis, as well as from 5 females/group at the scheduled necropsy.
Fetal examinations:
The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations

- External examinations: Yes: all per litter
The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Nonviable fetuses (if the degree of autolysis was minimal or absent) were examined, the crown-rump length measured, weighed, sexed, and tagged individually. Crown-rump measurements, degrees of autolysis and gross examinations, if possible, were recorded for late resorptions, and the tissues were discarded.

- Soft tissue examinations: Yes: all per litter
Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development.

- Skeletal examinations: Yes: all per litter
Following fixation in alcohol, each fetus was stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations

- Head examinations: Yes: all per litter. Heads from all fetuses were examined bya midcoronal slice.
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit.

Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA9 to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test10 was used to compare the test substance-treated groups to the control group. Histopathological findings in the test substance-treated groups were compared to the control group using a two-tailed Fisher’s Exact test.
Indices:
Intrauterine data were summarized using 2 methods of calculation.

1) Group Mean Litter Basis:

Post implantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group / No. Gravid Females/Group)

2) Proportional Litter Basis:

Summation Per Group (%) = (Sum of Post implantation Loss/Litter (%) / No. Litters/Group)

Where:
Post implantation Loss/Litter (%) = No. Dead Fetuses, Resorptions (Early/Late)/Litter / No. Implantation Sites/Litter) x 100

Fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:

Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%) / No. Litters/Group)

Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter / No. Viable Fetuses/Litter) x 100
Historical control data:
Charles River Ashland historical control data was used (Appendix 6 of the study report)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Female in the 500 mg/kg/day group showed incidences of decreased defecation at the daily examinations during Gestation Days 20–26. Additional adverse clinical observations included rales, labored respiration, decreased respiration rate, and a thin body were noted at the daily examinations and/or postdosing observations during Gestation Days 23–26.

In the 500 mg/kg/day group, Female was found dead on Gestation Day 26 with clinical observations of rales, labored respiration, and red material around the nose at the daily examinations and/or postdosing observations on Gestation Day 25. At necropsy, Female had dark red discoloration and firmness of all lobes of the left lung, which correlated microscopically with acute inflammation. The trachea was also noted to be filled with dark red contents, which microscopically was characterized by hemorrhage, edema, and dilatation of the submucosa. The cause of death of this female was considered acute inflammation of the lungs, most likely due to an intubation error, and not considered test substance-related.In the 100 mg/kg/day group, Female was euthanized with clinical observations of rales, labored respiration, a thin body, and/or clear discharge from the eyes and nose were noted for this female at the daily examinations and/or postdosing observations during Gestation Days 24–25. At necropsy, mottling and failure to fully inflate were observed in all lobes of the lungs, which correlated with acute inflammation microscopically. Acute inflammation was centered on the bronchi and bronchioles and was consistent with an intubation error; therefore, the cause of moribundity was acute inflammation of the lungs, and not considered test substance-related.

Test substance-related incidences of decreased defecation was noted for 4 surviving females in the 500 mg/kg/day group at the daily examinations during Gestation Days 15–29, which corresponded to and were considered secondary to the reduced food consumption noted in this group. There were no other test substance-related clinical observations noted at the daily examinations or approximately 1, 2, or 4 hours following dose administration. Observations
noted in the treated groups, including rales, labored respiration, and/or decreased respiration rate, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Test substance-related effects on survival were noted in the 500 mg/kg/day group.

One female in the 500 mg/kg/day group was euthanized in extremis on Gestation Day 26 following a severe body weight loss (18.3% during Gestation Days18–26) and markedly reduced food consumption (0 to 10 g/day during Gestation Days 19–26), with corresponding incidences of decreased defecation at the daily examinations during Gestation Days 20–26. In addition, Female in the 500 mg/kg/day group aborted 1 late resorption (with no apparent malformations) on Gestation Day 25, and was subsequently euthanized; however, no clinical observations or remarkable effects on body weights and food consumption were noted for this female prior to aborting. At necropsy, neither female was noted with remarkable macroscopic findings. Due to the adverse clinical observations, body weight loss, reduced food consumption, and no clear cause of moribundity or abortion at necropsy, these findings were considered test substance-related and adverse.

No other test substance-related effects on survival were noted during the study. In the 500 mg/kg/day group, Female was found dead on Gestation Day 26 following a severe body weight loss (16.0% during Gestation Days 20–25) and markedly reduced food consumption (0 to 7 g/day during Gestation Days 21–25), and clinical observations of rales, labored respiration, and red material around the nose at the daily examinations and/or postdosing observations on Gestation Day 25. At necropsy, Female No. 2474 had dark red discoloration and firmness of all lobes of the left lung, which correlated microscopically with acute inflammation. The trachea was also noted to be filled with dark red contents, which microscopically was characterized by hemorrhage, edema, and dilatation of the submucosa. The cause of death of this female was considered acute inflammation of the lungs, most likely due to an intubation error, and not considered test substance-related.


In the 100 mg/kg/day group, Female was euthanized in extremis on Gestation Day 25 following a body weight loss (13.6% during Gestation Days 14–25), reduced food consumption (0 to 44 g/day during Gestation Days 16–25), and corresponding incidences of decreased defecation at the daily examinations during Gestation Days 19–20. Additional clinical observations of rales, labored respiration, a thin body, and/or clear discharge from the eyes and nose were noted for this female at the daily examinations and/or postdosing observations during Gestation Days 24–25. At necropsy, mottling and failure to fully inflate were observed in all lobes of the lungs, which correlated with acute inflammation microscopically. Acute inflammation was centered on the bronchi and bronchioles and was consistent with an intubation error; therefore, the cause of moribundity was acute inflammation of the lungs, and not considered test substance-related. In addition, Female in the 100 mg/kg/day group was found dead on Gestation Day 13. No remarkable effects on body weight or food consumption were noted prior to death, but a clinical observation of rales was noted for this female at the daily examinations and postdosing observations up to 2 days prior to death. At necropsy, oily contents in all lung lobes and a perforation of the trachea at approximately the level of cervical vertebra No. 3 were noted. Microscopically, the lungs showed acute inflammation. Perforation of the trachea was not confirmed histologically, but findings of edema, hemorrhage, dilatation, and mixed inflammatory cell infiltrates in the tracheal submucosa were likely related. Therefore, the cause of death for this female was acute inflammation of the lungs due to an intubation error, which was not considered test substance-related. Female in the 100 mg/kg/day group was euthanized in extremis on Gestation Day 24; no clinical observations or noteworthy changes in body weight or food consumption were noted prior to euthanasia. At necropsy, oily contents in all lung lobes and multiple, dark red, irregularly shaped areas in all lobes were noted for this female. Microscopically, the lungs showed acute inflammation and hemorrhage. Intubation error was determined to be the cause of debility and euthanasia, and therefore was not considered test substance-related. All other females survived to the scheduled euthanasia.


Test substance-related incidences of decreased defecation was noted for 4 surviving females in the 500 mg/kg/day group at the daily examinations during Gestation Days 15–29, which corresponded to and were considered secondary to the reduced food consumption noted in this group. There were no other test substance-related clinical observations noted at the daily examinations or approximately 1, 2, or 4 hours following dose administration. Observations
noted in the treated groups, including rales, labored respiration, and/or decreased respiration rate, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean body weight gains or body weight losses were noted in the 500 mg/kg/day group generally during Gestation Days 13–29, resulting in a lower mean body weight gain when the entire treatment period (Gestation Days 7–29) was evaluated compared to the control group. Although mean body weights in the 500 mg/kg/day group were comparable to the control group throughout the treatment period, these changes were still considered adverse as they led to the mortality or abortion of 2 females within the group.

Test substance-related lower mean body weight gains or body weight losses were noted in the 500 mg/kg/day group compared to the control group beginning on Gestation Day 13, and continuing sporadically throughout the remainder of the treatment period, resulting in lower mean body weight gains in this group when the Gestation Days 13–20 and 20–29 cumulative intervals and when the entire treatment period (Gestation Days 7–29) were evaluated. Although
none of the differences were statistically significant when compared to the control group and were not of sufficient magnitude to affect absolute mean body weights in this group, these changes were considered adverse as they led to the euthanasia or abortion of 2 females within the group. A mean net body weight loss (not statistically significant) was noted in the 500 mg/kg/day group compared to the control group, while mean net body weight and gravid
uterine weight in this group were comparable to the control group. Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100 and 250 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. Significant (p < 0.05) mean body weight losses were noted in the 100 mg/kg/day group compared to the control group during Gestation Days 15–16 and 20–21; however, these transient differences did not occur in a dose-related manner, and were therefore not considered test substance-related. Mean net body weight losses were also noted in the 100 and 250 mg/kg/day groups; however, the differences were not statistically significant and did not occur in a dose-related manner, and were therefore not considered test substance-related.

Mean net body weight loss was also noted compared to the control group at 500 mg/kg/day. Although mean body weights in the 500 mg/kg/day group were
comparable to the control group throughout the treatment period, these changes were still considered adverse as they led to the mortality or abortion of 2 females within the group. Mean net body weight and gravid uterine weight were comparable to the control group at 500 mg/kg/day. In the 100 and 250 mg/kg/day groups, mean body weights, body weight changes, net body weights, net body weight changes, gravid uterine weights, and food consumption were unaffected by test substance administration.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption was also lower in this group during Gestation Days 13–29, which resulted in slightly lower mean food consumption for the overall dosing period (Gestation Days 7–29), and corresponded to the decreased mean body weight gains noted in this group.

Mean maternal food consumption, evaluated as g/animal/day or g/kg/day, in the 500 mg/kg/day group was comparable to the control group during the first week of treatment (Gestation Days 7–10 and 10–13), but generally lower than the control group during the remainder of the treatment period (Gestation Days 13–20 and 20–29); differences were significant (p < 0.05 or p < 0.01) during Gestation Days 21–23 and 24–25. The lower mean food consumption corresponded to the lower mean body weight gains noted in this group during the same period, and resulted in a slightly lower mean food consumption when the entire treatment period (Gestation Days 7–29) was evaluated, and in the presence of other adverse effects noted at this dosage level, was considered test substance-related and adverse.
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):
Hematology and serum chemistry parameters were unaffected by test substance administration at all dosage levels.

