Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
December 12th, 2020 to January 19th, 2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose for cross-reference:
other: Main study
Reason / purpose for cross-reference:
other: validation of method used for the quantification of the substance
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on June 25, 2018.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility of testing laboratory
- Age at study initiation: At the initiation of the acclimatisation female rats were 12-13 weeks old
- Weight at study initiation: 218.6 – 254.6 g
- Housing: Throughout the experimental period, rats were housed individually except during the mating period. During the mating period, rats were housed in a group of two rats/cage (one male and one female). Mated female rats were housed individually. During the study, rats were housed in solid floor polypropylene rat cages. Each cage was fitted with a stainless steel top grille having provision for keeping rat pellet food and a polypropylene water bottle with stainless steel drinking nozzle. A wooden chew block was provided as enrichment material. Nesting material (wood shaving) was provided to mated female rats towards the end of pregnancy (from GD 14 to 20). Cages were placed on 5 tier racks. The bottom of the cages was layered with clean sterilised rice husk as the bedding. Cages with beddingand enrichment material and water bottles were changed at least twice a week.
- Diet: standard rodent pellet diet (Teklad Certified Global 14% Protein Rodent Diet) ad libitum
- Water: unlimited supply of clean and filtered drinking water (Reverse Osmosis water filter system)
- Acclimation period: 5 days prior to the mating. During the acclimatisation period, rats were observed daily, at least once, for clinical signs
- Other: Each day, the floor of the experimental room was swept and all work tops and floor was cleaned with a disinfectant solution

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 64-67
- Air changes (per hr): 21 air changes with filtered fresh air per hour
- Photoperiod: 12 hours fluorescent light and 12 hours darkness
- Mean light intensity: 141-165 Lux
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item was formulated in the vehicle. Once the dose formulation was prepared, it was thoroughly mixed using a magnetic stirrer. The dose formulations were prepared on each day of dosing and was dosed to rats immediately after preparation.

VEHICLE
- Justification for use and choice of vehicle: Corn oil is selected as a vehicle based on solubility check. Test item was found to be stable in vehicle (corn oil) for 4 days from time of preparation.
- Lot/batch no.: MKCH1635

Dose volume for the administration will be 4 mL/kg body weight. Doses will be adjusted according to the most recent body weight determinations. Gavage was performed using a stainless steel cannula attached to a syringe.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Two sets of samples (10 samples per set) were collected by sampling three aliquots (top,
middle and bottom layers) from each concentration except the control (only one aliquot from the
middle layer). Sampling was performed before the start of treatment (15/12/2020) and once during the treatment period (29/12/2020) for dose formulation analyses. One set of samples was used for analyses, and the other set of samples was stored at 2 - 8 °C. The mean concentration was determined and compared to the nominal value and the coefficient of variation calculated. The acceptance criteria used for analysis are ±10% from nominal value and %CV < 10.
Details on mating procedure:
After completion of the acclimatisation period, female rats were cohabitated with untreated male rats (1:1) until the requisite numbers of mated female rats (8/group) were obtained. Detection of mating was confirmed by the evidence of a copulatory plug in the vagina or by a vaginal lavage for sperm. After confirmation of mating, female rats were returned to an individual cage, randomly assigned to a group, and the day was designated as day 0 of gestation (GD 0).
Mated females were assigned to dose groups by stratified randomisation on the basis of GD 0 body weights. Body weights of GD 0 were arranged in descending order, from the heaviest to the lightest on the first GD 0 date, and assigned in the same order on the subsequent days. Beginning with the heaviest weight, one rat was randomly assigned to each stratified group. In the event that the total number of rats mated on a given day was not an even multiple of the number of treatment groups, on the subsequent day the mated female rats were assigned to complete the last incomplete stratification group and then assigned to the next stratification group until all GD 0 females for that day were assigned.
Duration of treatment / exposure:
from the 5th day of gestation to the 19th day of gestation
Frequency of treatment:
daily
Duration of test:
from the 5th day of gestation to the 19th day of gestation
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
8 mated females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: selected based on the available information on dibutyl phosphonate; LD50 oral (rat): > 3000 mg/kg b. wt., obtained in an acute oral toxicity study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Mortality and Morbidity: Rats were observed twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- During the study period, visible clinical signs, such as changes in the skin, fur, eye, mucous
membranes, as well as the behaviour pattern of the pregnant female rats were recorded twice daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of all the mated female rats was recorded individually on days 0, 3, 5, 8, 11, 14, 17, and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The fix amount of food wasbe offered to mated female rats on gestation day 0, 3, 5, 8, 11, 14, 17 and food leftover was determined on gestation day 3, 5, 8, 11, 14, 17, 20. The food consumption was calculated for the gestation days 0–3, 3–5, 5–8, 8–11, 11–14, 14–17, 17–20 and 5-20.

