Dibutyltin oxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 January 2017 to 31 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 2001

Deviations:

yes

Remarks:

The dose interval was extended allowing exposure through the pre-implantation period by dosing from gestation day 0 through to 19. To evaluate the known target organ toxicity, the thymus gland was collected and weighed for each dam.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyltin oxide

EC Number:

212-449-1

EC Name:

Dibutyltin oxide

Cas Number:

818-08-6

Molecular formula:

C8H18OSn

IUPAC Name:

dibutylstannanone

Test material form:

solid: particulate/powder

Remarks:

Light, white powder

Details on test material:

- Expiration Date: Stable through use on study.
- Storage: Ambient (18 to 29 °C), protected from light.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd:Sprague Dawley® SD®

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 9 to 9.5 weeks of age at time of receipt.
- Weight at study initiation: 158 to 215 g, at randomisation.
- Housing: The animals were individually housed in solid bottom cages with nonaromatic bedding.
- Diet: Ad libitum
- Water: Ad libitum tap water was available via an automatic watering system.

ENVIRONMENTAL CONDITIONS
- Temperature: 68 to 79 °F
- Humidity (%): 30 to 70 %
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was provided for approximately 12 hours per day.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations of the test material were prepared by mixing the appropriate amount of vehicle with the appropriate amount of test material at nominal concentrations of 0.15, 0.6, and 1.2 mg/mL. Formulations were prepared as needed and were stored at room temperature.
The control group received the vehicle in the same manner as the treated groups. The vehicle and test material formulations were continually stirred prior to and throughout dose administration. Individual doses were based on the most recent body weights.

VEHICLE
- Amount of vehicle: Dose volume of 5 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples were collected using a positive displacement pipette, while stirring, and placed into amber glass scintillation vials. Upon receipt of the authorisation to finalise the main study report, the retained backup samples were discarded.
Dosing formulation samples were collected as follows:
Concentration/homogeneity analysis: 0.5 mL from 0.15 and 1.2 mg/mL dose concentrations were sampled at weekly intervals. Six samples were collected from each of the top, middle and bottom, with two analysed and four back up samples.
The averaged result from homogeneity analysis served as concentration verification.
Concentration analysis: 0.5 mL from 0, 0.15, 0.6 and 1.2 mg/mL dose concentrations were sampled at weekly and 3-week intervals. Six samples were collected from the middle stratum, with two analysed and four back up samples.
The samples, including backup samples, were stored at ambient temperature pending analyses or final disposition.
Stability analysis of the dosing formulations was not conducted because the stability of the dosing formulations at the concentrations used on study has been established for 13 days at ambient temperature.

All analytical work was conducted using an analytical method developed and validated under MPI Research. Dose formulations were verified to be the concentrations stated within the acceptable range of variability, for homogeneity and concentration analytical results.
Reference Standard: Test material (Correction Factor: 1.028 for purity)
LC-MS Conditions: Liquid chromatography system equipped with a Thermo BETASIL C18 column, 50 x 2.1 mm, with a gradient flow of 88:12 water:acetic acid with 0.05 % trimethylamine (TEA) (mobile phase A), and 88:12 isopropyl alcohol (IPA):acetic acid with 0.05 % TEA (mobile phase B) at a flow rate of 0.6 mL/minute. The analyte was detected using an LC-MS/MS system operated in the positive ionisation mode at a mass to charge (m/z) ratio of 293 for the precursor ion and 251 for the product ion.
Analysis Description: Prior to analysis, samples were diluted with 88:12 IPA:acetic acid (diluent) to within the range of the calibration curve. Vehicle samples were diluted with diluent using a dilution factor of 10. An aliquot of each sample was injected into the LC-MS/MS system for analysis.
Regression Type: Linear, unweighted
Study Sample Storage Conditions: Ambient, protected from light.
Storage Stability: All samples were analysed within the established storage stability.

Run Acceptance Criteria
System Suitability Test Standards: Injection repeatability (peak area and retention time) ≤10% RSD (relative standard deviation)
Calibration Standards:
1. Accuracy within ±10 % of the nominal concentration
2. Coefficient of determination (R^2) ≥0.99
Performance Check Standards (Same preparation as the System Suitability Standard):
1. Periodically injected so that no more than 10 samples are bracketed by performance check standards. Samples are considered valid if bracketed by passing performance check standard injections.
2. Accuracy within ±10 % of the nominal concentration
Blank Injections: ≤20 % of the limit of quantitation (LOQ)

Assessments
- Homogeneity:
1. Average concentration within ±15 % of the nominal concentration
2. Precision ≤15 % RSD
- Concentration:
1. Average concentration within ±15 % of the nominal concentration
2. Precision ≤15 % RSD
3. Vehicle (control) samples < effective limit of quantitation (ELOQ)

