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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July 2018 to 19 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 414, adopted on 25 June 2018
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALSTESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Hordaphos MDGB


CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies: OECD 422


FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design: The test item, Hordaphos MDGB, was administered by the oral route at 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19.TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Hordaphos MDGB


Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on 25 June 2018
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Phosphoric acid, mixed esters with Bu alc. and ethylene glycol
EC Number:
284-716-0
EC Name:
Phosphoric acid, mixed esters with Bu alc. and ethylene glycol
Cas Number:
84962-20-9
IUPAC Name:
Reaction mass of Phosphoric acid mono-(2-hydroxy-ethyl) ester and Phosphoric acid monobutyl ester and Phosphoric acid bis-(2-hydroxy-ethyl) ester
Constituent 2
Reference substance name:
Phosphoric acid, mixed with Ethylene glycol and Butanol
IUPAC Name:
Phosphoric acid, mixed with Ethylene glycol and Butanol
Test material form:
other: liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Clariant Plastics and Coatings (Deutschland) GmbH and DEH8006819
- Expiration date of the batch: 28 October 2019
- Purity test date: 01 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: Stable for 6 hours at Ambient (21 to 29°C)
- Solubility and stability of the test substance in the solvent/vehicle: The test item was clearly miscible with distilled water at the concentration of 35 mg/mL (highest dose concentration) and stable for 6 hours at Ambient (21 to 29°C).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulated

FORM AS APPLIED IN THE TEST: Liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: 200 to 300 g
- Fasting period before study: No
- Housing: Mating - three rats (one male and two females) were housed in standard polypropylene cages.
- Post mating - After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polypropylene cages.
- Diet (ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG
- Water (ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: minimum period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 to 22.9o C
- Humidity (%): 45 to 67%
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 18 July 2018; To: 30 August 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks on MMAD:
Not applicable
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle : The test item was clearly miscible with distilled water at the concentration of 35 mg/mL (highest dose concentration). Hence, distilled water was selected as a vehicle for test item formulation preparations.
- Concentration in vehicle: G1: 0 mg/mL; G2: 5 mg/mL; G3: 13 mg/mL; G4: 35 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation analysis for dose concentration verification was done by Analytical Chemistry department of Bioneeds India Private Limited. Sampling and analysis of formulations was performed during first week and last week (since treatment was from GD 5 to 19 only two treatment weeks has been considered) of the treatment. The samples were collected in duplicates (60 mL each) from vehicle control, low, mid and high dose concentrations
The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose concentration analysis. One set of aliquot of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples were discarded, as the analysis results of first set of samples were within the limits.
Details on mating procedure:
- Impregnation procedure: [cohoused]

- M/F ratio per cage: 1:2
- Length of cohabitation: 14 days
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Gestation day 5 to 19 for each presumed pregnant female
Frequency of treatment:
Once daily
Duration of test:
5 months
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control group
Dose / conc.:
50 mg/kg bw/day
Remarks:
Low dose group
Dose / conc.:
130 mg/kg bw/day
Remarks:
Mid dose group
Dose / conc.:
350 mg/kg bw/day
Remarks:
High dose group
No. of animals per sex per dose:
25 presumed pregnant females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses of 50, 130 and 350 mg/kg body weight for low, mid and high dose were selected as the dose range finding study (100, 300 and 1000 mg/kg body weight/day; duration: 14 days pre-mating, 14 days mating and gestation days 0 to 19) revealed mortality within the first week of dosing at 1000 mg/kg body weight/day. An abnormal breathing and attenuated body weight gain was reported at 300 mg/kg body weight/day. The number of implantation sites was slightly reduced in all groups. No treatment related observations were made in the course of visceral and skeletal examination.

The result of a subsequently performed OECD 422 study was observed with mortality beginning in week 2 of treatment, tracheal changes most probably related to local irritation at 200 mg/kg. The signs of discomfort (piloerection and abnormal breathing), tendency to decreased food consumption at 50 mg/kg was noted. The reproductive performance and developmental parameters were not affected in any dose group.

Based on the above mentioned observations the doses of 50, 130 and 350 mg/kg body weight for low, mid and high dose were selected

- Rationale for animal assignment: The body weight of mated females on each day of gestation day ‘0’ was recorded and arranged in the ascending order of their body weight until required number of mated females acquired for each group. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups and permanent identification numbers were assigned



Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section)

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Individual animal feed intake of mated females was recorded for gestation days
0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All organs grossly with special attention to uterus, ovaries and thyroid
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Data was subjected to various statistical analysis using SPSS software version 22. All analyses and comparisons were evaluated at the 95% level of confidence (P<0.05),

Data of non-pregnant females was not included in mean calculation and statistical analysis.
One-way ANOVA with Dunnett’s post test - Gestation body weight (g), Percent change in gestation
body weight, Corrected body weight (g & %), Gravid uterus weight (g), Feed consumption (g), Fetal
weights (g), Anogenital distance of all live fetuses (mm), Crown-rump length of fetuses (mm), serum
T4, T3 and TSH lelvels.