There were no test substance-related changes in hematology parameters on Gestation Day 29. Increases in the percentage and the absolute number of monocytes were noted at 500 mg/kg/day compared to the control group. However, the differences were not statistically significant and could be attributed to a single female in this group, and in the absence of any other effects on hematology parameters, were considered to be the result of normal biological variation. There were no other effects on hematology parameters; there was no dose-response relationship, the changes were of minimal magnitude, and were not considered to be of toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematology and serum chemistry parameters were unaffected by test substance administration at all dosage levels.

There were no test substance-related changes in serum chemistry parameters on Gestation Day 29. There was no dose-response and the changes were of minimal magnitude. These differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related adrenal cortical hypertrophy was noted for females in the 100, 250, and 500 mg/kg/day groups; however, due to the minimal to mild severity and the lack of other related microscopic and gross findings, this finding was considered nonadverse. There were no other test-substance-related microscopic observations at any dosage level.

Adrenal cortical hypertrophy was considered a nonadverse finding due to the minimal to mild severity and the lack of other related microscopic and gross findings. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There were no test substance-related alterations in the prevalence, severity, or histologic character of those incidental tissue alterations.

Dosage (mg/kg/day): Females
0 100 250 500
Adrenal cortex 5 5 5 5
Hypertrophy 0 1 3 3
Minimal - 0 1 1
Mild - 1 2 2
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg/day group, 1 (4.2%) abortion was observed. However, no clinical observations or remarkable effects on body weights and food consumption were noted for this female prior to aborting. No apparent malformations and in female no remarkable macroscopic findings were observed. There were total 22 (91.7%), 23 (95.8%), 23 (95.8%), and 23 (95.8%) gravid females at the time of scheduled necropsy in Group 1, 2, 3, and 4 respectively (See Table 4).
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Preimplantation loss was observed in 19 fetus in group 1, 13 fetus in group 2, 20 fetus in group 3 and 14 fetus in group 4.Post-implantation loss was observed in 10 fetus in group 1, 6 fetus in group 2, 11 fetus in group 3 and 6 fetus in group 4. The mean percentages for Preimplantation loss were 8.3,6.5, 8.6 and 6.2% in Group 1,2,3 and 4. The mean percentages for Post implantation loss were 5.8,3.2, 4.3 and 3.1% in Group 1,2,3 and 4. Differences from the control group were not statistically significant.

Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and group 4 is 500 mg/kg/d. Non significant changes were observed (Refer attached tables) .

Parameters evaluated included postimplantation loss, number and percentage of viable fetuses, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
Total 32 resorptions were observed in out of total 760 fetus. Total 10 resorptions in Group 1, and 6 resorptions in Group 2 and 10 resorptions in Group 3 and 6 resorptions in Group 4 were observed. The mean percentages for total resorption were 5.8, 3.2, 3.9 and 3.1% in Group 1, 2, 3, and 4 where Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and Group 4 is 500 mg/kg/d. The changes were non significant in comparisn to controls. Differences from the control group were slight and not statistically significant (See Tables 4 and 7) .
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Early resorption was observed in 9 fetus in group 1, 6 fetus in group 2, 2 fetus in group 3 and 3 fetus in group 4. Late resorption was observed in 1 fetus in group 1, 0 fetus in group 2, 8 fetus in group 3 and 3 fetus in group 4. The mean percentages for early resorption were 0.5, 0.0, 3.0 and 1.5% in Group 1,2,3 and 4. Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and group 4 is 500 mg/kg/d. The changes were non significant in comparison to controls.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
1 fetus was found dead in group 3 (250 mg/kg/d). There were total 88 male viable fetus and 117 female viable fetus. out of total 205 viable fetus one dead fetus was found and which was non dose related. The mean percentages were 0.0,0.0,0.4 and 0.0 % for group 1,2,3 and 4. The values were not significantly different from control group.Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and group 4 is 500 mg/kg/d.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Total 22 (100 %), 23 (91.7%) 23 (91.7%) and 23 (91.7%) number of females were gravid in Group 1,2,3 and 4. The non gravid preganicies were 2 (8.3%), 1 (4.8%), 1 (4.2%), 1 (4.8%) in Group 1,2,3 and 4.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant (Attached summary table below).
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
mortality
Abnormalities:
not specified
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male, female, and combined fetal weights in the 500 mg/kg/day group were 8.9%, 11.8%, and 10.5% lower, respectively, compared to the control group, which corresponded with maternal toxicity observed at this dosage level. The differences were generally significant (p < 0.05 or p < 0.01) and were considered test substance-related and adverse. Mean fetal body weights in the 100 and 250 mg/kg/day groups were similar to the control group.

The fetal weight in group 1 was 42.1 in grams and in group 2 was 42.0 gm and group 3 was 41.4 gm and in group three was 37.7 gm in 500 mg/kg/d. The group 4 was statistically significant different from the control group (See Table 7).
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Viable fetus in group 1 were 192 and group 2 were 178 and in group 3 were 205 and group 4 were 185. The results were comparable to controls and were non significant. The viable percentages values were 94.2, 96.8, 95.7 and 96.9 in Group 1, 2 , 3, and 4. Group 1 is 0 mg/kg/d; Group 2 is 100 mg/kg/d; Group 3 is 250 mg/kg/d; and Group 4 is 500 mg/kg/d. The mean values for vaible fetus in group 1, 2, 3, and 4 were 8.7 (0 mg/kg/d) , 8.9 (100 mg/kg/d), 8.9 (250 mg/kg/d) and 9.3 (500 mg/kg/d). The values showed that there were no treatment related effcts and values were comparable to controls.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio was similar across all groups
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations were observed in fetuses (litters) in dose groups and were considered spontaneous in origin.

No test substance-related external malformations were noted for fetuses at any dosage level. In the 500 mg/kg/day group, one fetus was noted with a short tail (approximately 7 mm in length), one fetus was noted with exencephaly with open eyelids, and other fetus in same group was noted with a malpositioned umbilicus (located more lateral than normal). Skeletally, the short tail consisted of fused, malpositioned, and absent caudal vertebrae, and exencephaly consisted of small and misshapen frontal and parietal bones, an absent interparietal bone, and an unossified supraoccipital bone. These findings were noted in single fetuses, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data; therefore, they were not considered test substance-related. No other external malformations were noted in the test substance-treated groups. In the control group, fetus noted with carpal flexure (right) and polydactyly (6 digits present on the right forepaw), and fetus was noted with macroglossia. Skeletally, polydactyly consisted of one extra digit with only the distal phalanx ossified. There were no other external malformations observed on this study. No external developmental variations were noted in the test substance-treated groups. The only finding observed (excessive fat pads) was noted in a single fetus in the control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Because the aforementioned malformations were noted infrequently or at a similar frequency in the control group, were not observed in a clear dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data, they were not considered test substance-related. There were no other skeletal malformations observed on this study.

Severely malaligned sternebrae, resulting in fused or attached sternebrae, were noted for 1 and 3 fetuses in the 100 and 500 mg/kg/day groups, respectively. In addition, sternoschisis (sternal band No. 2 not joined) and a vertebral centra anomaly (fused sacral centra) were noted for two Fetus in the 500 mg/kg/day group, respectively. Furthermore, one Fetus in the 250 mg/kg/day group was noted with a rib anomaly (left rib Nos. 3 and 4 were fused proximally through medially). A vertebral anomaly with or without associated rib anomaly, consisting of extra ribs and centra, malpositioned centra and arches, fused centra, arches, and ribs, bipartite centra, absent centra and arches, and malproportioned arches and centra, was noted for 1 and 2 fetuses in the 100 and 500 mg/kg/day groups, respectively. In addition costal cartilage anomalies were noted for three Fetus in the control, 100, 250, and 500 mg/kg/day groups, respectively; 2 of these fetuses were also noted with other skeletal malformations, as noted above. Because the aforementioned malformations were noted infrequently or at a similar frequency in the control group, were not observed in a clear dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data, they were not considered test substance-related. There were no other skeletal malformations observed on this study.


A higher mean litter proportion of the skeletal developmental variation pubis unossified was noted in the 500 mg/kg/day group (1.4% per litter) compared to the control group (0.0% per litter). The value in this group also exceeded the maximum mean value in the Charles River Ashland historical control data (0.58% per litter). This reduction in ossification was considered secondary to the lower mean fetal body weights noted at this dosage level . Other findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related visceral malformations were noted for fetuses at any dosage level.

Lobular agenesis of the lungs (absent right accessory lobe) was noted for 2 fetuses in the 250 mg/kg/day group. In the 100 mg/kg/day group, a bulbous aortic arch, vestigial pulmonary trunk, and interventricular defect (a 1 mm in diameter opening in the anterior portion of the septum) were noted for one fetus . Because these visceral malformations were noted in single fetuses, in the control group at similar or higher frequencies (in five fetus), and/or were not observed in the high-dose group, they were not considered test substance-related. No other visceral malformations were noted in the test substance-treated groups. In the control group, one fetus was also noted with a three chambered heart (2 atria and 1 ventricle with an absent tricuspid valve) and one fetus was also noted with a small right ventricle in the heart. There were no other visceral malformations observed on this study.

There were no test substance-related developmental variations noted at any dose level. A higher mean litter proportion of the visceral developmental variation pale spleen was noted in the 500 mg/kg/day group (3.1% per litter) compared to the control group (0.7% per litter). The value in this group exceeded the maximum mean value in the Charles River Ashland historical control data (1.77% per litter). However, because this finding was only noted for 6(3) fetuses (litters), and was not statistically significantly different from the control group, it was not considered test substance-related. Other findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River
Ashland historical control data.