POST-MORTEM EXAMINATIONS: Yes
- On day 20 of gestation, i.e., one day prior to the expected day of delivery, all female rats were weighed, euthanised by carbon dioxide asphyxiation, dissected and examined macroscopically for any structural abnormalities.
- Organ Weight and Histopathology: At the time of terminal sacrifice, the weight of the thyroid was recorded from all-female rats and preserved for possible histopathology. As histopathology was not performed, all preserved organs will be disposed of upon finalisation of the study
Ovaries and uterine content:
The non-gravid uterus was further examined (5% ammonium sulphide staining method, Salewski,
1964) for confirming the non-pregnant status.
Ovaries and gravid uteri, including the cervix, were removed and examined immediately for the
following parameters mentioned below:
1. Gravid uterus including the cervix weight (g)
2. Number of corpora lutea (CL)
3. Number of implantation sites (IS)
4. Number of live foetuses (LF)
5. Number of dead foetuses (DF)
6. Number of early resorptions (ER)
7. Number of late resorptions (LR)
Ovaries, foetuses and non-gravid uteri were preserved in 10% formalin solution and identified
Fetal examinations:
The foetuses were examined for gross external anomalies. After external examination, foetuses were preserved in 10% formalin solution.

Foetal Parameters
1. Number of male foetuses
2. Number of female foetuses
3. Body weight of male foetuses (g)
4. Body weight of female foetuses (g)
5. Foetal sex (based on anogenital distance)
6. Anogenital distance (mm)
Statistics:
Raw data were processed to determine group means and standard deviations with statistical
significance between the control and treatment groups, using in-house developed, validated statistical software. The data were summarised in tabular form. The parametric data (body weight, body weight change, food consumption, organ weight, relative organ weight, male sex ratio, percent preimplantation loss, percent post-implantation loss, percent live foetus, and percent resorption) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. The non-parametric data (mortality rate, pregnancy rate, fetal observations etc.) were analysed using the Chi-square test.
Count data (foetal count, number of corpora lutea, number of implants, number of live foetuses,
number of pre-implantation loss, number of post-implantation loss, number of resorptions) were
subjected to non-parametric Kruskal-Wallis test.
AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected to
Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance ANOVA). Non-pregnant female rat was not subjected to statistical analysis.
Indices:
1. Number and percent of pre-implantation loss
2. Number and percent of post-implantation loss
3. % of the live foetus
4. % of the dead foetus
5. % of resorption
6. Male sex ratio
7. 20th day corrected body weight
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical sign of toxicity was observed up to a dose level of 300 mg/kg b. wt./day.
In 1000 mg/kg b. wt./day dose group, salivation was observed approximately 5 to 10 minutes after dosing in all female rats from gestation days 10 to 19 and persisted for approximately 20 to 25 minutes.
This finding was considered a response to the dose formulation and toxicologically insignificant, hence considered as non-adverse in nature
Mortality:
no mortality observed
Description (incidence):
No mortality and morbidity were observed during the study period in the control, and the test item treated groups
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight, percent body weight change, and 20th day corrected body weight of pregnant rats of 100, 300, and 1000 mg/kg b. wt./day dose groups were comparable with that of the control group
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of pregnant rats of 100, 300, and 1000 mg/kg b. wt./day dose groups were comparable with that of the control group
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean absolute and relative weights of thyroid of pregnant rats was comparable with that of the
control group
Gross pathological findings:
no effects observed
Description (incidence and severity):
External and internal gross examination of terminally sacrificed female rats did not reveal any
abnormality
Number of abortions:
no effects observed
Description (incidence and severity):
comparable between the control, and the test item treated groups.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
comparable between the control, and the test item treated groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
comparable between the control, and the test item treated groups.
Early or late resorptions:
no effects observed
Description (incidence and severity):
comparable between the control, and the test item treated groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
comparable between the control, and the test item treated groups.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The pregnancy rate was 100.0% in control, 100 mg/kg b. wt./day, and 1000 mg/kg b. wt./day dose
groups whereas 87.5% in 300 mg/kg b. wt./day dose group
Other effects:
no effects observed
Description (incidence and severity):
The mean absolute and relative weights of the uterus of pregnant female rats were comparable between the control and the test item treated groups.
Details on maternal toxic effects:
The mean number of corpora lutea, implants, live foetuses, resorptions (early, late, and total), preimplantation loss, and post-implantation loss were comparable between the control, and the test item treated groups. The mean percent pre-implantation loss, post-implantation loss, live foetuses, and total resorptions were comparable between the control, and the test item treated groups
Dose descriptor:
NOAEL
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of male, female, and total (male + female) foetuses was comparable between
the control and the test item treated groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean foetal count of male, female, and total foetuses (male + female) was comparable between
the control and the test item treated groups
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male sex ratio was comparable to that of the control group
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
The mean anogenital distance (AGD) of male and female foetuses was comparable between the control and the test item treated groups
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 95, 96, 88, and 94 foetuses were examined from 0, 100, 300, and 1000 mg/kg b. wt./day
dose groups, respectively. No treatment-related external anomaly was observed in foetuses of the
control, and the test item treated groups up to the dose level 1000 mg/kg b. wt./day. However, one runt foetus was observed in the 1000 mg/kg b. wt./day dose group. This finding is considered common and unrelated to the test item treatment
Dose descriptor:
NOAEL
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
No maternal or developmental toxicity was observed up to the highest tested dose (1000 mg/kg bw/day)
Executive summary:

The test item was administered to three groups of 8 pregnant female rats via gavage from the 5th to 19th day of gestation at 100-300 and 1000 mg/kg bw/day. A control group received corn oil only on the same dosing schedule. Maternal and developmental toxicity were evaluated.