Kruskal-Wallis (Non-parametric) - No. of corpora lutea, implantations, resorptions, litter size, sex ratio, implantation lossess, live/dead fetuses.

Cross Tabs Chi-square test/ Fischer's Exact Test - Pregnancy rate, No. of dams with/without live/dead fetuses, No. of dams with/without resorptions
Indices:
Corrected Body weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight.

Percent of Live/Dead Fetuses = (Number of Live/Dead Fetuses / Litter Size) x 100.

Percent of Early/Late Resorptions (%) = (Number of Early/late Resorptions/ Number of Implantations) x 100.

Early Resorptions per Group (%) = (Number of Dams with Early Resorptions in the group / Total Number of Dams in the Group) x 100.

Pre-implantation Loss (%) = (Number of Corpora lutea - Number of implants) / Number of Corporalutea x 100.

Post-implantation Loss (%) per Dam = (Number of Dams with Post-implantation loss / Total Number of Dams) x 100

Male/Female Sex Ratio = Number of live male fetuses / Number of live female fetuses.

Male/Female Fetuses (%) = (Number of live male/female fetuses / Total number of live fetuses) x 100.

Group/Litter Fetal Observation for External, Visceral and Skeletal Examinations:

Fetal incidence (%) = (Number of Fetuses with a particular observation / Total number of Fetuses in a group) x 100
Historical control data:
Not applicable

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity and no mortality/morbidity observed at any of the tested dose group animals during the experimental period.
Dermal irritation (if dermal study):
not specified
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any of the tested dose group animals during the experimental period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in body weight during gestation period across all the tested dose groups when compared with the vehicle control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase in average feed consumption during gestation day 3 to 5 in G4 group animals and statistically significant increase in average feed consumption during gestation day 17 to 19 and 19 to 20 in G2 group animals was observed when compared with vehicle control group. This sporadic incidence is considered as incidental and unrelated to treatment with test item due to unaffected body weights during this period
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The absolute thyroid along with parathyroid weights (g) were 0.0232, 0.0220, 0.0236 and 0.0235 and relative thyroid along with parathyroid weights (%) were 0.0062, 0.0058, 0.0063 and 0.0062 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg) respectively. There were no statistically significant differences in mean absolute and relative thyroid along with parathyroid weight and no treatment related changes for this parameter across the dose groups when compared to controls
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes noted in any of the dose group and vehicle control group animals during conduct of the necropsy
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The thyroid along with parathyroid was collected and preserved in 10% neutral buffer formalin for microscopic examination. The histopathology of thyroid along with parathyroid was conducted for all the dose group dams. There were no treatment related histopathological findings noticed in the any of the dose group animals during conduct of the histopathological examination in the study.

However, the observations like, hemorrhage in thyroids at G1 group (1 animal); MNC infiltration in thyroids at G2 group (1 animal); Ultimobranchial cyst at G1 group (1 animal), G2 group (3 animals) and G3 group (3 animals), Ectopic thymus at G3 group (2 animals) and G4 group (1 animal) were noted. These lesions were considered as background lesions.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The serum T3 levels (ng/mL) were 2.510, 2.572, 2.370 and 2.358 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg) respectively. There were no statistically significant differences in these values and no treatment related changes for this parameter across the dose groups when compared to controls.

The serum T4 levels (ng/mL) were 66.724, 72.036, 73.684 and 79.023 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg) respectively. There were no changes in serum T4 levels (ng/mL) across all the tested dose groups when compared with the vehicle control group. The values obtained across groups were comparable.

A statistically significant increase in serum T4 levels (ng/mL) in G4 group animals was observed. This incidence is considered as incidental and unrelated to treatment with test item due to comparable values across groups and the values obtained were within biological control range.

The serum TSH levels (µIU/mL) were 4.351, 4.142, 3.767 and 4.439 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg) respectively. There were no statistically significant differences in serum TSH levels and no treatment related changes for this parameter across the dose groups when compared to controls.
Details on results:
The Test item, Hordaphos MDGB was administered via oral route at a dose volume of 10 mL/kg body weight to mated female rats from gestation day 5 (GD5) through gestation day 19 (GD19). The dose levels were 0 mg/kg, 50 mg/kg, 130 mg/kg and 350 mg/kg for the control (G1), low (G2), mid (G3) and high (G4) groups, respectively. The results of the experiment support the conclusion that the NOAEL [No Observed Adverse Effect Level] for the test item, Hordaphos MDGB for maternal toxicity is 350 mg/kg, the high dose, as there were no effects noted on maternal parameters at all the tested dose groups.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
not applicable
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The mean percentage pre-implantation loss was 0.00, 0.00, 0.00 and 0.37 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg) respectively. There were no statistically significant differences in these values and no treatment related changes for this parameter across the dose groups when compared to controls.