Results:
Analytical run 1: System sustainability test (SST) injection 1: Retention time (tR): 1.816 min, peak area 7596
Analytical run 1: SST injection 2: tR: 1.823 min, peak area 7537
Analytical run 1: SST injection 3: tR: 1.816 min, peak area 7456
Mean for analytical run 1: tR: 1.816 min, peak area 7530
% Relative standard deviation (RSD): tR 0.218 min, peak area 0.933

Analytical run 2: SST injection 1: tR: 1.806 min, peak area 11684
Analytical run 2: SST injection 2: tR: 1.812 min, peak area 11658
Analytical run 2: SST injection 3: tR: 1.806 min, peak area 11601
Mean for analytical run 2: tR: 1.808 min, peak area 11648
% RSD: tR 0.219 min, peak area 0.367

Analytical run 1: Performance check 1: 9.987354 μg/mL nominal concentration, 9.850389 μg/mL calculated concentration; 98.6 % recovery
Analytical run 1: Performance check 2: 9.987354 μg/mL nominal concentration, 9.646217 μg/mL calculated concentration; 96.6 % recovery
Analytical run 1: Performance check 3: 9.987354 μg/mL nominal concentration, 9.878215 μg/mL calculated concentration; 98.9 % recovery

Analytical run 3: Performance check 1: 9.994358 μg/mL nominal concentration, 9.866436 μg/mL calculated concentration; 98.7 % recovery
Analytical run 3: Performance check 2: 9.994358 μg/mL nominal concentration, 10.063415 μg/mL calculated concentration; 100.7 % recovery

Conclusion:
A total of 32 samples were analysed (24 reported results) for the test material in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results.
Deviation: Analytical Method Deviation: In analytical run 2, the performance check 2 failed to meet acceptance criterion for recovery (±10 %), therefore the results of the run were not reportable as samples were not bracketed by passing performance check injections. The backup samples were analysed in run 3 (on a different instrument) and the run met all acceptance criteria.

Details on mating procedure:

- Impregnation procedure: Purchased timed pregnant.

Duration of treatment / exposure:

GD 0 to 19

Frequency of treatment:

Once daily at approximately the same time each day (± 2 h from the GD 0 dose).

Duration of test:

GD 0 to 19

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.75 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

6 mg/kg bw/day

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route is one of the potential routes of human exposure to this test material.
The dose levels were selected by the Sponsor on the basis of available data from a previously conducted pilot developmental toxicity study. The pilot study showed that at the dose level of 9.5 mg/kg/day, 40 % of the dams were unable to tolerate the dosing schedule and had to be euthanised. In addition, dams exposed to 9.5 mg/kg/day weighed significantly less than controls in the early stages of gestation. The severity of these endpoints on the dam was the basis for selecting the high dose of 6 mg/kg/day, with the middle and low doses of 3.0 and 0.75 mg/kg/day being selected as following a geometric progression factor of 4 and 2, respectively.
- Rationale for animal assignment: Using a standard, by weight, randomidation procedure, 100 female animals (weighing 158 to 215 g, at randomisation) were assigned to the control and treatment groups. All animals were given a detailed clinical examination and body weights were recorded prior to selection and before dosing on GD 0. The Study Director reviewed GD 0 body weight and detailed observation data for all animals and gave final approval for assignment to study.
Animals assigned to study had body weights within ± 20 % of the mean body weight. Extra animals obtained, but not placed on study, were euthanised by carbon dioxide inhalation followed by an MPI Research SOP approved method to ensure death. The carcasses were discarded.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: All animals were observed for morbidity, mortality, injury, and the availability of food and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from GD 0 through 20 (75 minutes, ±15 minutes, post-dose on dosing days), each animal was removed from the cage and given a detailed clinical examination. On occasion, clinical observations were recorded at unscheduled intervals. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.
Veterinary Observations: On occasion, veterinary observations were conducted during the course of the study. All treatments and observations were recorded. The medical treatments and observations are not reported but are maintained in the study file.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights for all animals were measured and recorded on GD 0, 3, 6, 9, 12, 15, 18, and 20. Individual body weight change was calculated for the following GD intervals: GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20 and 0-20. Adjusted body weight (GD 20 body weight minus gravid uterine weight) and adjusted body weight change (GD 0 to 20) were also calculated.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured and recorded on the corresponding body weight days and calculated for the same intervals.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: On GD 20, each surviving female was euthanised by carbon dioxide inhalation followed by an SOP approved method to ensure death and immediately subjected to a cesarean section.
- Organs examined: Maternal necropsies were conducted without knowledge of the treatment groups in order to minimise bias. The skin was reflected from a ventral midline incision to examine mammary tissue and locate any subcutaneous masses. The abdominal cavity was then opened, and the uterus was exposed. The uterus was excised, and the gravid uterine weight was recorded. Beginning at the distal end of the left uterine horn, the location of viable and nonviable foetuses, early and late resorptions for each uterine horn, and the total number of implantations were recorded. The number of corpora lutea on each ovary was also recorded.
The foetuses were removed by making a dorsal incision longitudinally along both uterine horns. The embryonic membrane of each foetus was gently removed, and each foetus was pulled away from the placenta, fully extending the umbilical cord. The placentae were examined grossly.
Uteri from females that appeared nongravid were opened and placed in 10 % ammonium sulfide solution for detection of implantation sites. If no foci were detected, the female was considered to be nonpregnant.
A complete necropsy was performed on all euthanised in extremis and surviving dams under procedures approved by a veterinary pathologist. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced the pregnancy. The presence of lesions or other abnormal conditions in the dam were noted and described in the study records. Additionally, the thymus gland from each animal was collected, weighed, and preserved in 10 % neutral buffered formalin for possible further evaluation.