Administration of dibutyl phosphonate at dose levels 100, 300 and 1000 mg/kg b. wt./day to female rats from 5th day of gestation to 19th day of gestation did not produce any effect on various maternal and foetal parameters; hence following dose levels are suggested for the definitive prenatal developmental oral toxicity study of dibutyl phosphonate:
• 100 mg/kg b. wt./day (Low)
• 300 mg/kg b. wt./day (Mid)
• 1000 mg/kg b. wt./day (High)

Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
other: validation of method used for the quantification of the substance

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on June 25, 2018.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibutyl phosphonate
EC Number:
217-316-1
EC Name:
Dibutyl phosphonate
Cas Number:
1809-19-4
Molecular formula:
C8H19O3P
IUPAC Name:
dibutyl phosphonate
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility of the testing laboratory
- Age at study initiation: female rats were 11-13 weeks old and male rats at least 12 weeks old.
- Weight at study initiation: body weight range: 162.85 – 244.28 g. Mean: 203.56 g. The body weight variation among the female rats was within ±20% of the mean body weight at the beginning of the acclimatisation.
- Housing: Throughout the experimental period, rats were housed individually except during the mating period. During the mating period, rats were housed in a group of two rats/cage (one male and one female). Mated female rats were housed individually. Enrichment material (wooden chew blocks) was provided to all the rats. During the study, rats were housed in solid floor polypropylene rat cages (size: 427 x 287 x 198 mm). Each cage was fitted with a stainless-steel top grille having provision for keeping rat pellet food and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5 tier racks. The bottom of the cages was layered with clean, sterilised rice husk as the bedding. Cages with bedding and enrichment material (wooden chew block throughout the gestation period and nesting material towards the end of gestation, i.e., from GD 14) were changed at least twice a week. Water bottles were refilled daily and changed at least twice a week.
Chemical and microbial contaminant analysis of samples of bedding and enrichment material are checked regularly (microbial analysis at testing laboratory and chemical analysis by supplier or vendor)
- Diet: ad libitum with a standard rodent pelleted diet (Certified Teklad Global 14% Protein Rodent Diet, Batch No 2014SC-090920MA, procured from Envigo, Inc., USA)
- Water: unlimited supply of clean and filtered drinking water (Reverse Osmosis water filter system)
- Acclimation period: 6 days prior to the cohabitation. During the acclimatisation period, rats were checked once daily for clinical signs of toxicity that would preclude use in the study.
- Other: Each day, the floor of the experimental room and all worktops were cleaned with a disinfectant solution.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 65-67
- Air changes (per hr): 16-17 per hour
- Photoperiod: 12 hours fluorescent light and 12 hours darkness
- Mean light intensity: 170-190 lux

FOOR AND WATER CONTAMINANT ANALYSIS: Every food consignment received is accompanied by a certificate of analysis of nutrient content and chemical contaminant from the food supplier. The quality of food (microbial) and water (microbial and chemical) is monitored regularly (microbial analysis at testing laboratory and chemical analysis by supplier or vendor). The levels of potential contaminants (chemical as well as microbial) were within acceptable limits. It was considered that there were no known contaminants in the food or water that would interfere with the objectives of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was first mixed with 4-5 mL of corn oil in a beaker for 2-3 minutes on a magnetic stirrer. On complete mixing of the test item in corn oil, the formulation was transferred to a calibrated measuring cylinder, and the required volume was made up using corn oil. This procedure was followed for all dose groups sequentially from low dose to high dose. Once the final dose formulation was prepared, it was thoroughly mixed using a magnetic stirrer for at least 10 minutes before dosing. The dose formulations were prepared on each day of dosing and were dosed to rats immediately after preparation.

VEHICLE
- Justification for use and choice of vehicle: Corn oil is selected as a vehicle based on solubility check. Test item was found to be stable in vehicle (corn oil) for 4 days from time of preparation.