The mean percentage of post-implantation loss was 6.70, 4.99, 9.28 and 4.64 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg) respectively. There were no statistically significant differences in these values and no treatment related changes for this parameter across the dose groups when compared to controls.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
not applicable
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of early resorptions per dam was 0.57, 0.50, 0.95 and 0.43 and percentage of early resorptions was 5.71, 3.89, 8.14 and 3.25 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg) respectively. There were no statistically significant differences observed in the number and percentage of early resorptions per dam across dose groups when compared to the vehicle control.

The mean number of late resorptions per dam was 0.14, 0.15, 0.14 and 0.19 and percentage of early resorptions was 1.00, 1.10, 1.13 and 1.39 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg) respectively. There were no statistically significant differences observed in the number and percentage of late resorptions per dam across dose groups when compared to the vehicle control.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no dead fetuses noted for any litter in any of the test dose group or the vehicle control litters
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
not applicable
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): not applicable
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
not applicable
Details on maternal toxic effects:
No effects were noted at all the tested dose groups

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 350 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical signs
dead fetuses
early or late resorptions
effects on pregnancy duration
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
other: Thyroid hormonal levels

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
cervix
ovary
uterus
vagina

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (350 mg/kg), respectively, the male mean fetal weights were 4.03 g, 4.15 g, 4.22 g and 4.14 g; the female mean fetal weights were 3.84 g, 3.85 g, 3.94 g and 3.88 g. There were no changes in mean fetal weights across all the tested dose groups when compared with the vehicle control group. The values obtained across groups were comparable.

A statistically significant increase in mean male fetal weights in G3 group was observed. This incidence is considered as incidental and unrelated to treatment with test item due to comparable values across groups and missing dose-response relationship. Also other fetal parameters are unaffected

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): For groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (150 mg/kg) and G4 (350 mg/kg), respectively, the male mean fetal weights were 4.03 g, 4.15 g, 4.22 g and 4.14 g; the female mean fetal weights were 3.84 g, 3.85 g, 3.94 g and 3.88 g. There were no changes in mean fetal weights across all the tested dose groups when compared with the vehicle control group. The values obtained across groups were comparable.

A statistically significant increase in mean male fetal weights in G3 group was observed. This incidence is considered as incidental and unrelated to treatment with test item due to comparable values across groups and missing dose-response relationship. Also other fetal parameters are unaffected
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
not applicable
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio calculated as the mean percentage of male fetuses was 1.36, 1.54, 1.09 and 1.19 for G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg) respectively. There were no statistically significant differences in these values and no treatment related changes for this parameter across the dose groups when compared to controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean litter sizes, assessed as the in utero total of fetuses [live plus dead] per dam were 12.52, 12.20, 11.95 and 12.10 for groups G1 (0 mg/kg) ,G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg) respectively. There were no dead fetuses within any litter in any group; therefore, the numbers of live fetuses per dam across groups were the same. There were no statistically significant differences noted across dose groups when compared to the control.
Changes in postnatal survival:
not examined
Description (incidence and severity):
not applicable
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 263 (21), 244 (20), 251 (21) and 254 (21) fetuses (litters) with a mean of 12.52, 12.20, 11.95 and 12.10 fetuses per dam were available for gross external examination from groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg), respectively.
No treatment related external abnormalities were noted within any group during external examination of these fetuses. However, the following variations were noted from all the group fetuses during fetal external examinations:
In G1 (vehicle control) group, haemorrhagic spot on the hind limb unilaterally in one fetus was noted.
In G3 (130 mg/kg) group, haemorrhagic spot on ventral side of trunk in one fetus was noted.
In G4 (350 mg/kg) group, haemorrhagic spot on hind limb unilaterally in one fetus, on head in one fetus and on forelimb unilaterally in one fetus were noted.
These findings are common for fetuses of this species and strain and cannot be considered as treatment related.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The skeletal malformations reflect variations in the maturation rate of the ossified tissues of rat fetuses. They are unlikely to be detectable in juvenile animals after continued growth postnatally. These skeletal malformations did not occur in a dose-dependent pattern, nor was the severity or the incidence of the findings increased with dose.