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

Each implant was categorised according to the following criteria. Viable foetuses responded to touch. Nonviable foetuses did not respond to touch and had no signs of autolysis. Late resorptions were characterised by recognisable foetal form, but undergoing autolysis. Early resorptions were characterised as implantation sites that had no recognisable foetal characteristics.

Fetal examinations:

Foetal examinations were conducted without knowledge of the treatment groups in order to minimise bias. Each foetus was individually weighed, sexed, tagged, and examined for external malformations and variations. Foetuses were then euthanised by intraperitoneal injection of euthanasia solution.
Approximately one-half of the foetuses in each litter were placed in Bouin’s solution and the remaining foetuses were fixed in alcohol.

- External examinations: Yes: All per litter.
- Soft tissue examinations: Yes: Approximately half per litter. All foetuses fixed in Bouin’s solution were examined for soft tissue defects using the Wilson razor-blade sectioning technique.
- Skeletal examinations: Yes: Approximately half per litter. The foetuses fixed in alcohol were macerated in potassium hydroxide, stained with Alizarin Red S, and cleared with glycerin by a method similar to that described by Dawson for subsequent skeletal examination.
- Head examinations: No

Foetal findings were classified as malformations or developmental variations under procedures approved by a developmental toxicologist. On occasion, additional information to clarify or identify a visceral or skeletal observation was documented. These comments are not reported, but are maintained in the study data. Mechanical artifacts (i.e., tail removed and discarded) occurred during examination and processing of the foetuses. These artifacts are not reported, but are maintained in the study data.

Statistics:

The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group.

Parental In-life Data
Group pair-wise comparisons: Gestation body weights, gestation body weight changes, gestation food consumption, adjusted body weights, adjusted body weight changes (GD 0 - 20) and thymus weight (absolute and relative to adjusted GD 20 bodyweight).

Uterine and Ovarian Examinations
Fischer’s exact test: Pregnancy index, malformations by finding and exam type (external, visceral and skeletal)-litter incidence and variations by finding and exam type (external, visceral and skeletal)-litter incidence. Foetal and litter incidences are reported, but only the litter incidences were statistically analysed.
Group pair-wise comparison: Gravid uterine weights, corpora lutea/dam, total implantations/dam, litter size/dam, viable foetuses/dam, total number resorptions/dam, number early resorptions/dam and number late resorptions/dam.
Arcsin-square-root transformation: Foetal sex ratio (% males/litter), % pre-implantation loss and % post-implantation loss.
Descriptive statistics: Non-viable foetuses/dam.
Covariate analysis: Mean foetal body weights.

Indices:

Pregnancy Index = (No. females pregnant / No. females with evidence of mating) x 100

Post-implantation Loss = ((No. implantations - No. viable foetuses) / No. implantations) x 100

Pre-implantation Loss = ((No. corpora lutea - No. implantations) / No. corpora lutea) x 100

Historical control data:

No. of studies: 2
No. females: 135
No. pregnant: 124
No. not pregnant: 1
No. died pregnant: 0
No. females with all resorptions: 0
No. pregnant by stain: 0
No. females with viable foetuses: 124
No. of litters evaluated: 124
No. external foetuses evaluated: 1 588
No. visceral foetuses evaluated: 794
No. skeletal foetuses evaluated: 794

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No adverse effect of the test material at 0.75 and 3.0 mg/kg/day was observed from the detailed clinical examinations. Red material around the nose was observed at least once in 3/25 and 5/25 animals in the 0.75 and 3.0 mg/kg/day dose groups, respectively and was also observed in 6/25 animals in the 6.0 mg/kg/day group. A similar finding was not observed among the control animals. Due to its sporadic occurrence among the treated animals generally observed only on 1 or 2 days during the study and in many instances only during the first week, its presence while likely test material related, was not considered adverse. Other clinical findings observed in the 0.75 and 3.0 mg/kg/day animals occurred at low incidence or with similar frequency to controls and were considered unrelated to the test material. In the 6.0 mg/kg/day group, clinical findings observed with increased frequency and considered test material related and adverse included low body carriage, red material around the nose, thin appearance, loss of skin elasticity, and pale body colour.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