The dose-volume for the administration was 4 mL/kg body weight. The doses were adjusted according to the most recently recorded body weight determinations.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Two sets of samples (10 samples per set) were collected by sampling three aliquots (top, middle and bottom layers) from each concentration except the control (only one aliquot from the middle layer). Sampling was performed before the start of treatment and once during the treatment period for dose formulation analyses. One set of samples was used for analyses, and the other set of samples was refrigerated. The second set of samples will be disposed of upon report finalisation. The mean concentration was determined and compared to the nominal value, and the coefficient of variation was calculated. The acceptance criteria used for analysis were ±10% from nominal value and % CV < 10.
Details on mating procedure:
After completion of the acclimatisation period, each female rat was cohabitated with an untreated male rat (1:1) until the requisite numbers of mated female rats (25/group) were obtained. Detection of mating was confirmed by the evidence of a copulatory plug in the vagina or by vaginal lavage for the presence of sperm. After confirmation of mating, each female rat was returned to an individual cage, assigned to a group, and that day was designated as day 0 of gestation (GD 0).
Mated female rats were assigned to dose groups by stratified randomisation on the basis of GD 0 body weights. Body weights of GD 0 were arranged in descending order, from the heaviest to the lightest on the first GD 0 date and assigned in the same order on the subsequent days. Beginning with the heaviest weight, one rat was randomly assigned to each stratified group. In the event that the total number of rats mated on a given day was not an even multiple of the number of treatment groups, on a subsequent day, the mated female rats were assigned to complete the last incomplete stratification group and then assigned to the next stratification group until all GD 0 females for that day were assigned.
Duration of treatment / exposure:
from the 5th day of gestation to the 19th day of gestation
Frequency of treatment:
daily
Duration of test:
from the 5th day of gestation to the 19th day of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 mated females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a dose range-finding prenatal developmental toxicity study with the substance Wistar rats, in which no adverse effects were observed up to the dose level of 1000 mg/kg b. wt./day
- Time of day for (rat) dam blood sampling: in the morning

Examinations

Maternal examinations:
MORTALITY AND MORBIDITY
Rats were observed twice daily for mortality and morbidity.


CAGE SIDE OBSERVATIONS: Yes
Rats will be observed at least twice a day for the mortality and morbidity. Female rats which show
marked signs of reaction to the treatment or premature delivery/abortion will be sacrificed and
subjected to macroscopic examined. Females which die or are found moribund during the study will be weighed and subjected to the post-mortem examination/necropsy..

DETAILED CLINICAL OBSERVATIONS
During the study period, visible clinical signs, such as changes in the skin, fur, eye, mucous membranes, as well as the behavioural pattern of the mated female rats, were recorded twice daily.

BODY WEIGHT
- Time schedule for examinations: Body weights for the mated female rats were recorded individually on days 0, 3, 5, 8, 11, 14, 17, and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE
A fixed amount of pelleted rat food was presented to each mated female rat, and the food remaining at the end of the specified interval was recorded. The food consumption was determined for the following intervals: Days 0–3, 3–5, 5–8, 8–11, 11–14, 14–17, 17-20, and 5-20.

POST-MORTEM EXAMINATIONS
- Necropsy: On day 20 of gestation, all female rats were weighed, euthanised by carbon dioxide asphyxiation, dissected, and examined macroscopically for any structural abnormalities. During the macroscopic examination, findings were noted in the stomach. For this reason, the stomach was collected and preserved with the concurrent control group for possible histopathological examination.
- Blood collection: At the time of terminal sacrifice on gestation day 20, blood (approximately 1 mL) was collected from all the female rats under anaesthesia (isoflurane) by orbital plexus puncture within a short timeframe in the morning. Separated serum samples were stored at -70 ± 10 °C till analysis.
- Thyroid Hormone Analysis: Serum thyroid hormones (T4, TSH, and T3) were analysed from all dams during the terminal sacrifice. Serum thyroid hormones T3 and T4 were analysed at the laboratory using a validated bioanalytical method and TSH was analysed by an ELISA method.
- Organ weight and histopathology: At the time of terminal sacrifice, the thyroid gland was collected and weighed (post-fixation) from all female rats and preserved for histopathology. Stomach with gross lesions was also collected along with concurrent control group and preserved for histopathology. Detailed histological examination of the thyroid gland was performed for all dams and stomach with gross lesions was performed.
Ovaries and uterine content:
Immediately after termination, the uteri were removed, and the pregnancy status of rats was ascertained. The non-gravid uteri were further examined by immersing in 5% ammonium sulphide solution to confirm non-pregnant status.
Ovaries and gravid uteri, including the cervix, were removed and examined immediately for the parameters mentioned below. Ovaries, foetuses, and non-gravid uteri were preserved and identified.
The parameters mentioned below were examined:
1. Gravid uterine including the cervix weight (g)
2. Number of corpora lutea (CL) determined for pregnant rats
3. Number of implantation sites (IS)
4. Number of live foetuses (LF)
5. Number of dead foetuses (DF)
6. Number of early resorptions (ER)
7. Number of late resorptions (LR)
8. Number of total resorptions (TR)
Blood sampling:
At the time of terminal sacrifice on day 20 of gestation, blood (approximately 1 mL) will be collected from all surviving dams under anesthesia (isoflurane) by orbital plexus puncture within a short timeframe (e.g., two hours) on the morning of the day of necropsy. From humanely euthanised/moribund, or aborted rats, blood will not be collected. Serum samples will be stored at -70 ± 10 °C till analysis.
Serum thyroid hormones (T4, TSH, and T3) will be analysed from all surviving dams during terminal sacrifice. Serum thyroid hormones T3 and T4 will be analysed using validated bioanalytical method and TSH will be analysed using ELISA method given in kit (BioVendor - Rat Thyroid Stimulating Hormone ELISA kit).
Fetal examinations:
Foetal Parameters
1. Number of male foetuses
2. Number of female foetuses
3. Body weight (g) of male foetuses
4. Body weight (g) of female foetuses
5. Foetus sex (based on gross observation of anogenital distance and internal examination of gonads)
6. Ano-genital distance (mm)
- Foetus Allocation: Allocation of foetuses for the skeletal and soft tissue evaluation was done by selecting alternate live foetuses. The first live foetus was considered for skeletal evaluation, and the second live foetus was considered for soft tissue evaluation. Late resorptions were not considered for foetal distribution
- External evaluation: All foetuses (100%) were examined externally. External foetal sex (as determined by gross examination of ano-genital distance) was compared with internal (gonadal) sex in all foetuses (examined for both skeletal and soft tissue malformations). In addition, male foetuses were also observed for incomplete testicular descent/cryptorchidism. Particular attention was paid to the reproductive tract which was examined for signs of altered development.
- Skeletal evaluation: Approximately 50% of the foetuses in each litter were subjected to the detailed skeletal evaluation after processing and staining with alizarin red. Foetuses were preserved in isopropyl alcohol and glycerin solution and the skeletal evaluation was performed.
- Soft Tissue Evaluation: The remaining 50% (approximately) of the foetuses in each litter were subjected to the detailed visceral evaluation by microdissection. The cephalic evaluation was done by head razor sectioning after fixing in the aceto-alcohol formalin solution (i.e., Davidson’s fixative).
Statistics:
Raw data were processed to obtain group means and standard deviations with significance between the vehicle control and treated groups at 95 or 99% confidence levels, using laboratory's-developed statistical software.