A cystic oviduct was noted for 1 and 2 fetuses in the 100 and 250 mg/kg/day groups, respectively, renal papilla(e) not fully developed (Woo and Hoar Grade 1) was noted for 1 and 2 fetuses in the 250 and 500 mg/kg/day groups, respectively, and red fluid in the abdominal cavity was noted for 1 fetus in the 500 mg/kg/day group. These findings were not classified as either a malformation or developmental variation and were not considered to be test substance-related because they occurred infrequently and/or in a manner that was not dose-related.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
not specified
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Table 4. Summary of Maternal Survival and Pregnancy Status

Dose Group

(mg/Kg/day)

0 (Control)

100

250

500

No.

%

No.

%

No.

%

No.

%

Females on Study

24

 

24

 

24

 

24

 

Females that aborted or delivered

0

0.0

0

0.0

0

0.0

1

4.2

 

Females that Died

0

0.0

1

4.2

0

0.0

1

4.2

    Females that aborted

0

0.0

0

0.0

0

0.0

0

0.0

    Non Gravid

0

0.0

0

0.0

0

0.0

0

0.0

    Gravid

0

0.0

1

100.0

0

0.0

1

100.0

 

Females that were euthanized

0

0.0

2

8.3

0

0.0

1

4.2

    Gravid

0

0.0

0

0.0

0

0.0

0

0.0

    Non Gravid

0

0.0

2

100.0

0

0.0

1

100.0

 

Females examined at Scheduled

Necropsy

24

100.0

21

87.5

24

100.0

21

87.5

    Non Gravid

2

8.3

1

4.8

1

4.2

1

4.8

    Gravid

22

91.7

20

95.2

23

95.8

20

95.2

       With resorptions only

0

0.0

0

0.0

0

0.0

0

0.0

       With viable foetuses

22

100.0

20

100.0

23

100.0

20

100.0

Total Females Gravid

22

91.7

23

95.8

23

95.8

23

95.8

Table 5. Summary of Clinical Findings: Total Occurrence / No. of Animals

 

Dose Group (mg/Kg/day)

0 (Control)

100

250

500

Normal – No significant clinical observations

646/24

623/24

652/24

611/24

 

Disposition

 

 

 

 

  - Found dead

0/0

1/1

0/0

1/1

  - Sent to necropsy in extremis

0/0

2/2

0/0

1/1

  - Aborted

0/0

0/0

0/0

1/1

  - Scheduled Euthanasia; Gestation Day 29

24/24

21/21

24/24

21/21

 

Body/Integument

 

 

 

 

- Hair loss forelimb (s)

0/0

2/1

0/0

6/2

- Thin

0/0

1/1

0/0

2/2

- Hair loss Hindlimb (s)

0/0

0/0

0/0

1/1

 

Cardio-Pulmonary

 

 

 

 

- Rales

3/1

4/3

3/2

9/3

- Increases Respiration Rate

0/0

0/0

1/1

0/0

- Laboured Respiration

0/0

2/1

1/1

4/2

- Pale Body

0/0

0/0

1/1

0/0

 

Eyes / Ears / Nose

 

 

 

 

- Clear Discharge Left Eye

0/0

2/1

0/0

0/0

- Clear Discharge Right Eye

0/0

2/1

0/0

0/0

- Dried Red Material Around Nose

0/0

0/0

0/0

1/1

- Clear Nasal Discharge

0/0

1/1

0/0

0/0

 

Excreta – Decreased Defaecation

0/0

3/2

1/1

21/5

 

Body / Integ II

 

 

 

 

- Scabbing Dorsal Trunk

3/1

0/0

0/0

0/0

- Wet Brown Material Anogenital Area

1/1

0/0

0/0

0/0

- Dried Brown Material Anogenital Area

3/3

0/0

0/0

2/2

Table 6. Summary of Body Weight Changes during Gestation (g)

Parameter

 

Group (mg/Kg/day)

0 (control)

100

250

500

Initial Body Weight

Mean

3314.

3357.

3321.

3348.

S.D.

160.7

232.7

192.1

205.2

S.E.

34.2

52.0

40.1

45.9

N

22

20

23

20

 

Terminal Body Weight

Mean

3826.

3816.

3811.

3735.

S.D.

229.0

236.2

254.4

344.9

S.E.

48.8

52.8

53.1

77.1

N

22

20

23

20

 

Gravid Uterine Weight

Mean

497.9

510.0

516.2

487.4

S.D.

113.98

79.44

98.45

87.61

S.E.

24.30

17.76

20.53

19.59

N

22

20

23

20

 

Net Body Weight

Mean

3328.3

3306.1

3295.2

3247.1

S.D.

235.97

222.21

235.63

285.10

S.E.

50.31

49.69

49.13

63.75

N

22

20

23

20

 

Net Body Weight Change

Mean

14.7

-51.3

-25.3

-100.5

S.D.

191.80

191.90

146.80

275.20

S.E.

40.89

42.91

30.61

61.54

N

22

20

23

20

Mean Differences Calculated from Individual Differences

Non Gravid Weight(s) Not Included in Calculation of Mean

Table 7. Summary of Fetal Data at Scheduled Necropsy

Group

(mg/Kg/day)

 

Sex

Viable

Fetuses

Dead

Fetuses

Resorptions

Post Implantation Loss

Implantation Sites

Corpora Lutea

Pre-Implantation

Loss

Fetal Weight in Grams

No. of Gravid Females

Male

Female

Early

Late

0 (Control)

Total

92

100

192

0

9

1

10

202

221

19

NA

22

Mean

4.2

4.5

8.7

0.0

0.4

0.0

0.5

9.2

10.0

0.9

42.1

S.D.

2.08

1.63

2.57

0.00

0.80

0.21

0.86

2.22

1.68

1.78

5.98

S.E.

0.44

0.35

0.55

0.00

0.17

0.05

0.18

0.47

0.36

0.38

1.27

 

100

Total

91

87

178

0

6

0

6

184

197

13

NA

20

Mean

4.6

4.4

8.9

0.0

0.3

0.0

0.3

9.2

9.9

0.7

42.0

S.D.

2.09

2.21

1.74

0.00

0.92

0.00

0.92

1.54

1.39

0.99

3.79

S.E.

0.47

0.49

0.39

0.00

0.21

0.00

0.21

0.34

0.31

0.22

0.85

 

250

Total

88

117

205

1

2

8

11

216

236

20

NA

23

Mean

3.8

5.1

8.9

0.0

0.1

0.3

0.5

9.4

10.3

0.9

41.4

S.D.

1.67

1.70

1.83

0.21

0.29

0.71

0.85

2.13

2.12

0.92

4.57

S.E.

0.35

0.36

0.38

0.04

0.06

0.15

0.18

0.44

0.44

0.19

0.95

 

1000

Total

88

97

185

0

3

3

6

191

205

14

NA

20

Mean

4.4

4.9

9.3

0.0

0.2

0.2

0.3

9.6

10.3

0.7

37.7*

S.D.

1.10

1.46

1.33

0.00

0.37

0.37

0.57

1.32

1.55

0.98

6.35

S.E.

0.24

0.33

0.30

0.00

0.08

0.08

0.13

0.29

0.35

0.22

1.42

* = Significantly different from the control group at 0.05

NA = Not applicable

Mean Number of Viable Fetuses; Mean Number of Implantation Sites; Mean Number of Corpora Lutea; Fetal Weights compared using Dunnett’s Test.

Table 8. Summary of Litter Proportions of Malformations (% per Litter)

Dose Group (mg/Kd/day)

 

0 (Control)

100

250

500

Number of Litters Examined

22

20

23

20

 

Total Malformations

 

Percent per Litter with External Malformations

Mean

1.0

0.0

0.0

1.4

S.D.

3.27

0.00

0.00

3.33

S.E.

0.70

0.00

0.00

0.74

 

Percent per Litter with Soft Tissue Malformations

Mean

2.9

0.5

0.8

0.0

S.D.

8.25

2.03

2.75

0.00

S.E.

1.76

0.45

0.57

0.00

 

Percent per Litter with Skeletal Malformations

Mean

1.2

1.6

0.4

3.0

S.D.

3.77

5.04

2.09

7.03

S.E.

0.80

1.13

0.43

1.57

 

Total Percent per Litter with Malformations

Mean

5.0

2.1

1.3

4.0

S.D.

10.62

6.73

4.50

7.17

S.E.

2.26

1.51

0.94

1.60

Modified Statistics Used

Table 9. Summary of Fetuses and Litters with Variations (Absolute No.)

Dose Group

(mg/Kg/day)

Fetuses

Litters

0 (Control)

100

250

500

0 (Control)

100

250

500

Number Examined Externally

192

178

205

185

22

20

23

20

  Excessive Fat Pads

1

0

0

0

1

0

0

0

 

Number Examined Viscerally

192

178

205

185

22

20

23

20

   Major Blood Pressure Variation

20

15

14

19

8

7

9

9

   Retrocaval Ureter

7

2

2

1

4

1

2

1

   Accessory Spleen (S)

29

21

20

23

15

8

11

11

   Heart Extra Papillary Muscle

4

3

4

3

2

2

3

2

   Spleen – Small

0

0

0

3

0

0

0

2

   Gall Bladder – Absent or Small

3

6

1

2

3

3

1

1

   Liver – Accessory Lobule (s)

1

0

0

0

1

0

0

0

   Spleen – Pale

2

0

0

6

1

0

0

3

   Renal Papillae(s) Not Developed and/or Distended Ureters

1

0

0

0

1

0

0

0

   Liver – Pale

0

0

0

1

0

0

0

1

 

Number Examined Skeletally

192

178

205

185

22

20

23

20

   13thRudimentary Rib(s)

36

28

41

22

15

13

18

15

   13thFull Rib(s)

64

64

52

48

18

16

18

14

   27 Presacral Vertebrae

10

20

5

7

7

9

3

5

   Sternebra(E) Malaligned (slight or moderate)

2

7

4

3

2

6

4

3

   Sternebra(E) #5 and/or #6 Unossified

31

27

34

28

14

13

13

10

   Hyoid Arch (es) Bent

4

3

2

3

4

2

2

3

   Extra Site of Ossification Anterior to Sternebrae(E) # 1

7

0

8

2

4

0

7

2

   Sternebrae with Thread like Attachment

1

3

0

2

1

2

0

1

   7thCervical Ribs

1

1

5

1

1

1

4

1

   Pubis Unossified

0

0

0

3

0

0

0

1

   Vertebral Centra not Fully Ossified

0

1

0

0

0

1

0

0

   Accessory Skull Bone(s)

0

0

1

2

0

0

1

2

   7thSternebrae

0

0

0

1

0

0

0

1

Table 10. Summary of Fetuses and Litters with Malformations (Absolute No.)