All animals in the control, 0.75, and 3.0 mg/kg/day groups survived to scheduled termination on GD 20. In the 6.0 mg/kg/day group, animals 4512 and 4525 were euthanised in extremis on GD 12 and GD 9, respectively. Both animals had clinical signs of toxicity that included decreased activity, low body carriage, red material around the nose/mouth, hunched posture, pale body color, and thin unkempt appearance. Likewise both animals lost weight from the start of treatment and had low food consumption. The moribundity observed in these animals was considered test material-related. All other animals in the 6.0 mg/kg/day group survived to scheduled termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No effect of the test material at dose levels of 0.75 and 3.0 mg/kg/day was observed on gestation body weights and body weight change. At 6.0 mg/kg/day, mean body weights were statistically lower than mean control values on GD 18 (-8 %) and 20 (-9 %). Mean body weight change in this group was lower than mean control values over much of gestation and statistically lower over GD 0 to 3 (-24 %), GD 6 to 9 (-34 %) and over the entire GD 0 to 20 treatment period (-19 %). Considerable variability in gestation body weight change was observed in the 6.0 mg/kg/day dose group. This was attributed to low weight gain and/or weight loss in several animals (4510, 4511, 4516, and 4524) that failed to retain pregnancies with foetuses and at GD 20 had uterine implantations comprised entirely of resorbing foetuses (100 % post-implantation loss). These effects on gestation body weights and body weight change at 6.0 mg/kg/day were considered test material related correlating with adverse pregnancy outcomes in several animals.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean gravid uterine weights, adjusted GD 20 body weights and adjusted weight change GD 0 to 20 in the test material-treated groups were comparable to mean control values.

Lower thymus weights, absolute and relative to the adjusted GD 20 body weight, were observed in the test material-treated groups. Mean absolute thymus weights were 19 %, 34 %, and 44 % lower than the mean control value in the 0.75, 3.0, and 6.0 mg/kg/day dose groups, respectively and relative to the adjusted GD 20 body weights, were 20 %, 35 %, and 37 % lower, respectively. These differences in thymus weights, absolute and relative to adjusted GD 20 body weight in the test material-treated groups relative to controls were statistically significant and considered test material related. In the context of this study, the toxicological significance of this decrease in thymus weight in the absence of histopathological examination is unclear.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No effect of the test material at 0.75 and 3.0 mg/kg/day was observed from the maternal macroscopic examinations. The few findings observed among these animals occurred at low incidence or with similar frequency as controls and were considered unrelated to the test material. At 6.0 mg/kg/day, small thymus was observed with increased frequency. This was observed in the two animals euthanised in extremis in this group and 4/23 animals that survived to scheduled termination. Other maternal macroscopic findings observed in the 6.0 mg/kg/day group occurred at low incidence or similar to controls and were considered unrelated to the test article.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- Food consumption: No effect of the test material at dose levels of 0.75 and 3.0 mg/kg/day was observed on gestation food consumption. At 6.0 mg/kg/day, mean food consumption was statistically lower than mean control values over GD 6 to 9 (-14 %), GD 9 to 12 (-13 %), and GD 15 to 18 (-17 %). The considerable variability in food consumption observed in this group was largely attributable to low food consumption in those females with failed pregnancies as discussed previously. Mean food consumption in the 6.0 mg/kg/day group over the entire treatment period (GD 0 to 20) was 15.47g/animal/day and 9 % lower (not statistically significant) than the 17.09 g/animal/day observed in controls.

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

No effect of the test material at 0.75 and 3.0 mg/kg/day was observed on GD 20 uterine implantation parameters.
Statistically significant decreases in mean post-implantation loss in the 0.75 mg/kg/day dose group relative to controls were considered incidental, not adverse, and unrelated to the test material.
In the 6.0 mg/kg/day group, no statistically significant changes in corpora lutea count or uterine implantation data were observed relative to controls.
In the 6.0 mg/kg/day group, notable increases in mean post-implantation loss (25.70 %) were observed. Changes in this parameter in the 6.0 mg/kg/day dose group were also outside the range of recent historical control data for the laboratory maximum study values were 5.35 % for post-implantation loss. Changes were largely attributable to the four females in the group with all resorption sites in utero (100 % post-implantation loss).

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Four females in the 6.0 mg/kg/day group (4510, 4511, 4516, and 4524) had uterine implantations comprised entirely of resorbing foetuses (100 % post-implantation loss).
The increased incidence of females with all resorption sites in utero was considered test material related and adverse.
In the 6.0 mg/kg/day group, notable increases in mean number of resorption sites (total and early)/dam (2.7) relative to controls (5.40 % and 0.7, respectively) were observed. Changes in these parameters in the 6.0 mg/kg/day dose group were also outside the range of recent historical control data for the laboratory maximum study values were 0.7 resorption sites (total and early)/dam. Changes were largely attributable to the four females in the group with all resorption sites in utero (100 % post-implantation loss).