The parametric data (body weight, body weight gain/change, food consumption, organ weight, organ weight ratio, male sex ratio, and prenatal data etc.) were subjected to Shapiro-Wilk’s test for normality check wherever applicable, followed by Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. If the data did not meet the normality, data were transformed to check the normality again. If transformed data did not meet the normality, the Kruskal-wallis/Mann Whitney test was performed to calculate significance. If the data did not meet the homogeneity of variance, statistical analysis was extended following decision tree (Gad, S.C., 2007). The non-parametric data (mortality rate, pregnancy rate, fetal observations etc.) was analysed using Chi-square test.
Count data (foetal count, number of corpora lutea, number of implants, number of live foetuses,
number of dead foetuses, number of pre-implantation loss, number of post-implantation loss, number of resorptions) were subjected to non-parametric Kruskal-Wallis test. AGD will be normalised (the ratio of AGD to the cube root of body weight) and then subjected to statistical analysis.
Non-pregnant rats were not subjected to statistical analysis
Indices:
1. Number and percent of pre-implantation loss = [(CL-IS)/CL*100]
2. Number and percent of post-implantation loss = [(IS-LF)/IS*100]
3. Live foetus (%) = [(LF/IS) *100]
4. Dead foetus (%) = [(DF/IS) *100]
5. Male sex ratio = [(N° of male foetuses/ Total N° foetuses) * 100]
6. Corrected body weight = Body weight on gestation day 20th – gravid uterine weight
7. Percent of resorptions
Historical control data:
Species: Rattus Norvegicus (Rat) Strain: Wistar

Summary of pregnancy data:
Number of positive female rats: mean: 25.00; st.dev.: 0.00; n. of studies: 27; min 25; max 25
None pregnant females: mean: 2.37; st.dev.: 1.60; n. of studies: 27; min: 0; max: 5
Pregnant female: mean: 22.63; st.dev.: 1.60; n. of studies: 27; min 20; max 25.

Summary of External Observation:
Total numer of studies: 28
Total number of foetuses: 6935
Total number of litters: 628

Observations runt:
Abnormal foetus: n° 5; %: 0.07; min: 0; max: 1
Abnormal litter: n° 5; %: 0.80; min: 0; max: 1

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs of toxicity were observed up to a dose level of 300 mg/kg b. wt./day.
In 1000 mg/kg b. wt./day dose group, salivation was observed approximately 5 to 10 minutes post- dosing in all-female rats from around gestation day 9 to 19 and persisted for approximately 20 to 25 minutes post-dosing. Salivation was not seen during morning observation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity (Wook-Joon Yu at al, 2011). Therefore, salivation observed in this study was of no toxicological significance
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality or morbidity was observed during the study period in the control group and the test item treated groups
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights and 20th day corrected body weights of the pregnant female rats were comparable between the control and the test item treated groups up to a dose level of 300 mg/kg b. wt./day.
For the 1000 mg/kg b. wt./day dose group, a statistically significant decrease in the mean body weight of the pregnant female rats was observed on gestation days 17 and 20. Reduced mean body weight was also observed during gestation day 14 without statistical significance. A decrease in the 20th day corrected mean body weight was also observed without statistical significance.