Dose Group

(mg/Kg/day)

Fetuses

Litters

0 (Control)

100

250

500

0 (Control)

100

250

500

Number Examined Externally

192

178

205

185

22

20

23

20

   Short-tail

0

0

0

1

0

0

0

1

   Carpal and/or Tarsal Flexure

1

0

0

0

1

0

0

0

   Polydactyly

1

0

0

0

1

0

0

0

   Exencephaly with or without Open Eyelid

0

0

0

1

0

0

0

1

   Macroglossia

1

0

0

0

1

0

0

0

   Malpositioned Umbilicus

0

0

0

1

0

0

0

1

 

Number Examined Viscerally

192

178

205

185

22

20

23

20

   Lungs – Lobular Agenesis

4

0

2

0

2

0

2

0

   Vestigial Pulmonary Trunk

1

1

0

0

1

1

0

0

  Bulbous Aorta

2

1

0

0

2

1

0

0

  Interventricular Septal Defect

0

1

0

0

0

1

0

0

  Heart – Three Chambered

1

0

0

0

1

0

0

0

  Heart – Small Ventricle

1

0

0

0

1

0

0

0

 

Number Examined Skeletally

192

178

205

185

22

20

23

20

  Vertebral Anomaly with or without associated Rib Anomaly

1

1

0

2

1

1

0

2

  Rib Anomaly

0

0

1

0

0

0

1

0

  Coastal Cartilage Anomaly

1

1

1

1

1

1

1

1

  Sternebra (E) Malaligned (Severe)

0

1

0

3

0

1

0

3

  Sternoschisis

0

0

0

1

0

0

0

1

  Vertebral Centra Anomaly

0

0

0

1

0

0

0

1

 

Total number with malformations

 

   External

2

0

0

3

2

0

0

3

   Soft Tissue

5

1

2

0

3

1

2

0

   Skeletal

2

3

1

6

2

2

1

4

 

 

   Combined

9

4

3

8

6

2

2

6

Conclusions:
The no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal developmental toxicity was 250 mg/kg bw/day.
Executive summary:

Only screening reproductive/developmental toxicity data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

A Prenatal Developmental GLP Toxicity Study was conducted according to test guideline 414. The test substance, dipropyleneglycol dibenzoate (DPGDB), in the vehicle (0.5% carboxymethylcellulose in deionized water) was administered orally by gavage to 3 groups of 24 time-mated female New Zealand White [Hra:(NZW)SPF] rabbits once daily from Gestation Days 7–28. Dosage levels were 100, 250, and 500 mg/kg/day administered at a dose volume of 5 mL/kg. Adverse effects on maternal survival, mean body weight changes, and food consumption were noted in the 500 mg/kg/day group; therefore, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on lower mean fetal weights at 500 mg/kg/day, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity when dipropyleneglycol dibenzoate was administered orally by gavage to time-mated New Zealand White rabbits. Since no severe developmental toxicity effects were observed and fetal weights reduction was considered to be due to maternal toxicity effect at 500 mg/kg/d group, the substance is not categorised as developmental toxicant under GHS classification criteria.

Test substance-related effects on survival were noted in the 500 mg/kg/day group, as 1 female was euthanized in extremis on Gestation Day 26 following a severe body weight loss and markedly reduced food consumption, with corresponding incidences of decreased defecation noted at the daily examinations. Adverse clinical observations of rales, labored respiration, decreased respiration rate, and a thin body were also noted for this female at the daily examinations and/or postdosing observations. An additional female in the 500 mg/kg/day group aborted on Gestation Day 25 and was subsequently euthanized. At necropsy, neither female had remarkable macroscopic findings. There were no other test-substance related effects on survival. In the 100 and 500 mg/kg/day groups, 3 and 1 females, respectively, were found dead or euthanized in extremis during Gestation Days 13–26; following macroscopic/microscopic examination, the cause of mortality/moribundity of these females were attributed to or considered likely related to intubation error. All other females survived to the scheduled necropsy. In the 500 mg/kg/day group, there was a slight increase in the incidence of decreased defecation at the daily examinations, which was considered secondary to the reduced mean food consumption noted in this group. Test substance-related lower mean body weight gains or body weight losses were noted in the 500 mg/kg/day group generally during Gestation Days 13–29, resulting in a lower mean body weight gain when the entire treatment period (Gestation Days 7–29) was evaluated compared to the control group. Mean food consumption was also lower in this group during Gestation Days 13–29, which resulted in slightly lower mean food consumption for the overall dosing period (Gestation Days 7–29), and corresponded to the decreased mean body weight gains noted in this group. In addition, mean net body weight loss was also noted compared to the control group at 500 mg/kg/day. Although mean body weights in the 500 mg/kg/day group were comparable to the control group throughout the treatment period, these changes were still considered adverse as they led to the mortality or abortion of 2 females within the group. Mean net body weight and gravid uterine weight were comparable to the control group at 500 mg/kg/day. In the 100 and 250 mg/kg/day groups, mean body weights, body weight changes, net body weights, net body weight changes, gravid uterine weights, and food consumption were unaffected by test substance administration. Hematology and serum chemistry parameters were unaffected by test substance administration at all dosage levels.

Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance. Test substance-related adrenal cortical hypertrophy was noted for females in the 100, 250, and 500 mg/kg/day groups; however, due to the minimal to mild severity and the lack of other related microscopic and gross findings, this finding was considered non adverse. There were no other test-substance-related microscopic observations at any dosage level. Mean fetal body weights (male, female, and combined) were 8.9% to 11.8% lower in the 500 mg/kg/day group compared to the control group, and were considered test substance-related and adverse. Mean fetal body weights in the 100 and 250 mg/kg/day groups were similar to the control group. Intrauterine survival was unaffected by test substance administration at all dosage levels. No test substance-related effects on fetal morphology were noted at any dosage level.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Oxydipropyl dibenzoate
EC Number:
248-258-5
EC Name:
Oxydipropyl dibenzoate
Cas Number:
27138-31-4
Molecular formula:
C20H22O5
IUPAC Name:
oxydipropyl dibenzoate
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Eastman Chemical Company (United States); Batch no. V988701802
- Expiration date of the lot/batch: None
- Purity test date: 2017-01-25

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (18°C to 24°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: previously shown to be stable over the range of concentrations used on this studyfor at least 10 days under refrigerated (2°C to 8°C) conditions

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid

OTHER SPECIFICS:
Purity: > 99.0%

Test animals

Species:
rabbit
Strain:
New Zealand White
Remarks:
Hra:(NZW)SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc. (Denver, PA)
- Age at study initiation: approximately 7 months old at the time of mating by the supplier
- Weight at study initiation: 2949 g to 3901 g on Gestation Day 0
- Fasting period before study: No
- Housing: Upon arrival, all rabbits were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel-O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). The bedding was changed at least twice weekly. Nesting material was not required because the females were euthanized prior to the date of expected parturition. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rabbit LabDiet®5322 - The basal diet was offered in 25-g increments 3 times per dayon the dayof arrival and in increased amounts over the next few days, until the animals gradually achieved ad libitum status prior to the dose administration period; basal diet was offered ad libitum thereafter. Kale (1 leaf at each occasion) was provided to each animal daily for environmental enrichment and to aid in maintaining the animal's gastrointestinal health, beginning upon animal receipt and continuing throughout the duration of the study. Kale present in the cage at the time of providing a new leaf was discarded. The diet was supplemented with extra kale, celery sticks, and haycubes as necessary.
- Water (e.g. ad libitum): Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum
- Acclimation period: time-mated rabbits received on Gestation Day 1, 2, 3, or 4.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 61°F to 71°F (16°C to 22°C)
- Humidity (%): 30% to 70%
- Air changes (per hr): minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours) / 12-hour dark photoperiod

IN-LIFE DATES: From: 2017-06-30 To: 2017-07-28

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
medium viscosity, 400–800 cps, USP
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle and test substance formulations were administered orally by gavage, via appropriately sized rubber catheters, once daily during Gestation Days 7–28. The dose volume for all groups was 5 mL/kg. Following administration of each dose, the catheter was flushed with 5 mL of deionized water to ensure delivery of the entire dose.

VEHICLE
- Justification for use and choice of vehicle (if other than water): carboxymethylcellulose (CMC); reason for choice not specified
- Concentration in vehicle: 0, 20, 50, or 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required): Lot Nos. 2GA0044 and 1GE0858, retest dates: 31 Jul 2017 and 16 Apr 2018, respectively
- Purity: 0.5% carboxymethylcellulose in deionized water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A validated HPLC/UV method was used and extended for the determination of DPGDB concentration in suspension formulations. Method specificity/selectivity, calibration reproducibility, precision, and accuracy and were assessed and validated, satisfying SOP specified criteria. In addition, the results of the assessment of test substance homogeneity and, following 10 days of refrigerated storage, resuspension homogeneity in formulations prepared at target concentrations of 20 and 100 mg DPGDB/mL met the applicable protocol-specified acceptance criteria.
Details on mating procedure:
One hundred six time-mated female New Zealand White rabbits were received in good health from Covance Research Products, Inc., Denver, PA, on 30 Jun 2017. The time-mated rabbits were received on Gestation Day 1, 2, 3, or 4.
Duration of treatment / exposure:
The vehicle and test substance formulations were administered orally by gavage, via appropriately sized rubber catheters, once daily during Gestation Days 7–28. The dose volume for all groups was 5 mL/kg. Dosage levels of 100, 250, and 500 mg/kg/day were selected for the current study.
Frequency of treatment:
once daily during Gestation Days 7–28
Duration of test:
during Gestation Days 7–28.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle Control (Group 1)
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
500 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
The experimental design consisted of 3 test substance-treated groups and 1 control group, composed of 24 rabbits/group.