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increases in mean number of resorption sites (total and early) in the 0.75 mg/kg/day dose group relative to controls were considered incidental, not adverse, and unrelated to the test material.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases in mean number of viable foetuses and litter size in the 0.75 mg/kg/day dose group relative to controls were considered incidental, not adverse, and unrelated to the test material.
In the 6.0 mg/kg/day group a decrease in number of viable foetuses/dam (9.7 vs. 12.5 in controls) were observed. Changes in these parameters in the 6.0 mg/kg/day dose group were also outside the range of recent historical control data for the laboratory maximum study values were 13.5 viable foetuses/dam). Changes were largely attributable to the four females in the group with all resorption sites in utero (100 % post-implantation loss).

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

Pregnancy index in the control, 0.75, 3.0, and 6.0 mg/kg/day groups was 96 %, 96 %, 92 %, and 88 %, respectively. There were one, one, two, and three nonpregnant females in the control, 0.75, 3.0, and 6.0 mg/kg/day groups, respectively. Two of the nonpregnant females in the 6.0 mg/kg/day group were euthanised in extremis.
Overall, there were 24, 24, 23, and 18 litters with GD 20 foetuses for evaluation in the control, 0.75, 3.0, and 6.0 mg/kg/day dose groups, respectively.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

mortality

pre and post implantation loss

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No effect of the test material was observed on foetal body weights. Mean foetal body weights distinguished by sex and for the combined sexes, in the test material-treated groups were comparable to mean control values.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No effect of the test material was observed on foetal sex ratios (percent male foetuses/animal). Mean sex ratios ranged from 45.2 % to 48.8 % in the treated groups and were comparable to the mean of 47.1 % in controls.

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No effect of the test material was observed from the foetal external examinations. No malformations were observed among foetuses in the 0.75 and 6.0 mg/kg/day groups. In the 3.0 mg/kg/day group, the only external malformation observed was misshapen tongue. The litter incidence for this finding was 17.4 % (4/23 litters affected) and comparable to the 12.5 % (3/24 litters affected) in controls. Filamentous tail and absence of an anus were observed in one control foetus. No external developmental variations were observed among the control and test material-treated foetuses.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No effect of the test material was observed from the foetal skeletal examinations. No skeletal malformations were observed among foetuses from the control and the test material-treated groups. Litter incidences for ossification variations observed in the test material-treated groups were comparable to control and no effect of treatment was observed from these data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No effect of the test material was observed from the foetal visceral examinations. No malformations were observed among foetuses in the control, 0.75, and 6.0 mg/kg/day dose groups. In the 3.0 mg/kg/day group the only visceral malformation observed was folded retina. This was observed in a single foetus and in the absence of a similar malformation among foetuses in the 6.0 mg/kg/day group, its occurrence on study was considered spontaneous and unrelated to the test material. The litter incidence of an irregular palatal rugae pattern, a visceral variation, in the control, 0.75, and 3.0 mg/kg/day treated groups was 4.2 %, 12.5 %, and 26.1 %, respectively. The higher incidence of this variation in these treated groups did not differ statistically from controls and in the absence of a similar finding among foetuses in the 6.0 mg/kg/day group, was not considered test material related. The occurrence of a smaller than normal thyroid, a visceral variation in one foetus at 6.0 mg/kg/day was considered incidental and unrelated to test article.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

not specified

Any other information on results incl. tables

Summary of Maternal and Developmental Observations at Uterine Examination

Endpoint	0 mg/kg/day	0.75 mg/kg/day	3.0 mg/kg/day	6.0 mg/kg/day
No. females on study	25	25	25	25
No. not pregnant	1	1	2	3
No. pregnant	24	24	23	22
Pregnancy index percent	96.0	96.0	92.0	88.0
No. females with resorptions	0	0	0	4
No. females with viable foetuses GD 20	24	24	23	18

 

Summary of Maternal and Developmental Observations at Uterine Examination

Endpoint		0 mg/kg/day	0.75 mg/kg/day	3.0 mg/kg/day	6.0 mg/kg/day
Corpora lutea No. per animal	Mean	15.4	16.1	16.0	15.4
	SD	2.30	2.02	3.01	2.43
	N	24	24	23	18
Implantation sites No. per animal	Mean	13.2	14.3	14.2	12.5
	SD	1.89	2.35	1.67	2.42
	N	24	24	23	22
Pre-implantation loss % per animal	Mean	12.91	11.03	9.80	14.66
	SD	14.050	9.759	10.112	15.271
	N	24	24	23	18
Viable foetuses	Mean	12.5	14.1*	13.5	9.7
	SD	1.96	2.36	1.78	5.42
	N	24	24	23	22
Foetal sex ration % males per animal	Mean	47.1	45.2	46.6	48.8
	SD	16.45	15.21	11.34	11.35
	N	24	24	23	18
Post-implantation loss % per anima	Mean	5.40	1.46*	4.89	25.70
	SD	5.626	2.952	5.687	39.370
	N	24	24	23	22
Nonviable Foetuses No. per animal	Mean	0	0	0	0
	SD	0	0	0	0
	N	24	24	23	22
Litter size No. per animal	Mean	12.5	14.1*	13.5	9.7
	SD	1.96	2.36	1.78	5.42
	N	24	24	23	22
Resorptions: early + late No. per animal	Mean	0.7	0.2*	0.7	2.7
	SD	0.75	041	0.82	4.26
	N	24	24	23	22
Resorptions: early No. per animal	Mean	0.7	0.2*	0.7	2.7
	SD	0.75	0.41	0.83	4.22
	N	24	24	23	22
Resorptions: late No. per animal	Mean	0	0	0	0
	SD	0	0	0.21	0.21
	N	24	24	23	22