The mean body weight change of the pregnant female rats was comparable between the control and the test item treated groups up to a dose level of 300 mg/kg b. wt./day.
For the 1000 mg/kg b. wt./day dose group, a statistically significant decrease in mean body weight change was observed during the gestation days 5-8, 14-17, and 5-20. Reduced mean body weight change was also observed during the gestation days 11-14 and 17-20 without statistical significance.
Decreased mean body weight and body weight change were correlated with the decreased mean food consumption and were considered as adverse effects of the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the pregnant female rats was comparable between the control and the test item treated groups up to a dose level of 300 mg/kg b. wt./day.
For the 1000 mg/kg b. wt./day dose group, a statistically significant decrease in mean food consumption was observed during the gestation days 5-8, 8-11, 11-14, and 5-20. Reduced mean food consumption was also observed during the gestation days 14-17 and 17-20 without statistical significance.
Decreased mean food consumption was considered as adverse effects of the test item treatment
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically, a significant decrease in serum TSH level was observed in pregnant female rats belonging to 100 mg/kg b. wt./day dose group. This finding was considered incidental and unrelated to the test item treatment due to lack of dose-dependency.
A statistically significant increase in serum levels of T3 and T4 were observed in pregnant female rats belonging to 100 mg/kg b. wt./day dose group. These effects were considered incidental and unrelated to the test item treatment due to lack of dose-dependency and absence of other supporting findings in thyroid weight and thyroid histopathology
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Statistically, a significant decrease in the terminal body weight (GD 20) of the pregnant female rats was observed in 1000 mg/kg b. wt./day dose group .
Test item treatment did not lead to any alteration in absolute and relative weights of the thyroid gland.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
External (gross) examination of terminally sacrificed female rats did not reveal any lesion. Internal (gross) examination of terminally sacrificed female rats revealed the increased thickness of non-glandular stomach (in 23 out of 25 female rats) in 1000 mg/kg b. wt./day dose group. This effect was related to the test item treatment but considered insignificant due to lack of human relevance as the non-glandular stomach is absent in humans (Mckee and Gass, 2011)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histological examination of the stomach (with gross lesions) revealed mucosal hyperkeratosis and hypertrophy/hyperplasia as well as infiltrate of inflammatory cells in 1000 mg/kg b. wt./day dose group. These lesions were related to the test item treatment but considered insignificant due to lack of human relevance as the non-glandular stomach is absent in humans (Mckee and Gass, 2011).
Histological examination of thyroid gland did not reveal any lesion in rats of the control as well as the test item treated groups except ectopic thymic tissue (in rat N° 19, 71, and 82 ) and ultimobranchial cyst/s (in rat N° 90 and 93), which were considered as spontaneous changes commonly observed in the rat thyroid gland.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
comparable between the control, and test item treated groups
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
comparable between the control, and test item treated groups
Early or late resorptions:
no effects observed
Description (incidence and severity):
comparable between the control, and test item treated groups
Description (incidence and severity):
comparable between the control, and test item treated groups
Changes in pregnancy duration:
not examined
Description (incidence and severity):
The pregnancy rate for all the test item treated groups was comparable to that of the control group.
Pregnancy rate was 92.0% in the control and 300 mg/kg b. wt./day dose group whereas 100% in 100 and 88.0% in 1000 mg/kg b. wt./day dose groups
Description (incidence and severity):
The mean absolute and relative uterine weight of the pregnant female rats were comparable between the control and the test item treated groups up to a dose level of 300 mg/kg b. wt./day.
For the 1000 mg/kg b. wt./day dose group, a statistically significant decrease in mean absolute uterine weight (-13.01% than the control group) was observed. This lowered uterine weight was correlated with decreased maternal body weight and was considered secondary to the maternal toxicity and adverse effects of the test item.
Details on maternal toxic effects:
The mean absolute and relative uterine weight of the pregnant female rats were comparable between the control and the test item treated groups up to a dose level of 300 mg/kg b. wt./day.
For the 1000 mg/kg b. wt./day dose group, a statistically significant decrease in mean absolute uterine weight (-13.01% than the control group) was observed. This lowered uterine weight was correlated with decreased maternal body weight and was considered secondary to the maternal toxicity and adverse effects of the test item.
The mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, the mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions were comparable between the control, and test item treated groups

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: general toxicity (adverse effects observed on body weight, food consumption, and absolute uterine weight at the dose level of 1000 mg/kg b. wt./day)