Group Number Treatment Dosage/Dose Level Number of
Females
(mg/kg/day)

1 Vehicle Control 0 24
2 DPGDB 100 24
3 DPGDB 250 24
4 DPGDB 500 24







Control animals:
yes
Details on study design:
- Dose selection rationale:
Dosage levels were selected based on results of a previous dose range-finding prenatal developmental toxicity study in rabbits with DPGDB, and were provided by the Sponsor
Representative after consultation with the Charles River Study Director. In the previous study, time-mated rabbits were dosed (via oral gavage) with 100, 250, 500, and 1000 mg/kg/day of DPGDB during Gestation Days 7–28. Significant toxicity was noted at the 1000 mg/kg/day dosage level as evidenced by abortions, body weight losses, and reduced food consumption, resulting in early termination of this dosage group. At the 500 mg/kg/day dosage level, excreta-related findings were noted without significant effects on body weights or food consumption. Dosage levels of 100 and 250 mg/kg/day were well tolerated. Therefore, dosage levels of 100, 250, and 500 mg/kg/day were selected for the current study to provide the evaluation of potential dose-response and determination of a no-observed-adverse-effect level (NOAEL).

The selected route of administration for this studywas oral (gavage) because this is a potential route of exposure for humans.

- Rationale for animal assignment (if not random): The bred females were assigned to groups using a WTDMS™ computer program, which randomized the animals based on stratification of the Gestation Day 0 body weights in a block design.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations were recorded daily from the dayof receipt through Gestation Day 29 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1, 2, and 4 hours following dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on Gestation Days 0 (by supplier under conditions that were not compliant with GLPs, but in accordance with the supplier’s SOPs), 5, and 7–29. Group mean body weights were calculated for each of these days. Mean bodyweight changes were calculated for each corresponding interval and also for Gestation Days 7–10, 10–13, 13–20, 20–29, and 7–29. Gravid uterine weight was collected and net bodyweight (the Gestation Day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Individual food consumption was recorded on Gestation Days 5–29. Food intake was reported as g/animal/dayand g/kg/dayfor the corresponding bodyweight change intervals.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Gestation Day 29 Laparohysterectomy: The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovarywas recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss. Maternal tissues with gross lesions were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. In addition, the adrenal glands, stomach, heart, and kidneys were preserved in 10% neutral-buffered formalin for microscopic examination.

Unscheduled Deaths: For females that were found dead, euthanized in extremis, or aborted during the course of the study, maternal tissues with gross lesions were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Inaddition, the adrenal glands, stomach, heart, and kidneys were preserved in 10% neutral-buffered formalin for microscopic examination.The number and location of implantation sites, corpora lutea, and viable fetuses were recorded.

OTHER:

CLINICAL PATHOLOGY:
Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 females/group from all groups at necropsy (Gestation Day29), and from females that were euthanized in extremis or exhibited signs of toxicity during the study. For females euthanized in extremis, blood was collected prior to euthanasia, and animals were euthanized immediatelyfollowing blood collection by an intravenous injection of sodium pentobarbital via a marginal ear vein. The animals were not fasted prior to blood collection. Blood samples were collected from a marginal ear vein (or other site as deemed necessary). Blood was collected in tubes containing K2EDTA (hematology) or no anticoagulant (serum chemistry).

Parameters listed in Table 2. and Table 3. were evaluated for Haematology and Clinical Chemistry, respectively.
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

The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. Selected tissues were examined microscopically from all females found dead and euthanized in extremis, as well as from 5 females/group at the scheduled necropsy.
Fetal examinations:
The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations

- External examinations: Yes: all per litter
The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Nonviable fetuses (if the degree of autolysis was minimal or absent) were examined, the crown-rump length measured, weighed, sexed, and tagged individually. Crown-rump measurements, degrees of autolysis and gross examinations, if possible, were recorded for late resorptions, and the tissues were discarded.

- Soft tissue examinations: Yes: all per litter
Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development.

- Skeletal examinations: Yes: all per litter
Following fixation in alcohol, each fetus was stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations

- Head examinations: Yes: all per litter. Heads from all fetuses were examined bya midcoronal slice.
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit.

Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA9 to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test10 was used to compare the test substance-treated groups to the control group. Histopathological findings in the test substance-treated groups were compared to the control group using a two-tailed Fisher’s Exact test.
Indices:
Intrauterine data were summarized using 2 methods of calculation.

1) Group Mean Litter Basis:

Post implantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group / No. Gravid Females/Group)

2) Proportional Litter Basis:

Summation Per Group (%) = (Sum of Post implantation Loss/Litter (%) / No. Litters/Group)

Where:
Post implantation Loss/Litter (%) = No. Dead Fetuses, Resorptions (Early/Late)/Litter / No. Implantation Sites/Litter) x 100

Fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:

Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%) / No. Litters/Group)

Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter / No. Viable Fetuses/Litter) x 100
Historical control data:
Charles River Ashland historical control data was used (Appendix 6 of the study report)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Female in the 500 mg/kg/day group showed incidences of decreased defecation at the daily examinations during Gestation Days 20–26. Additional adverse clinical observations included rales, labored respiration, decreased respiration rate, and a thin body were noted at the daily examinations and/or postdosing observations during Gestation Days 23–26.

In the 500 mg/kg/day group, Female was found dead on Gestation Day 26 with clinical observations of rales, labored respiration, and red material around the nose at the daily examinations and/or postdosing observations on Gestation Day 25. At necropsy, Female had dark red discoloration and firmness of all lobes of the left lung, which correlated microscopically with acute inflammation. The trachea was also noted to be filled with dark red contents, which microscopically was characterized by hemorrhage, edema, and dilatation of the submucosa. The cause of death of this female was considered acute inflammation of the lungs, most likely due to an intubation error, and not considered test substance-related.In the 100 mg/kg/day group, Female was euthanized with clinical observations of rales, labored respiration, a thin body, and/or clear discharge from the eyes and nose were noted for this female at the daily examinations and/or postdosing observations during Gestation Days 24–25. At necropsy, mottling and failure to fully inflate were observed in all lobes of the lungs, which correlated with acute inflammation microscopically. Acute inflammation was centered on the bronchi and bronchioles and was consistent with an intubation error; therefore, the cause of moribundity was acute inflammation of the lungs, and not considered test substance-related.

Test substance-related incidences of decreased defecation was noted for 4 surviving females in the 500 mg/kg/day group at the daily examinations during Gestation Days 15–29, which corresponded to and were considered secondary to the reduced food consumption noted in this group. There were no other test substance-related clinical observations noted at the daily examinations or approximately 1, 2, or 4 hours following dose administration. Observations
noted in the treated groups, including rales, labored respiration, and/or decreased respiration rate, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Test substance-related effects on survival were noted in the 500 mg/kg/day group.

One female in the 500 mg/kg/day group was euthanized in extremis on Gestation Day 26 following a severe body weight loss (18.3% during Gestation Days18–26) and markedly reduced food consumption (0 to 10 g/day during Gestation Days 19–26), with corresponding incidences of decreased defecation at the daily examinations during Gestation Days 20–26. In addition, Female in the 500 mg/kg/day group aborted 1 late resorption (with no apparent malformations) on Gestation Day 25, and was subsequently euthanized; however, no clinical observations or remarkable effects on body weights and food consumption were noted for this female prior to aborting. At necropsy, neither female was noted with remarkable macroscopic findings. Due to the adverse clinical observations, body weight loss, reduced food consumption, and no clear cause of moribundity or abortion at necropsy, these findings were considered test substance-related and adverse.

No other test substance-related effects on survival were noted during the study. In the 500 mg/kg/day group, Female was found dead on Gestation Day 26 following a severe body weight loss (16.0% during Gestation Days 20–25) and markedly reduced food consumption (0 to 7 g/day during Gestation Days 21–25), and clinical observations of rales, labored respiration, and red material around the nose at the daily examinations and/or postdosing observations on Gestation Day 25. At necropsy, Female No. 2474 had dark red discoloration and firmness of all lobes of the left lung, which correlated microscopically with acute inflammation. The trachea was also noted to be filled with dark red contents, which microscopically was characterized by hemorrhage, edema, and dilatation of the submucosa. The cause of death of this female was considered acute inflammation of the lungs, most likely due to an intubation error, and not considered test substance-related.


In the 100 mg/kg/day group, Female was euthanized in extremis on Gestation Day 25 following a body weight loss (13.6% during Gestation Days 14–25), reduced food consumption (0 to 44 g/day during Gestation Days 16–25), and corresponding incidences of decreased defecation at the daily examinations during Gestation Days 19–20. Additional clinical observations of rales, labored respiration, a thin body, and/or clear discharge from the eyes and nose were noted for this female at the daily examinations and/or postdosing observations during Gestation Days 24–25. At necropsy, mottling and failure to fully inflate were observed in all lobes of the lungs, which correlated with acute inflammation microscopically. Acute inflammation was centered on the bronchi and bronchioles and was consistent with an intubation error; therefore, the cause of moribundity was acute inflammation of the lungs, and not considered test substance-related. In addition, Female in the 100 mg/kg/day group was found dead on Gestation Day 13. No remarkable effects on body weight or food consumption were noted prior to death, but a clinical observation of rales was noted for this female at the daily examinations and postdosing observations up to 2 days prior to death. At necropsy, oily contents in all lung lobes and a perforation of the trachea at approximately the level of cervical vertebra No. 3 were noted. Microscopically, the lungs showed acute inflammation. Perforation of the trachea was not confirmed histologically, but findings of edema, hemorrhage, dilatation, and mixed inflammatory cell infiltrates in the tracheal submucosa were likely related. Therefore, the cause of death for this female was acute inflammation of the lungs due to an intubation error, which was not considered test substance-related. Female in the 100 mg/kg/day group was euthanized in extremis on Gestation Day 24; no clinical observations or noteworthy changes in body weight or food consumption were noted prior to euthanasia. At necropsy, oily contents in all lung lobes and multiple, dark red, irregularly shaped areas in all lobes were noted for this female. Microscopically, the lungs showed acute inflammation and hemorrhage. Intubation error was determined to be the cause of debility and euthanasia, and therefore was not considered test substance-related. All other females survived to the scheduled euthanasia.