* Significantly different from control (p<0.05).

 

Summary of Gestation Body Weight Values (g)

Study Interval (Day)	0 mg/kg/day			0.75 mg/kg/day			3.0 mg/kg/day			6.0 mg/kg/day
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0	187.7	13.87	24	188.1	14.5	24	187.3	14.93	23	186.3	13.16	22
3	205.0	15.09	24	205.2	13.75	24	206.1	16.25	23	199.5	15.07	22
6	215.4	14.72	24	216.3	14.23	24	218.3	17.18	23	210.0	16.60	22
9	232.4	15.6	24	232.5	14.84	24	234.7	18.18	23	221.1	22.80	22
12	248.2	16.57	24	247.2	17.2	24	250.4	20.35	23	233.6	29.55	22
15	265.5	16.66	24	265.7	17.17	24	270.0	21.28	23	248.5	38.22	22
18	303.9	17.65	24	310.0	20.51	24	311.5	26.81	23	280.3*	51.34	22
20	334.4	19.82	24	344.6	24.61	24	344.9	30.34	23	305.4*	62.95	22

* Significantly different from control (p<0.05).

 

Summary of Gestation Body Weight Change Values (g)

Study Interval (Day)	0 mg/kg/day			0.75 mg/kg/day			3.0 mg/kg/day			6.0 mg/kg/day
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0 - 3	17.3	4.80	24	17.1	4.10	24	18.8	3.55	23	13.2*	5.95	22
3 – 6	10.5	4.57	24	11.1	3.60	24	12.2	2.96	23	10.5	4.68	22
6 – 9	17.0	3.85	24	16.3	3.12	24	16.4	4.18	23	11.2*	8.96	22
9 – 12	15.8	3.20	24	14.7	4.19	24	15.7	3.70	23	12.5	8.40	22
12 – 15	17.4	5.43	24	18.5	4.65	24	19.5	6.71	23	15.0	11.03	22
15 – 18	38.4	6.25	24	44.4	6.66	24	41.6	7.71	23	31.7	15.13	22
18 – 20	30.5	4.75	24	34.5	6.61	24	33.3	4.71	23	25.1	13.74	22
0 - 20	146.7	11.73	24	156.5	17.38	24	157.5	21.08	23	119.1†	56.38	22

*Significantly different from control (p<0.05).

† Significantly different from control (p<0.01).

 

Summary of Gestation Food Consumption Values (g/animal/day)

Study Interval (Day)	0 mg/kg/day			0.75 mg/kg/day			3.0 mg/kg/day			6.0 mg/kg/day
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0 - 3	12.28	2.037	24	12.89	1.562	24	13.16	1.875	23	12.21	4.377	22
3 – 6	14.97	2.567	24	15.22	1.817	24	16.54	2.886	23	15.20	3.306	22
6 – 9	16.74	2.852	24	16.81	1.702	24	16.19	1.729	23	14.44*	4.538	22
9 – 12	16.93	1.624	24	16.69	1.322	24	17.22	1.719	23	14.77*	5.286	22
12 – 15	17.61	1.531	24	17.53	1.985	24	18.38	1.876	23	15.89	6.273	22
15 – 18	22.25	2.497	24	21.97	2.113	24	22.14	3.270	23	18.55†	7.008	22
18 – 20	19.69	2.453	24	20.83	2.268	24	21.15	2.822	23	18.11	7.265	22
0 - 20	17.09	1.530	24	17.25	1.182	24	17.66	1.798	23	15.47	4.692	22

* Significantly different from control (p<0.05).

† Significantly different from control (p<0.01).

Summary of GD 20 Maternal Thymus Weights

Group (mg/kg/day)	Thymus Weight (g)	Thymus/ Adjusted GD20 Body Weight (%)
0	0.239	0.0891
0.75	0.193*	0.0716*
3.0	0.158*	0.0581*
6.0	0.134*	0.0558*

* Significantly different from control (p < 0.01).