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean weight of the male foetus, female foetus, and total foetuses (male + female) was comparable between the control, and the test item treated groups up to the dose level of 300 mg/kg b. wt./day.
In the 1000 mg/kg b. wt./day dose group, a statistically significant decrease in the mean foetal weight of male foetuses (-7.43% than the control group), female foetuses (-7.83% than the control group), and a composite of foetuses of both sexes (-6.74% than the control group) was observed when compared to the control group. These lowered foetal body weights were correlated with decreased maternal body weight as well as absolute uterus weight. Hence, these findings were considered secondary to the maternal toxicity and adverse effects of the test item.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male sex ratio was comparable between the control, and the test item treated groups
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
The mean anogenital distance of male and female foetuses was comparable between the control, and the test item treated groups.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 249, 270, 252, and 202 foetuses were examined in 0, 100, 300, and 1000 mg/kg b. wt./day dose groups, respectively.
No treatment-related external anomaly was observed in foetuses of the treatment groups up to the dose level of 1000 mg/kg b. wt./day except one runt foetus in the 1000 mg/kg b. wt./day dose group. However, as the number of runt foetus was within the range of historical control data , this finding was considered incidental.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 131, 141, 132, and 107 foetuses were observed for the skeletal evaluation in 0, 100, 300, and 1000 mg/kg b. wt./day dose groups, respectively. Incidences of skeletal malformations were not observed in any of the dose groups. Incidences of skeletal variations were observed in all dose groups. The type of skeletal variations and the incidence of the number of foetuses and number of litters affected with these variations were recorded.
Statistically, a significant decrease in the number of foetuses with 14th rib: extra ossification centre and 3rd sternebrae: misaligned was observed in 300 mg/kg b. wt./day dose group. Statistically significant decrease in the number of foetuses with xiphisternum: incomplete ossification was also observed in 100 and 300 mg/kg b. wt./day dose groups. These findings were only because of a higher number of incidences in the control group compared to treatment and had no toxicological relevance .
Statistically significant increase in the number of foetuses and litters with xiphisternum: unossified was observed in 1000 mg/kg b. wt./day dose group. Statistically significant increase in the number of foetuses 5th sternebrae: unossified was also observed in 1000 mg/kg b. wt./day dose group. These findings indicated a delayed ossification associated with reduced foetal weights, i.e., a delay in foetal development rather than a direct effect on bone tissue and considered as secondary to the maternal toxicity and adverse effects of the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 118, 129, 120, and 95 foetuses were examined in 0, 100, 300, and 1000 mg/kg b. wt./day dose groups, respectively.
No treatment-related visceral anomaly was observed in foetuses of the treatment groups up to the dose level of 1000 mg/kg b. wt./day. Dilated ureters were observed in four foetuses from each of the control and 100 mg/kg b. wt./day dose group, three foetuses from the 300 mg/kg b. wt./day dose group, and one foetus from the 1000 mg/kg b. wt./day dose group. The incidence of this finding was comparable between control and the test item treated groups
Other effects:
no effects observed
Description (incidence and severity):
Head Razor Observations
A total of 118, 129, 120, and 95 foetuses were examined in 0, 100, 300, and 1000 mg/kg b. wt./day dose groups, respectively.
No treatment-related anomaly was observed in the head razor section up to the dose level of 1000 mg/kg b. wt./day

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Remarks on result:
other: The developmental NOAEL was also found to be 300 mg/kg b. wt./day, based on the adverse effects observed on foetal body weight and skeletal variations at the dose level of 1000 mg/kg b. wt./day.

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: skeletal variations: 14th rib; foetuses with xiphisternum: incomplete ossification; with xiphisternum: unossified; 5th sternebrae: unossified

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Active Ingredient Concentration and Homogeneity of Dose Formulation


The mean percent recovery obtained for the test doses were within the acceptance level of ±10% of the nominal concentration, demonstrating that the exposure concentrations were as intended in the study plan and the % CV was less than 10, suggesting that the test doses were homogeneously prepared

Applicant's summary and conclusion

Conclusions:
NOAEL (maternal toxicity) = 300 mg/kg bw/day, based on the adverse effects observed on body weight, food consumption, and absolute uterine weight at the dose level of 1000 mg/kg bw/day

NOAEL (developmental) = 300 mg/kg bw/day, based on the adverse effects observed on foetal body weight and skeletal variations at the dose level of 1000 mg/kg bw/day
Executive summary:

This study was performed to evaluate the potential prenatal developmental and maternal toxicity of dibutyl phosphonate when administered orally, through gavage, to pregnant female rats as per the OECD 414 and under GLP conditions. The substance was administered orally from gestation day (GD) 5 to 19, through gavage, to 25 mated female rats per group at dose levels 100, 300, and 1000 mg/kg b. wt./day. The control group received the vehicle, corn oil, only. Rats were observed twice daily for mortality and clinical signs. Maternal body weights and food consumption were recorded throughout the gestation period. All rats were sacrificed on GD 20 and assessed for gross pathological changes. The uteri were excised, weighed, and examined for the numbers of implantation sites, early and late resorptions, and numbers of live and dead foetuses. The ovaries were excised, and the number of corpora lutea was counted. The foetuses were identified for sex, weighed, and examined for external findings. The anogenital distance was measured for all foetuses. At the time of terminal sacrifice, the weight of the thyroid gland was recorded from all-female rats and preserved for histopathology. Stomach with gross lesions was also collected along with concurrent control group and preserved for histopathology. Detailed histological examination of the thyroid gland and stomach was performed. Serum thyroid hormones T3 (liothyronine), T4 (levothyroxine), and TSH (thyroid-stimulating hormone) were analysed from all pregnant female rats during the terminal sacrifice. Following appropriated fixation, foetuses were examined for visceral abnormalities, including razor sectioning of the head and for skeletal abnormalities. Dose formulations were analysed for the test item concentration and homogeneity prior to the initiation of the treatment and once during the treatment period.


Results:


At 100 and 300 mg/kg b. wt./day: No treatment-related effects were seen in the 100 and 300 mg/kg b. wt./day dose groups for any evaluated parameter.


At 1000 mg/kg b. wt./day


No treatment-related effects were seen in the 1000 mg/kg b. wt./day dose group for the below-mentioned parameters:



  • Mortality and morbidity

  • Salivation was observed approximately 5 to 10 minutes after dosing in all-female rats from around gestation days 9 to 19 and persisted for approximately 20 to 25 minutes post-dosing. Salivation was not seen during morning observation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity (Wook-Joon Yu at al, 2011). Therefore, salivation observed in this study was of no toxicological significance.

  • Pregnancy rate

  • Mean relative uterine weights

  • T3, T4, and TSH (Thyroid Stimulating Hormone)

  • External (gross) examination of the terminally sacrificed female rats

  • Internal (gross) examination of terminally sacrificed female rats revealed increased thickness of non-glandular stomach (in 23 out of 25 female rats). This effect was related to the test item treatment but was considered insignificant due to lack of human relevance as the non-glandular stomach is absent in humans.

  • The absolute and relative weight of the thyroid gland

  • Histological examination of the thyroid gland

  • Histological examination of the stomach (with gross lesions) revealed mucosal hyperkeratosis and hypertrophy/hyperplasia as well as infiltrate of inflammatory cells. These lesions were related to the test item treatment but were considered insignificant due to lack of human relevance as the non-glandular stomach is absent in humans.

  • Mean foetal count of male, female, and total foetuses (male + female)

  • Mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss

  • Mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions

  • Anogenital distance of male and female foetuses

  • Male sex ratio

  • Incidence of malformation/variation for external, visceral, and head razor examination of foetuses


The following treatment-related adverse effects were seen in the 1000 mg/kg b. wt./day dose group:



  • A statistically significant decrease in mean body weight of the pregnant female rats was observed on gestation days 17 and 20. Reduced mean body weight was also observed during gestation day 14 without statistical significance. A decrease in 20th day corrected mean body weight was also observed without statistical significance.

  • A statistically significant decrease in the mean body weight change was observed during the gestation days 5-8, 14-17, and 5-20. A reduced mean body weight change was also observed during the gestation days 11-14 and 17-20 without statistical significance.

  • A statistically significant decrease in mean food consumption was observed during the gestation days 5-8, 8-11, 11-14, and 5-20. Reduced mean food consumption was also observed during the gestation days 14-17 and 17-20 without statistical significance.

  • The decrease in the mean body weight, 20th day corrected body weight, and body weight change were correlated with the decreased mean food consumption and were considered as adverse effects of the test item.

  • A statistically significant decrease in mean absolute uterine weight (-13.01% than the control group) was observed. This finding was considered secondary to the maternal toxicity and adverse effect of the test item.

  • A statistically significant decrease in mean foetal weight of male foetuses (-7.43% than the control group), female foetuses (-7.83% than the control group), and the composite of foetuses of both sexes (-6.74% than the control group) was observed when compared to the control group. These lowered foetal body weights were correlated with decreased maternal body weight as well as absolute uterus weight. Hence, these findings were considered secondary to the maternal toxicity and adverse effects of the test item.

  • During foetal skeletal examination, statistically significant increase in the number of foetuses and litters with xiphisternum: unossified was observed. Statistically significant increase in the number of foetuses with 5th sternebrae: unossified was also observed. These variations indicated a delayed ossification associated with reduced foetal weights, i.e., a delay in foetal development rather than a direct effect on bone tissue and considered as secondary to the maternal toxicity and adverse effects of the test item.


Conclusion


NOAEL (maternal toxicity) = 300 mg/kg bw/day, based on the adverse effects observed on body weight, food consumption, and absolute uterine weight at the dose level of 1000 mg/kg bw/day


NOAEL (developmental) =  300 mg/kg bw/day, based on the adverse effects observed on foetal body weight and skeletal variations at the dose level of 1000 mg/kg bw/day