Test substance-related incidences of decreased defecation was noted for 4 surviving females in the 500 mg/kg/day group at the daily examinations during Gestation Days 15–29, which corresponded to and were considered secondary to the reduced food consumption noted in this group. There were no other test substance-related clinical observations noted at the daily examinations or approximately 1, 2, or 4 hours following dose administration. Observations
noted in the treated groups, including rales, labored respiration, and/or decreased respiration rate, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean body weight gains or body weight losses were noted in the 500 mg/kg/day group generally during Gestation Days 13–29, resulting in a lower mean body weight gain when the entire treatment period (Gestation Days 7–29) was evaluated compared to the control group. Although mean body weights in the 500 mg/kg/day group were comparable to the control group throughout the treatment period, these changes were still considered adverse as they led to the mortality or abortion of 2 females within the group.

Test substance-related lower mean body weight gains or body weight losses were noted in the 500 mg/kg/day group compared to the control group beginning on Gestation Day 13, and continuing sporadically throughout the remainder of the treatment period, resulting in lower mean body weight gains in this group when the Gestation Days 13–20 and 20–29 cumulative intervals and when the entire treatment period (Gestation Days 7–29) were evaluated. Although
none of the differences were statistically significant when compared to the control group and were not of sufficient magnitude to affect absolute mean body weights in this group, these changes were considered adverse as they led to the euthanasia or abortion of 2 females within the group. A mean net body weight loss (not statistically significant) was noted in the 500 mg/kg/day group compared to the control group, while mean net body weight and gravid
uterine weight in this group were comparable to the control group. Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100 and 250 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. Significant (p < 0.05) mean body weight losses were noted in the 100 mg/kg/day group compared to the control group during Gestation Days 15–16 and 20–21; however, these transient differences did not occur in a dose-related manner, and were therefore not considered test substance-related. Mean net body weight losses were also noted in the 100 and 250 mg/kg/day groups; however, the differences were not statistically significant and did not occur in a dose-related manner, and were therefore not considered test substance-related.

Mean net body weight loss was also noted compared to the control group at 500 mg/kg/day. Although mean body weights in the 500 mg/kg/day group were
comparable to the control group throughout the treatment period, these changes were still considered adverse as they led to the mortality or abortion of 2 females within the group. Mean net body weight and gravid uterine weight were comparable to the control group at 500 mg/kg/day. In the 100 and 250 mg/kg/day groups, mean body weights, body weight changes, net body weights, net body weight changes, gravid uterine weights, and food consumption were unaffected by test substance administration.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption was also lower in this group during Gestation Days 13–29, which resulted in slightly lower mean food consumption for the overall dosing period (Gestation Days 7–29), and corresponded to the decreased mean body weight gains noted in this group.

Mean maternal food consumption, evaluated as g/animal/day or g/kg/day, in the 500 mg/kg/day group was comparable to the control group during the first week of treatment (Gestation Days 7–10 and 10–13), but generally lower than the control group during the remainder of the treatment period (Gestation Days 13–20 and 20–29); differences were significant (p < 0.05 or p < 0.01) during Gestation Days 21–23 and 24–25. The lower mean food consumption corresponded to the lower mean body weight gains noted in this group during the same period, and resulted in a slightly lower mean food consumption when the entire treatment period (Gestation Days 7–29) was evaluated, and in the presence of other adverse effects noted at this dosage level, was considered test substance-related and adverse.
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):
Hematology and serum chemistry parameters were unaffected by test substance administration at all dosage levels.

There were no test substance-related changes in hematology parameters on Gestation Day 29. Increases in the percentage and the absolute number of monocytes were noted at 500 mg/kg/day compared to the control group. However, the differences were not statistically significant and could be attributed to a single female in this group, and in the absence of any other effects on hematology parameters, were considered to be the result of normal biological variation. There were no other effects on hematology parameters; there was no dose-response relationship, the changes were of minimal magnitude, and were not considered to be of toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematology and serum chemistry parameters were unaffected by test substance administration at all dosage levels.

There were no test substance-related changes in serum chemistry parameters on Gestation Day 29. There was no dose-response and the changes were of minimal magnitude. These differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related adrenal cortical hypertrophy was noted for females in the 100, 250, and 500 mg/kg/day groups; however, due to the minimal to mild severity and the lack of other related microscopic and gross findings, this finding was considered nonadverse. There were no other test-substance-related microscopic observations at any dosage level.

Adrenal cortical hypertrophy was considered a nonadverse finding due to the minimal to mild severity and the lack of other related microscopic and gross findings. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There were no test substance-related alterations in the prevalence, severity, or histologic character of those incidental tissue alterations.

Dosage (mg/kg/day): Females
0 100 250 500
Adrenal cortex 5 5 5 5
Hypertrophy 0 1 3 3
Minimal - 0 1 1
Mild - 1 2 2
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg/day group, 1 (4.2%) abortion was observed. However, no clinical observations or remarkable effects on body weights and food consumption were noted for this female prior to aborting. No apparent malformations and in female no remarkable macroscopic findings were observed. There were total 22 (91.7%), 23 (95.8%), 23 (95.8%), and 23 (95.8%) gravid females at the time of scheduled necropsy in Group 1, 2, 3, and 4 respectively (See Table 4).
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Preimplantation loss was observed in 19 fetus in group 1, 13 fetus in group 2, 20 fetus in group 3 and 14 fetus in group 4.Post-implantation loss was observed in 10 fetus in group 1, 6 fetus in group 2, 11 fetus in group 3 and 6 fetus in group 4. The mean percentages for Preimplantation loss were 8.3,6.5, 8.6 and 6.2% in Group 1,2,3 and 4. The mean percentages for Post implantation loss were 5.8,3.2, 4.3 and 3.1% in Group 1,2,3 and 4. Differences from the control group were not statistically significant.

Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and group 4 is 500 mg/kg/d. Non significant changes were observed (Refer attached tables) .

Parameters evaluated included postimplantation loss, number and percentage of viable fetuses, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
Total 32 resorptions were observed in out of total 760 fetus. Total 10 resorptions in Group 1, and 6 resorptions in Group 2 and 10 resorptions in Group 3 and 6 resorptions in Group 4 were observed. The mean percentages for total resorption were 5.8, 3.2, 3.9 and 3.1% in Group 1, 2, 3, and 4 where Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and Group 4 is 500 mg/kg/d. The changes were non significant in comparisn to controls. Differences from the control group were slight and not statistically significant (See Tables 4 and 7) .
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Early resorption was observed in 9 fetus in group 1, 6 fetus in group 2, 2 fetus in group 3 and 3 fetus in group 4. Late resorption was observed in 1 fetus in group 1, 0 fetus in group 2, 8 fetus in group 3 and 3 fetus in group 4. The mean percentages for early resorption were 0.5, 0.0, 3.0 and 1.5% in Group 1,2,3 and 4. Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and group 4 is 500 mg/kg/d. The changes were non significant in comparison to controls.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
1 fetus was found dead in group 3 (250 mg/kg/d). There were total 88 male viable fetus and 117 female viable fetus. out of total 205 viable fetus one dead fetus was found and which was non dose related. The mean percentages were 0.0,0.0,0.4 and 0.0 % for group 1,2,3 and 4. The values were not significantly different from control group.Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and group 4 is 500 mg/kg/d.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Total 22 (100 %), 23 (91.7%) 23 (91.7%) and 23 (91.7%) number of females were gravid in Group 1,2,3 and 4. The non gravid preganicies were 2 (8.3%), 1 (4.8%), 1 (4.2%), 1 (4.8%) in Group 1,2,3 and 4.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant (Attached summary table below).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
mortality

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male, female, and combined fetal weights in the 500 mg/kg/day group were 8.9%, 11.8%, and 10.5% lower, respectively, compared to the control group, which corresponded with maternal toxicity observed at this dosage level. The differences were generally significant (p < 0.05 or p < 0.01) and were considered test substance-related and adverse. Mean fetal body weights in the 100 and 250 mg/kg/day groups were similar to the control group.

The fetal weight in group 1 was 42.1 in grams and in group 2 was 42.0 gm and group 3 was 41.4 gm and in group three was 37.7 gm in 500 mg/kg/d. The group 4 was statistically significant different from the control group (See Table 7).
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Viable fetus in group 1 were 192 and group 2 were 178 and in group 3 were 205 and group 4 were 185. The results were comparable to controls and were non significant. The viable percentages values were 94.2, 96.8, 95.7 and 96.9 in Group 1, 2 , 3, and 4. Group 1 is 0 mg/kg/d; Group 2 is 100 mg/kg/d; Group 3 is 250 mg/kg/d; and Group 4 is 500 mg/kg/d. The mean values for vaible fetus in group 1, 2, 3, and 4 were 8.7 (0 mg/kg/d) , 8.9 (100 mg/kg/d), 8.9 (250 mg/kg/d) and 9.3 (500 mg/kg/d). The values showed that there were no treatment related effcts and values were comparable to controls.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio was similar across all groups
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations were observed in fetuses (litters) in dose groups and were considered spontaneous in origin.