Historical Control Data

Parameter	Mean	Minimum	Maximum
Pregnancy Index (%)	99.50	99.0	100.0
Corpora Lutea (Mean No. per Animal)	15.35	15.1	15.6
Implantation Sites (Mean No. per Animal)	13.7	13.3	14.1
Preimplantation Loss (Mean % per Animal)	9.950	8.82	11.08
Viable Foetuses (Mean No. per Animal)	13.05	12.6	13.5
Foetal Sex Ratio (Mean % Males per Animal)	49.20	47.4	51.0
Post-implantation Loss (Mean % per Animal)	4.755	4.16	5.35
Nonviable Foetuses (Mean No. per Animal)	0	0	0
Litter Size (Mean No. per Animal)	13.05	12.6	13.5
Resorptions: Early and Late (Mean No. per Animal)	0.65	0.6	0.7
Resorptions: Early (Mean No. per Animal)	0.65	0.6	0.7
Resorptions: Late (Mean No. per Animal)	0	0	0
Mean Foetal Body Weight Male (g)	4.05	4.02	4.08
Mean Foetal Body Weight Female (g)	3.850	3.83	3.87
Mean Foetal Body Weight Males and Females (g)	3.950	3.93	3.97

 

Historical Control Data - Litter Evaluations

Parameter	Total No.		Max % / Study
	Foetuses	Litters	Foetuses	Litters
External malformation
Body, umbilicus, ophalocele	2	2	0.2	2.0
Skeletal malformation
Cervical vertebra€, neural arch(es), misshapen	1	1	0.6	4.0
Visceral malformations
Head, retina, folded retina	1	1	0.2	1.0
Skeletal variation
Rib(s), rudimentary	158	65	29.8	68.0
Rib(s), unilateral full rib	5	4	1.8	8.0
Skull, hyoid, not ossified	1	1	0.2	1.0
Skull, palatine, irregular ossification	62	20	11.3	24.0
Sternum, sternebra(e), misaligned	10	10	1.6	10.1
Sternum, sternebra(e), not ossified	11	11	1.8	11.1

 

Analytical Calibration Results

Analytical Run	Standard Identification	Nominal Concentration (μg/mL)	Calculated Concentration (μg/mL)	% Recovery	Coefficient of Determination (R^2)
1	Standard 1	2.003113	2.053228	102.5	0.9984
	Standard 2	3.004669	2.925588	94.7
	Standard 3	6.00939	6.206982	103.3
	Standard 4	10.015564	9.868528	98.5
	Standard 5	12.519455	12.220998	97.6
	Standard 6	15.023346	15.300160	101.8
3	Standard 1	2000934	2.039972	102.0	0.9992
	Standard 2	3.001401	3.036667	101.2
	Standard 3	6.002802	6.047250	100.7
	Standard 4	10.004669	9.7400897	97.9
	Standard 5	12.505837	12.400897	99.2
	Standard 6	15.007004	15.206865	101.3

 

Analysis of the Test Material in Dosing Formulations – Week 1

Dose Level (mg/kg/day)	Sample Identification	Nominal Concentration (mg/mL)	Calculated Concentration (mg/mL)	Average Calculated Concentration (mg/mL)	% Recovery	Average % Recovery	% Relative Standard Deviation
0	Replicate 1	0.00	BLQ	BLQ	NA	NA	NA
	Replicate 2		BLQ
0.75	Top replicate 1	0.15	0.1501	0.1416	100.1	94.4	5.976
	Top replicate 2		0.1514		101.0
	Middle replicate 1		0.1296		86.4
	Middle replicate 2		0.1386		92.4
	Bottom replicate 1		0.1360		90.7
	Bottom replicate 2		0.1437		95.8
3.0	Replicate 1	0.60	0.6070	0.5916	101.2	98.6	3.691
	Replicate 2		0.5761		96.0
6.0	Top replicate 1	1.20	1.1520	1.1083	96.0	92.4	3.485
	Top replicate 2		1.1598		96.6
	Middle replicate 1		1.0905		90.9
	Middle replicate 2		1.0709		89.2
	Bottom replicate 1		1.1020		91.8
	Bottom replicate 2		1.0745		89.5

BLQ: Below the Limit of Quantitation (< 20.0000 μg/mL).

NA: Not Applicable/Not Available

 

Analysis of the Test Material in Dosing Formulations – Week 3

Dose Level (mg/kg/day)	Sample Identification	Nominal Concentration (mg/mL)	Calculated Concentration (mg/mL)	Average Calculated Concentration (mg/mL)	% Recovery	Average % Recovery	% Relative Standard Deviation
0	Backup replicate 1*	0.00	BLQ	BLQ	NA	NA	NA
	Backup replicate 2*		BLQ		NA
0.75	Backup replicate 1*	0.15	0.1362	0.1445	90.8	96.4	9.135
	Backup replicate 2*		0.1529		101.9
3.00	Backup replicate 1*	0.60	0.5932	0.6007	98.9	100.1	1.764
	Backup replicate 2*		0.6082		101.4
6.00	Backup replicate 1*	1.20	1.2518	1.2083	104.3	100.7	5.088
	Backup replicate 2*		1.1649		97.1

BLQ: Below the Limit of Quantitation (< 20.0000 μg/mL).