Overall, the skeletal morphologic observations reflect variations in the maturation rate of the ossified tissues of rat fetuses; they did not occur in a dose-dependent pattern, nor was the severity of the anomaly increased with dose. These result support the conclusion that the findings are incidental and that the test item did not produce an adverse effect on the skeletal system during fetal development at any dose in this study.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The visceral observations are common findings for fetuses of this species; they did not occur in a dose-dependent pattern, nor was the severity or the incidence of the findings increased with dose. This result supports the conclusion that the findings are incidental and that the test item, Hordaphos MDGB, did not produce an adverse effect on the soft tissues during fetal development.
Other effects:
no effects observed
Description (incidence and severity):
For groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg), respectively, the male fetal crown rump length was 39.02 mm, 39.77 mm, 39.90 mm and 39.77 mm; the female fetal crown rump length was 38.07 mm, 38.07 mm, 38.28 mm and 38.20 mm. There were no statistically significant differences in fetal crown rump length and no treatment related changes for this parameter across the dose groups when compared to controls.

For groups G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg), respectively, the mean male ano-genital distance ratio was 3.02 mm, 2.98 mm, 3.02 mm and 3.06 mm; the mean female ano-genital distance ratio was 1.40 mm, 1.41 mm, 1.39 mm and 1.43 mm. There were no statistically significant differences in fetal ano-genital distance ratio and no treatment related changes for this parameter across the dose groups when compared to controls.
Details on embryotoxic / teratogenic effects:
The Test item, Hordaphos MDGB was administered via oral route at a dose volume of 10 mL/kg body weight to mated female rats from gestation day 5 (GD5) through gestation day 19 (GD19). The dose levels were 0 mg/kg, 50 mg/kg, 130 mg/kg and 350 mg/kg for the control (G1), low (G2), mid (G3) and high (G4) groups, respectively. The results of the experiment support the conclusion that the NOAEL [No Observed Adverse Effect Level] for the test item, Hordaphos MDGB for developmental toxicity is 350 mg/kg, the high dose, as there were no effects noted on fetal parameters at all the tested dose groups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 350 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
other: Ano-genital distance and crown rump length

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: cranium
external: ear
external: eye
external: face
external: limb
external: paw
external: tail
external: trunk
external: anogenital distance
external: anus
external: genital tubercle
external: large intestine
external: thorax
external: umbilicus
external: pelvic region
skeletal: skull
skeletal: skull, fontanelles
skeletal: skull sutures
skeletal: clavicle
skeletal: scapule
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: supernumerary rib
skeletal: vertebra
skeletal: pelvic girdle
skeletal: hindlimb
visceral/soft tissue: integumentary
visceral/soft tissue: gastrointestinal tract
visceral/soft tissue: hepatobiliary
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
visceral/soft tissue: heamatopoietic
visceral/soft tissue: immune system
visceral/soft tissue: musculoskeletal system
visceral/soft tissue: nervous system
visceral/soft tissue: central nervous system
visceral/soft tissue: peripheral nervous system
visceral/soft tissue: somatic nervous system
visceral/soft tissue: autonomic nervous system
visceral/soft tissue: endocrine system
visceral/soft tissue: respiratory system
visceral/soft tissue: male reproductive system
visceral/soft tissue: female reproductive system
visceral/soft tissue: eye
visceral/soft tissue: ear
other:

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
50 mg/kg bw/day
Treatment related:
no

Any other information on results incl. tables

SUMMARY OF THE STUDY

Parameters↓

Group

G1

G2

G3

G4

Dose
(mg/kg body weight)

0

50

130

350

PREGNANCY DATA

Initial Animals per Group

25

25

25

25

No. of animals survived

25

25

25

25

No. of animals aborted

0

0

0

0

No. of animals delivering early

0

0

0

0

No. of animals confirmed with pregnancy at necropsy

21

20

21

21

Pregnancy rate (%)

84.0

80.0

84.0

84.0

MATERNAL BODY WEIGHT

Maternal Body Weight (g) on Gestation Day 5

Mean

272.42

278.74

273.34

274.52

±SD

16.95

21.58

14.87

11.68

Maternal Body Weight (g) on Gestation Day 20

Mean

374.77

383.01

374.23

377.60

±SD

24.23

30.70

25.35

16.48

Body Weight Change (g) during Gestation Day 5 to 20

Mean

102.35

104.27

100.89

103.07

±SD

20.71

24.57

18.23

13.18

Corrected Body Weight (g)

Mean

27.51

30.75

27.34

28.40

±SD

15.45

13.97

13.54

11.74

UTERINE OBSERVATION AND MATERNAL DATA

Gravid Uterus Weight (g)

Mean

74.84

73.52

73.55

74.67

±SD

18.15

16.22

16.60

11.53

Corpora Lutea (no.)

Mean

13.24

12.85

13.05

12.76

±SD

3.06

3.48

2.50

2.30

Implantation per Dam (no.)