No test substance-related external malformations were noted for fetuses at any dosage level. In the 500 mg/kg/day group, one fetus was noted with a short tail (approximately 7 mm in length), one fetus was noted with exencephaly with open eyelids, and other fetus in same group was noted with a malpositioned umbilicus (located more lateral than normal). Skeletally, the short tail consisted of fused, malpositioned, and absent caudal vertebrae, and exencephaly consisted of small and misshapen frontal and parietal bones, an absent interparietal bone, and an unossified supraoccipital bone. These findings were noted in single fetuses, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data; therefore, they were not considered test substance-related. No other external malformations were noted in the test substance-treated groups. In the control group, fetus noted with carpal flexure (right) and polydactyly (6 digits present on the right forepaw), and fetus was noted with macroglossia. Skeletally, polydactyly consisted of one extra digit with only the distal phalanx ossified. There were no other external malformations observed on this study. No external developmental variations were noted in the test substance-treated groups. The only finding observed (excessive fat pads) was noted in a single fetus in the control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Because the aforementioned malformations were noted infrequently or at a similar frequency in the control group, were not observed in a clear dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data, they were not considered test substance-related. There were no other skeletal malformations observed on this study.

Severely malaligned sternebrae, resulting in fused or attached sternebrae, were noted for 1 and 3 fetuses in the 100 and 500 mg/kg/day groups, respectively. In addition, sternoschisis (sternal band No. 2 not joined) and a vertebral centra anomaly (fused sacral centra) were noted for two Fetus in the 500 mg/kg/day group, respectively. Furthermore, one Fetus in the 250 mg/kg/day group was noted with a rib anomaly (left rib Nos. 3 and 4 were fused proximally through medially). A vertebral anomaly with or without associated rib anomaly, consisting of extra ribs and centra, malpositioned centra and arches, fused centra, arches, and ribs, bipartite centra, absent centra and arches, and malproportioned arches and centra, was noted for 1 and 2 fetuses in the 100 and 500 mg/kg/day groups, respectively. In addition costal cartilage anomalies were noted for three Fetus in the control, 100, 250, and 500 mg/kg/day groups, respectively; 2 of these fetuses were also noted with other skeletal malformations, as noted above. Because the aforementioned malformations were noted infrequently or at a similar frequency in the control group, were not observed in a clear dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data, they were not considered test substance-related. There were no other skeletal malformations observed on this study.


A higher mean litter proportion of the skeletal developmental variation pubis unossified was noted in the 500 mg/kg/day group (1.4% per litter) compared to the control group (0.0% per litter). The value in this group also exceeded the maximum mean value in the Charles River Ashland historical control data (0.58% per litter). This reduction in ossification was considered secondary to the lower mean fetal body weights noted at this dosage level . Other findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related visceral malformations were noted for fetuses at any dosage level.

Lobular agenesis of the lungs (absent right accessory lobe) was noted for 2 fetuses in the 250 mg/kg/day group. In the 100 mg/kg/day group, a bulbous aortic arch, vestigial pulmonary trunk, and interventricular defect (a 1 mm in diameter opening in the anterior portion of the septum) were noted for one fetus . Because these visceral malformations were noted in single fetuses, in the control group at similar or higher frequencies (in five fetus), and/or were not observed in the high-dose group, they were not considered test substance-related. No other visceral malformations were noted in the test substance-treated groups. In the control group, one fetus was also noted with a three chambered heart (2 atria and 1 ventricle with an absent tricuspid valve) and one fetus was also noted with a small right ventricle in the heart. There were no other visceral malformations observed on this study.

There were no test substance-related developmental variations noted at any dose level. A higher mean litter proportion of the visceral developmental variation pale spleen was noted in the 500 mg/kg/day group (3.1% per litter) compared to the control group (0.7% per litter). The value in this group exceeded the maximum mean value in the Charles River Ashland historical control data (1.77% per litter). However, because this finding was only noted for 6(3) fetuses (litters), and was not statistically significantly different from the control group, it was not considered test substance-related. Other findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River
Ashland historical control data.

A cystic oviduct was noted for 1 and 2 fetuses in the 100 and 250 mg/kg/day groups, respectively, renal papilla(e) not fully developed (Woo and Hoar Grade 1) was noted for 1 and 2 fetuses in the 250 and 500 mg/kg/day groups, respectively, and red fluid in the abdominal cavity was noted for 1 fetus in the 500 mg/kg/day group. These findings were not classified as either a malformation or developmental variation and were not considered to be test substance-related because they occurred infrequently and/or in a manner that was not dose-related.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Table 4. Summary of Maternal Survival and Pregnancy Status

Dose Group

(mg/Kg/day)

0 (Control)

100

250

500

No.

%

No.

%

No.

%

No.

%

Females on Study

24

 

24

 

24

 

24

 

Females that aborted or delivered

0

0.0

0

0.0

0

0.0

1

4.2

 

Females that Died

0

0.0

1

4.2

0

0.0

1

4.2

    Females that aborted

0

0.0

0

0.0

0

0.0

0

0.0

    Non Gravid

0

0.0

0

0.0

0

0.0

0

0.0

    Gravid

0

0.0

1

100.0

0

0.0

1

100.0

 

Females that were euthanized

0

0.0

2

8.3

0

0.0

1

4.2

    Gravid

0

0.0

0

0.0

0

0.0

0

0.0

    Non Gravid

0

0.0

2

100.0

0

0.0

1

100.0

 

Females examined at Scheduled

Necropsy

24

100.0

21

87.5

24

100.0

21

87.5

    Non Gravid

2

8.3

1

4.8

1

4.2

1

4.8

    Gravid

22

91.7

20

95.2

23

95.8

20

95.2

       With resorptions only

0

0.0

0

0.0

0

0.0

0

0.0

       With viable foetuses

22

100.0

20

100.0

23

100.0

20

100.0

Total Females Gravid

22

91.7

23

95.8

23

95.8

23

95.8

Table 5. Summary of Clinical Findings: Total Occurrence / No. of Animals

 

Dose Group (mg/Kg/day)

0 (Control)

100

250

500

Normal – No significant clinical observations

646/24

623/24

652/24

611/24

 

Disposition

 

 

 

 

  - Found dead

0/0

1/1

0/0

1/1

  - Sent to necropsy in extremis

0/0

2/2

0/0

1/1

  - Aborted

0/0

0/0

0/0

1/1

  - Scheduled Euthanasia; Gestation Day 29

24/24

21/21

24/24

21/21

 

Body/Integument

 

 

 

 

- Hair loss forelimb (s)

0/0

2/1

0/0

6/2

- Thin

0/0

1/1

0/0

2/2

- Hair loss Hindlimb (s)

0/0

0/0

0/0

1/1

 

Cardio-Pulmonary

 

 

 

 

- Rales

3/1

4/3

3/2

9/3

- Increases Respiration Rate

0/0

0/0

1/1

0/0

- Laboured Respiration

0/0

2/1

1/1

4/2

- Pale Body

0/0

0/0

1/1

0/0

 

Eyes / Ears / Nose

 

 

 

 

- Clear Discharge Left Eye

0/0

2/1

0/0

0/0

- Clear Discharge Right Eye

0/0

2/1

0/0

0/0

- Dried Red Material Around Nose

0/0

0/0

0/0

1/1

- Clear Nasal Discharge

0/0

1/1

0/0

0/0

 

Excreta – Decreased Defaecation

0/0

3/2

1/1

21/5

 

Body / Integ II

 

 

 

 

- Scabbing Dorsal Trunk

3/1

0/0

0/0

0/0

- Wet Brown Material Anogenital Area

1/1

0/0

0/0

0/0

- Dried Brown Material Anogenital Area

3/3

0/0

0/0

2/2

Table 6. Summary of Body Weight Changes during Gestation (g)

Parameter

 

Group (mg/Kg/day)

0 (control)

100

250

500

Initial Body Weight

Mean

3314.

3357.

3321.

3348.

S.D.

160.7

232.7

192.1

205.2

S.E.

34.2

52.0

40.1

45.9

N

22

20

23

20

 

Terminal Body Weight

Mean

3826.

3816.

3811.

3735.

S.D.

229.0

236.2

254.4

344.9

S.E.

48.8

52.8

53.1

77.1

N

22

20

23

20

 

Gravid Uterine Weight

Mean

497.9

510.0

516.2

487.4

S.D.

113.98

79.44

98.45

87.61

S.E.

24.30

17.76

20.53

19.59

N

22

20

23

20

 

Net Body Weight

Mean

3328.3

3306.1

3295.2

3247.1

S.D.

235.97

222.21

235.63

285.10

S.E.

50.31

49.69

49.13

63.75

N

22

20

23

20

 

Net Body Weight Change

Mean

14.7

-51.3

-25.3

-100.5

S.D.

191.80

191.90

146.80

275.20

S.E.

40.89

42.91

30.61

61.54

N

22

20

23

20

Mean Differences Calculated from Individual Differences

Non Gravid Weight(s) Not Included in Calculation of Mean

Table 7. Summary of Fetal Data at Scheduled Necropsy

Group

(mg/Kg/day)

 

Sex

Viable

Fetuses

Dead

Fetuses

Resorptions

Post Implantation Loss

Implantation Sites

Corpora Lutea

Pre-Implantation

Loss

Fetal Weight in Grams

No. of Gravid Females

Male

Female

Early

Late

0 (Control)

Total

92

100

192

0

9

1

10

202

221

19

NA

22

Mean

4.2

4.5

8.7

0.0

0.4

0.0

0.5

9.2

10.0

0.9

42.1

S.D.

2.08

1.63

2.57

0.00

0.80

0.21

0.86

2.22

1.68

1.78

5.98

S.E.

0.44

0.35

0.55

0.00

0.17

0.05

0.18

0.47

0.36

0.38

1.27

 

100

Total

91

87

178

0

6

0

6

184

197

13

NA

20

Mean

4.6

4.4

8.9

0.0

0.3

0.0

0.3

9.2

9.9

0.7

42.0

S.D.

2.09

2.21

1.74

0.00

0.92

0.00

0.92

1.54

1.39

0.99

3.79

S.E.

0.47

0.49

0.39

0.00

0.21

0.00

0.21

0.34

0.31

0.22

0.85

 

250

Total

88

117

205

1

2

8

11

216

236

20

NA

23

Mean

3.8

5.1

8.9

0.0

0.1

0.3

0.5

9.4

10.3

0.9

41.4

S.D.

1.67

1.70

1.83

0.21

0.29

0.71

0.85

2.13

2.12

0.92

4.57

S.E.