NA: Not Applicable/Not Available

* Backup samples were analysed due to the initial run failing to meet acceptance criteria for performance check 2.

Analysis of Dosing Formulations

- Homogeneity: Analyses of the low- and high-dose formulations (0.15 and 1.20 mg/mL) used for dosing the first week of study confirmed they were homogeneous as prepared meeting the laboratory’s acceptance criteria of 100 ± 15 % of nominal for the average percent recovery and percent Relative Standard Deviation (RSD) ≤ 10.

Dose Level (mg/kg/day)	Nominal Concentration (mg/mL)	Average Calculated Concentration (mg/mL)	Average % Recovery*	% Relative Standard Deviation
0.75	0.15	0.1416	94.4	5.976
6.0	1.20	1.1083	92.4	3.485

* Average % recovery was calculated from the nominal concentration.

- Concentration: Mean concentrations of formulations used for dosing in Weeks 1 and 3 ranged between 92.4 % and 100.7 % of nominal with percent RSDs ranging between 1.764 and 8.135, confirming that animals were receiving the appropriate dose levels when the formulations were administered at 5 mL/kg. No test material was detected in the control samples.

Dose Level (mg/kg/day)	Nominal Concentration (mg/mL)	Average Calculated Concentration* (mg/mL)	Average % Recovery*†	% Relative Standard Deviation*
0	0.00	BLQ	NA	NA
0.75	0.15	0.1416 – 0.1445	94.4 – 96.4	5.976 – 8.135
3.0	0.60	0.5916 – 0.6007	98.6 – 100.1	1.764 – 3.691
6.0	1.20	1.1083 – 1.2083	92.4 – 100.7	3.485 – 5.088

* Results are the range of values determined during Weeks 1 and 3.

† Average % recovery was calculated from the nominal concentration.

BLQ: Below the Limit of Quantitation (< 20.0000 μg/mL).

NA: Not applicable.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity with the test material was 3.0 mg/kg/day. At dose levels of 0.75, 3.0, and 6.0 mg/kg/day, the test material was not teratogenic in the rat.

Executive summary:

This study was conducted for to evaluate the possible adverse effects of the test article following repeated oral exposure to pregnant animals according to OECD Test Guideline 414 and in compliance with GLP.

This evaluation included systemic toxicity, female reproductive performance, and evaluation of the fetuses. The vehicle, peanut oil, or test material was administered to time-mated female Hsd:Sprague Dawley® SD® rats once daily via oral gavage from Gestation Day (GD) 0 through 19.

Observations of the animals included clinical signs, body weights, food consumption, and anatomical pathology including a uterine examination. All foetuses were given an external and visceral or skeletal examination.

Analytical results for formulations used for dosing in Weeks 1 and 3 confirmed homogeneity and concentration levels ranged between 92.4 % and 100.7 % of nominal. No test material was detected in the control samples.

All animals in the control, 0.75, and 3.0 mg/kg/day groups survived to scheduled termination on GD 20. At 6.0 mg/kg/day, two animals were euthanized in extremis with clinical signs of toxicity, low body weights, low body weight change, and low food consumption. A low incidence of red material around the mouth was observed in the test material-treated groups but not considered adverse due to its sporadic occurrence generally early in the study. No other maternal effects at dose levels ≤3.0 mg/kg/day were observed from clinical findings, gestation body weights, body weight change, food consumption, or maternal macroscopic findings. At 6.0 mg/kg/day, maternal effects were apparent from clinical findings (low body carriage, red material around the nose, thin appearance, loss of skin elasticity, and pale body color), lower gestation body weights, lower body weight change, and lower food consumption. No effect at dose levels ≤3.0 mg/kg/day was observed on pregnancy or uterine implantation data. At 6.0 mg/kg/day, 4/23 females at GD 20 had uterine implantation sites comprised entirely of resorbing foetuses (100 % post-implantation loss) and this contributed to decreases in mean number of viable foetuses and increases in mean post-implantation loss and mean number of resorption sites for the group. No effect of the test material was observed on foetal sex ratio, foetal body weight, or foetal external, visceral, or skeletal examinations. Lower maternal thymus weights were observed at all test material-treatment levels and an increased incidence of small thymus observed macroscopically in the 6.0 mg/kg/day animals.

In the context of this study, the toxicological significance of test material-related effects on thymus weights (decrease) in the absence of histopathological examination is unclear.

Under the conditions of the study the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity with the test material was 3.0 mg/kg/day. At dose levels of 0.75, 3.0, and 6.0 mg/kg/day, the test material was not teratogenic in the rat.