Mean

13.24

12.85

13.05

12.71

±SD

3.06

3.48

2.50

2.31

Male/female Sex Ratio (no.)

Mean

1.36

1.54

1.09

1.19

±SD

0.95

1.07

0.91

0.64

Litter Size (no.)

Mean

12.52

12.20

11.95

12.10

±SD

3.57

3.37

2.96

2.14

Live Fetuses per Dam (no.)

Mean

12.52

12.20

11.95

12.10

±SD

3.57

3.37

2.96

2.14

Percent of Live Fetuses (%)

Mean

100.00

100.00

100.00

100.00

±SD

0.00

0.00

0.00

0.00

Dead Fetuses per Dam (no.)

Mean

0.00

0.00

0.00

0.00

±SD

0.00

0.00

0.00

0.00

Percent of Dead Fetuses (%)

Mean

0.00

0.00

0.00

0.00

±SD

0.00

0.00

0.00

0.00

SUMMARY OF THE STUDY (Contd…).

Parameters↓

Group

G1

G2

G3

G4

Dose
(mg/kg body weight)

0

50

130

350

Early Resorptions per Dam (no.)

Mean

0.57

0.50

0.95

0.43

±SD

0.93

0.69

0.92

0.68

Percent of Early Resorptions (%)

Mean

5.71

3.89

8.14

3.25

±SD

10.08

5.52

8.36

5.17

Late Resorptions per Dam (no.)

Mean

0.14

0.15

0.14

0.19

±SD

0.48

0.37

0.36

0.40

Percent of Late Resorptions (%)

Mean

1.00

1.10

1.13

1.39

±SD

3.37

2.69

2.85

2.94

Pre-implantation Loss (%)

Mean

0.00

0.00

0.00

0.37

±SD

0.00

0.00

0.00

1.68

Post-implantation Loss (%)

Mean

6.70

4.99

9.28

4.64

±SD

10.54

6.69

8.41

5.09

MATERNAL THYROID ALONG WITH PARATHYROID WEIGHT

Thyroid along with parathyroid Weight (g)

Mean

0.0232

0.0220

0.0236

0.0235

±SD

0.0028

0.0024

0.0019

0.0031

MATERNAL THYROID HORMONE LEVELS

Serum T3 Levels (ng/mL)

Mean

2.510

2.572

2.370

2.358

±SD

0.253

0.250

0.183

0.182

Serum T4 Levels (ng/mL)

Mean

66.724

72.036

73.684

79.023*

±SD

8.319

9.857

11.366

12.286

Serum TSH Levels (µIU/mL))

Mean

4.351

4.142

3.767

4.439

±SD

2.948

2.997

1.247

2.174

LITTER DATA

Live Male Fetuses (no.)

Mean

6.43

6.60

5.48

6.24

±SD

2.42

1.90

2.18

2.17

Live Female Fetuses (no.)

Mean

6.10

5.60

6.48

5.86

±SD

2.70

2.64

2.62

1.65

Male Fetal Weight per Dam (g)

Mean

4.03

4.15

4.22*

4.14

±SD

0.25

0.33

0.21

0.27

Female Fetal Weight per Dam (g)

Mean

3.84

3.85

3.94

3.88

±SD

0.29

0.35

0.25

0.30

Male Fetal Crown Rump Length per Dam (mm)

Mean

39.020

39.768

39.899

39.774

±SD

1.45

2.00

1.39

2.75

Female Fetal Crown Rump Length per Dam (mm)

Mean

38.069

38.066

38.276

38.203

±SD

1.83

2.44

1.61

2.38

Mean Male Ano-genital Distance Ratio

Mean

3.02

2.98

3.02

3.06

±SD

0.18

0.19

0.16

0.16

Mean Female Ano-genital Distance Ratio

Mean

1.40

1.41

1.39

1.43

±SD

0.29

0.17

0.19

0.15

SUMMARY OF THE STUDY (Contd…).

Parameters↓

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

50

130

350

FETAL DATA

External Examination

Total No. of Fetuses for Gross External Examination

263

244

251

254

Number of fetuses observed with No Abnormality Detected

262

244

250

251

Number of fetuses observed with Malformations

0

0

0

0

Number of fetuses observed with Variations

1

0

1

3

Visceral Examination

Total No. of Fetuses for Visceral Examination

125

118

118

123

Number of fetuses observed with No Abnormality Detected

117

111

111

116

Number of fetuses observed with Malformations

0

1

1

0

Number of fetuses observed with Variations

8

6

6

7

Skeletal Examination

Total No. of Fetuses for Skeletal Examination

138

126

133

131

Number of fetuses observed with No Abnormality Detected

100

92

92

95

Number of fetuses observed with Malformations

18

16

17

16

Number of fetuses observed with Variations

20

18

24

20

TABLE 1.           SUMMARY OF CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