0.35

0.36

0.38

0.04

0.06

0.15

0.18

0.44

0.44

0.19

0.95

 

1000

Total

88

97

185

0

3

3

6

191

205

14

NA

20

Mean

4.4

4.9

9.3

0.0

0.2

0.2

0.3

9.6

10.3

0.7

37.7*

S.D.

1.10

1.46

1.33

0.00

0.37

0.37

0.57

1.32

1.55

0.98

6.35

S.E.

0.24

0.33

0.30

0.00

0.08

0.08

0.13

0.29

0.35

0.22

1.42

* = Significantly different from the control group at 0.05

NA = Not applicable

Mean Number of Viable Fetuses; Mean Number of Implantation Sites; Mean Number of Corpora Lutea; Fetal Weights compared using Dunnett’s Test.

Table 8. Summary of Litter Proportions of Malformations (% per Litter)

Dose Group (mg/Kd/day)

 

0 (Control)

100

250

500

Number of Litters Examined

22

20

23

20

 

Total Malformations

 

Percent per Litter with External Malformations

Mean

1.0

0.0

0.0

1.4

S.D.

3.27

0.00

0.00

3.33

S.E.

0.70

0.00

0.00

0.74

 

Percent per Litter with Soft Tissue Malformations

Mean

2.9

0.5

0.8

0.0

S.D.

8.25

2.03

2.75

0.00

S.E.

1.76

0.45

0.57

0.00

 

Percent per Litter with Skeletal Malformations

Mean

1.2

1.6

0.4

3.0

S.D.

3.77

5.04

2.09

7.03

S.E.

0.80

1.13

0.43

1.57

 

Total Percent per Litter with Malformations

Mean

5.0

2.1

1.3

4.0

S.D.

10.62

6.73

4.50

7.17

S.E.

2.26

1.51

0.94

1.60

Modified Statistics Used

Table 9. Summary of Fetuses and Litters with Variations (Absolute No.)

Dose Group

(mg/Kg/day)

Fetuses

Litters

0 (Control)

100

250

500

0 (Control)

100

250

500

Number Examined Externally

192

178

205

185

22

20

23

20

  Excessive Fat Pads

1

0

0

0

1

0

0

0

 

Number Examined Viscerally

192

178

205

185

22

20

23

20

   Major Blood Pressure Variation

20

15

14

19

8

7

9

9

   Retrocaval Ureter

7

2

2

1

4

1

2

1

   Accessory Spleen (S)

29

21

20

23

15

8

11

11

   Heart Extra Papillary Muscle

4

3

4

3

2

2

3

2

   Spleen – Small

0

0

0

3

0

0

0

2

   Gall Bladder – Absent or Small

3

6

1

2

3

3

1

1

   Liver – Accessory Lobule (s)

1

0

0

0

1

0

0

0

   Spleen – Pale

2

0

0

6

1

0

0

3

   Renal Papillae(s) Not Developed and/or Distended Ureters

1

0

0

0

1

0

0

0

   Liver – Pale

0

0

0

1

0

0

0

1

 

Number Examined Skeletally

192

178

205

185

22

20

23

20

   13thRudimentary Rib(s)

36

28

41

22

15

13

18

15

   13thFull Rib(s)

64

64

52

48

18

16

18

14

   27 Presacral Vertebrae

10

20

5

7

7

9

3

5

   Sternebra(E) Malaligned (slight or moderate)

2

7

4

3

2

6

4

3

   Sternebra(E) #5 and/or #6 Unossified

31

27

34

28

14

13

13

10

   Hyoid Arch (es) Bent

4

3

2

3

4

2

2

3

   Extra Site of Ossification Anterior to Sternebrae(E) # 1

7

0

8

2

4

0

7

2

   Sternebrae with Thread like Attachment

1

3

0

2

1

2

0

1

   7thCervical Ribs

1

1

5

1

1

1

4

1

   Pubis Unossified

0

0

0

3

0

0

0

1

   Vertebral Centra not Fully Ossified

0

1

0

0

0

1

0

0

   Accessory Skull Bone(s)

0

0

1

2

0

0

1

2

   7thSternebrae

0

0

0

1

0

0

0

1

Table 10. Summary of Fetuses and Litters with Malformations (Absolute No.)

Dose Group

(mg/Kg/day)

Fetuses

Litters

0 (Control)

100

250

500

0 (Control)

100

250

500

Number Examined Externally

192

178

205

185

22

20

23

20

   Short-tail

0

0

0

1

0

0

0

1

   Carpal and/or Tarsal Flexure

1

0

0

0

1

0

0

0

   Polydactyly

1

0

0

0

1

0

0

0

   Exencephaly with or without Open Eyelid

0

0

0

1

0

0

0

1

   Macroglossia

1

0

0

0

1

0

0

0

   Malpositioned Umbilicus

0

0

0

1

0

0

0

1

 

Number Examined Viscerally

192

178

205

185

22

20

23

20

   Lungs – Lobular Agenesis

4

0

2

0

2

0

2

0

   Vestigial Pulmonary Trunk

1

1

0

0

1

1

0

0

  Bulbous Aorta

2

1

0

0

2

1

0

0

  Interventricular Septal Defect

0

1

0

0

0

1

0

0

  Heart – Three Chambered

1

0

0

0

1

0

0

0

  Heart – Small Ventricle

1

0

0

0

1

0

0

0

 

Number Examined Skeletally

192

178

205

185

22

20

23

20

  Vertebral Anomaly with or without associated Rib Anomaly

1

1

0

2

1

1

0

2

  Rib Anomaly

0

0

1

0

0

0

1

0

  Coastal Cartilage Anomaly

1

1

1

1

1

1

1

1

  Sternebra (E) Malaligned (Severe)

0

1

0

3

0

1

0

3

  Sternoschisis

0

0

0

1

0

0

0

1

  Vertebral Centra Anomaly

0

0

0

1

0

0

0

1

 

Total number with malformations

 

   External

2

0

0

3

2

0

0

3

   Soft Tissue

5

1

2

0

3

1

2

0

   Skeletal

2

3

1

6

2

2

1

4

 

 

   Combined

9

4

3

8

6

2

2

6

Applicant's summary and conclusion

Conclusions:
The no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal developmental toxicity was 250 mg/kg bw/day.
Executive summary:

Only screening reproductive/developmental toxicity data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

A Prenatal Developmental GLP Toxicity Study was conducted according to test guideline 414. The test substance, dipropyleneglycol dibenzoate (DPGDB), in the vehicle (0.5% carboxymethylcellulose in deionized water) was administered orally by gavage to 3 groups of 24 time-mated female New Zealand White [Hra:(NZW)SPF] rabbits once daily from Gestation Days 7–28. Dosage levels were 100, 250, and 500 mg/kg/day administered at a dose volume of 5 mL/kg. Adverse effects on maternal survival, mean body weight changes, and food consumption were noted in the 500 mg/kg/day group; therefore, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on lower mean fetal weights at 500 mg/kg/day, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity when dipropyleneglycol dibenzoate was administered orally by gavage to time-mated New Zealand White rabbits. Since no severe developmental toxicity effects were observed and fetal weights reduction was considered to be due to maternal toxicity effect at 500 mg/kg/d group, the substance is not categorised as developmental toxicant under GHS classification criteria.

Test substance-related effects on survival were noted in the 500 mg/kg/day group, as 1 female was euthanized in extremis on Gestation Day 26 following a severe body weight loss and markedly reduced food consumption, with corresponding incidences of decreased defecation noted at the daily examinations. Adverse clinical observations of rales, labored respiration, decreased respiration rate, and a thin body were also noted for this female at the daily examinations and/or postdosing observations. An additional female in the 500 mg/kg/day group aborted on Gestation Day 25 and was subsequently euthanized. At necropsy, neither female had remarkable macroscopic findings. There were no other test-substance related effects on survival. In the 100 and 500 mg/kg/day groups, 3 and 1 females, respectively, were found dead or euthanized in extremis during Gestation Days 13–26; following macroscopic/microscopic examination, the cause of mortality/moribundity of these females were attributed to or considered likely related to intubation error. All other females survived to the scheduled necropsy. In the 500 mg/kg/day group, there was a slight increase in the incidence of decreased defecation at the daily examinations, which was considered secondary to the reduced mean food consumption noted in this group. Test substance-related lower mean body weight gains or body weight losses were noted in the 500 mg/kg/day group generally during Gestation Days 13–29, resulting in a lower mean body weight gain when the entire treatment period (Gestation Days 7–29) was evaluated compared to the control group. Mean food consumption was also lower in this group during Gestation Days 13–29, which resulted in slightly lower mean food consumption for the overall dosing period (Gestation Days 7–29), and corresponded to the decreased mean body weight gains noted in this group. In addition, mean net body weight loss was also noted compared to the control group at 500 mg/kg/day. Although mean body weights in the 500 mg/kg/day group were comparable to the control group throughout the treatment period, these changes were still considered adverse as they led to the mortality or abortion of 2 females within the group. Mean net body weight and gravid uterine weight were comparable to the control group at 500 mg/kg/day. In the 100 and 250 mg/kg/day groups, mean body weights, body weight changes, net body weights, net body weight changes, gravid uterine weights, and food consumption were unaffected by test substance administration. Hematology and serum chemistry parameters were unaffected by test substance administration at all dosage levels.

Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance. Test substance-related adrenal cortical hypertrophy was noted for females in the 100, 250, and 500 mg/kg/day groups; however, due to the minimal to mild severity and the lack of other related microscopic and gross findings, this finding was considered non adverse. There were no other test-substance-related microscopic observations at any dosage level. Mean fetal body weights (male, female, and combined) were 8.9% to 11.8% lower in the 500 mg/kg/day group compared to the control group, and were considered test substance-related and adverse. Mean fetal body weights in the 100 and 250 mg/kg/day groups were similar to the control group. Intrauterine survival was unaffected by test substance administration at all dosage levels. No test substance-related effects on fetal morphology were noted at any dosage level.