Group & Dose (mg/kg

 body weight)

No. of Animals

Clinical Signs of Toxicity
(No. of Animals revealed)

Mortality
(No. of Mortality /
No. of Animals dosed)

G1 & 0

25

N (25)

0/25

G2 & 50

25

N (25)

0/25

G3 & 130

25

N (25)

0/25

G4 & 350

25

N (25)

0/25

N: Normal

TABLE 2.           SUMMARY OF GESTATION BODY WEIGHT (g)RECORD

Group & Dose 
(mg/kg body weight)

Body Weight (g) on Gestation Day

0

3

5

8

11

14

17

19

20

G1 & 0

Mean

257.63

264.61

272.42

279.76

292.08

307.78

331.61

358.50

374.77

±SD

15.33

16.85

16.95

17.04

18.80

19.57

21.90

23.30

24.23

n

21

21

21

21

21

21

21

21

21

G2 & 50

Mean

264.13

272.42

278.74

286.78

300.98

318.11

345.75

369.81

383.01

±SD

16.51

18.48

21.58

19.70

21.61

22.68

28.87

31.76

30.70

n

20

20

20

20

20

20

20

20

20

G3 & 130

Mean

258.60

266.10

273.34

283.31

296.38

311.77

336.83

359.24

374.23

±SD

15.37

15.54

14.87

15.77

15.38

19.56

21.46

26.01

25.35

n

21

21

21

21

21

21

21

21

21

G4 & 350

Mean

258.30

266.35

274.52

282.45

293.93

310.29

337.05

361.51

377.60

±SD

10.06

9.96

11.68

14.11

14.47

15.82

15.29

17.06

16.48

n

21

21

21

21

21

21

21

21

21

SD: Standard Deviation; n: number of dams

Note: The data of non-pregnant females is not included in mean calculations and not subjected to statistical analysis.

TABLE 3.           SUMMARY OF PERCENT CHANGE IN GESTATION BODY WEIGHT (%) RECORD

Group & Dose
(mg/kg body weight)

Percent (%) Change in Body Weight during Gestation Day

0 to 3

3 to 5

5 to 8

8 to 11

11 to 14

14 to 17

17 to 19

19 to 20

0 to 20

5 to 20

G1 & 0

Mean

2.70

2.97

2.71

4.40

5.40

7.75

8.15

4.55

45.73

37.80

±SD

1.95

1.57

1.70

1.98

2.01

2.48

2.70

1.80

9.43

8.41

n

21

21

21

21

21

21

21

21

21

21

G2 & 50

Mean

3.13

2.27

2.96

4.95

5.74

8.64

6.97

3.63

45.09

37.69

±SD

2.14

1.81

1.91

1.83

3.45

3.47

2.53

1.88

8.90

9.63

n

20

20

20

20

20

20

20

20

20

20

G3 & 130

Mean

2.92

2.75

3.65

4.65

5.15

8.06

6.61

4.22

44.80

36.96

±SD

1.79

1.81

1.48

2.19

2.08

2.40

2.09

1.31

7.06

6.60

n

21

21

21

21

21

21

21

21

21

21

G4 & 350

Mean

3.15

3.06

2.87

4.08

5.58

8.67

7.28

4.48

46.26

37.64

±SD

2.15

1.94

1.71

1.53

1.94

2.18

2.52

1.64

5.42

5.41

n

21

21

21

21

21

21

21

21

21

21

SD: Standard Deviation; n: No. of dams

Note: The data of non-pregnant females is not included in mean calculations and not subjected to statistical analysis.

TABLE 4.        SUMMARY OFGRAVID UTERUS WEIGHT (g) AND MATERNAL BODYWEIGHT CHANGE CORRECTED FOR GRAVID UTERINE WEIGHT (g) RECORD

Group & Dose

 (mg/kg

body weight)

Gravid Uterus Weight

(g)

Body Weight Change (g) from GD 5 to 20

Corrected

Body weight

(g)

Corrected
Body weight

(%)

G1 & 0

Mean

74.84

102.35

27.51

10.30

±SD

18.15

20.71

15.45

5.99

n

21

21

21

21

G2 & 50

Mean

73.52

104.27

30.75

11.18

±SD

16.22

24.57

13.97

5.27

n

20

20

20

20

G3 & 130

Mean

73.55

100.89

27.34

10.03

±SD

16.60

18.23

13.54

4.92

n

21

21

21

21

G4 & 350

Mean

74.67

103.07

28.40

10.42

±SD

11.53

13.18

11.74

4.56

n

21

21

21

21

SD: Standard Deviation; n: No. of dams

Note: The data of non-pregnant females is not included in mean calculations and not subjected to statistical analysis.

TABLE 5.           SUMMARY OF AVERAGE GESTATION FEED CONSUMPTION (g/animal/day) RECORD

Group & Dose (mg/kg

 body weight)

Feed Consumption (g/animal/day) during Gestation Day

0 to 3

3 to 5

5 to 8

8 to 11

11 to 14

14 to 17

17 to 19

19 to 20

0 to 20

5 to 20

G1 & 0

Mean

18.68

18.89

19.88

21.75

22.38

24.66

25.23

25.60

22.13

23.25

±SD

2.84

3.74

3.84

3.15

3.93

4.18

4.96

8.28

1.94

2.37

n

21

21

21

21

21

21

21

21

21

21

G2 & 50

Mean

18.50

19.69

19.94

21.97

22.57

24.02

29.20*

35.08*

23.87

25.46

±SD

2.31

2.70

2.97

3.84

4.22

4.14

4.47

15.78

2.62

3.13

n

20

20

20

20

20

20

20

20

20

20

G3 & 130

Mean

17.74

19.94

20.73

21.80

23.01

24.77

27.92

31.07

23.37

24.88

±SD

2.32

2.72

3.28

3.32

3.08

4.05

3.28

10.98

1.62

1.88

n

21

21

21

21

21

21

21

21

21

21

G4 & 350

Mean

18.90

20.66*

20.22

21.55

22.68

23.94

28.69

32.07

23.59

24.86

±SD

2.71

3.25

3.72

3.96

3.85

3.62

3.66

13.82

2.67

2.97

n

21

21

21

21

21

21

21

21

21

21

SD: Standard Deviation; n: Number of dams;*. The mean difference is significant at the 0.05 level.

Note: The data of non-pregnant females is not included in mean calculations and not subjected to statistical analysis.

TABLE 6.           SUMMARY OF UTERI OBSERVATION RECORD       

Group & Dose

(mg/kg

body weight)

No. of

Corpora lutea

No. of Implantations

Litter

Size

No. of

Live Fetuses

No. of Male

Live Fetuses

No. of Female

Live

Fetuses

No. of

Dead Fetuses

No. of
Early Resorptions

No. of
Late Resorptions

G1 & 0

Mean

13.24

13.24

12.52

12.52

6.43

6.10

0.00

0.57

0.14

±SD

3.06

3.06

3.57

3.57

2.42

2.70

0.00

0.93

0.48

n

21

21

21

21

21

21

21

21

21

G2 & 50

Mean

12.85

12.85

12.20

12.20

6.60

5.60

0.00

0.50

0.15

±SD

3.48

3.48

3.37

3.37

1.90

2.64

0.00

0.69

0.37

n

20

20

20

20

20

20

20

20

20

G3 & 130

Mean

13.05

13.05

11.95

11.95

5.48

6.48

0.00

0.95

0.14

±SD

2.50

2.50

2.96

2.96

2.18

2.62

0.00

0.92

0.36

n

21

21

21

21

21

21

21

21

21

G4 & 350

Mean

12.76

12.71

12.10

12.10

6.24

5.86

0.00

0.43

0.19

±SD

2.30

2.31

2.14

2.14

2.17

1.65

0.00

0.68

0.40

n

21

21

21

21

21

21

21

21

21

SD: Standard Deviation; n: Number of dams;*. The mean difference is significant at the 0.05 level.

Note: The data of non-pregnant females is not included in mean calculations and not subjected to statistical analysis.

TABLE 7.           SUMMARY OF MATERNAL DATA RECORD

Group & Dose (mg/kg

body weight)

 

Pre-Implantation Loss

(%)

Post-Implantation Loss

(%)

Percent of Dead Fetus (%)

Percent of Early Resorptions (%)

Percent of Late Resorptions(%)

Male / Female

 Sex

ratio

Male Fetuses

(%)

Female Fetuses (%)

Percent of

Live

Fetuses

(%)

G1 & 0

Mean

0.00

6.70

0.00

5.71

1.00

1.36

52.15

47.85

100.00

±SD

0.00

10.54

0.00

10.08

3.37

0.95

15.35

15.35

0.00

n

21

21

21

21

21

21

21

21

21

G2 & 50

Mean

0.00

4.99

0.00

3.89

1.10

1.54

55.71

44.29

100.00

±SD

0.00

6.69

0.00

5.52

2.69

1.07

13.67

13.67

0.00

n

20

20

20

20

20

20

20

20

20

G3 & 130

Mean

0.00

9.28

0.00

8.14

1.13

1.09

45.81

54.19

100.00

±SD

0.00

8.41

0.00

8.36

2.85

0.91

16.45

16.45

0.00

n

21

21

21

21

21

21

21

21

21

G4 & 350

Mean

0.37

4.64

0.00

3.25

1.39

1.19

51.52

48.72

100.00

±SD

1.68

5.09

0.00

5.17

2.94

0.64

15.33

11.66

0.00

n

21

21

21

21

21

21

21

21

21

SD: Standard Deviation; n: Number of dams

Sex Ratio Calculation:

Male/Female Sex Ratio = Total Number of Live Male Fetuses / Total Number of Live Female Fetuses

Percent of Live Fetuses (%):

Percent of Male Fetuses (%) = Total Number of live male fetuses / Total number of live fetuses x 100

Percent of Female Fetuses (%) = Total Number of live female fetuses / Total number of live fetuses x 100

TABLE 8.           SUMMARY OFABSOLUTE ORGAN WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD

Group & Dose
(mg/kg body weight)

 

Absolute Weight of Thyroid along with parathyroid#

Terminal Body Weight (g)

Relative Thyroid along with parathyroid Weight (%)

G1 & 0

Mean

0.0232

374.77

0.0062

±SD

0.0028

24.23

0.0008

n

21

21

21

G2 & 50

Mean

0.0220

383.01

0.0058

±SD

0.0024

30.70

0.0008

n

20

20

20

G3 & 130

Mean

0.0236

374.23

0.0063

±SD

0.0019

25.35

0.0006

n

21

21

21

G4 & 350

Mean

0.0235

377.60

0.0062

±SD

0.0031

16.48

0.0009

n

21

21

21

SD: Standard Deviation; n: Number of dams; #: Weighed post fixation


TABLE 9.           SUMMARY OF SERUM TRIIODOTHYRONINE (T3) LEVELS (ng/mL) RECORD

Group & Dose
(mg/kg body weight)

 

Serum T3 Levels (ng/mL)

G1 & 0

Mean

2.510

±SD

0.253

n

21

G2 & 50

Mean

2.572

±SD

0.250

n

20

G3 & 130

Mean

2.370

±SD

0.183

n

21

G4 & 350

Mean

2.358

±SD

0.182

n

21

SD: Standard Deviation; n: Number of dams

TABLE 10.           SUMMARY OF SERUM THYROXINE (T4) LEVELS (ng/mL) RECORD

Group & Dose
(mg/kg body weight)

 

Serum T4 Levels (ng/mL)

G1 & 0

Mean

66.724

±SD

8.319

n

21

G2 & 50

Mean

72.036

±SD

9.857

n

20

G3 & 130

Mean

73.684

±SD

11.366

n

21

G4 & 350

Mean

79.023*

±SD

12.286

n

21

SD: Standard Deviation; n: Number of dams

*. The mean difference is significant at the 0.05 level.

TABLE 11.           SUMMARY OF SERUM THYROID STIMULATING HORMONE (TSH) LEVELS (µIU/mL) RECORD

Group & Dose
(mg/kg body weight)

 

Serum TSH Levels (µIU/mL)

G1 & 0

Mean

4.351

±SD

2.948

n

21

G2 & 50

Mean

4.142

±SD

2.997

n

20

G3 & 130

Mean

3.767

±SD

1.247

n

21

G4 & 350

Mean

4.439

±SD

2.174

n

21

SD: Standard Deviation; n: Number of dams

TABLE 12.           SUMMARY OF MEAN FETAL WEIGHT (g) RECORD

Group & Dose
(mg/kg body weight)

Mean Fetus Weight (g)

 

Mean Male Fetal Weight (g)

Mean Female Fetal Weight (g)

G1 & 0

Mean

4.03

3.84

±SD

0.25

0.29

n

21

21

G2 & 50

Mean

4.15

3.85

±SD

0.33

0.35

n

20

20

G3 & 130

Mean

4.22*

3.94

±SD

0.21

0.25

n

21

21

G4 & 350

Mean

4.14

3.88

±SD

0.27

0.30

n

21

21

SD: Standard Deviation; n: Number of dams

*. The mean difference is significant at the 0.05 level.

TABLE 13.           SUMMARY OF MEAN FETAL CROWN RUMP LENGTH (mm) RECORD

Group & Dose
(mg/kg body weight)

Mean Fetal Crown Rump Length (mm)

Mean Male Fetal Crown Rump Length

(mm)

Mean Female Fetal Crown Rump Length (mm)

G1 & 0

Mean

39.020

38.069

±SD

1.45

1.83

n

21

21

G2 & 50

Mean

39.768

38.066

±SD

2.00

2.44

n

20

20

G3 & 130

Mean

39.899

38.276

±SD

1.39

1.61

n

21

21

G4 & 350

Mean

39.774

38.203

±SD

2.75

2.38

n

21

21

SD: Standard Deviation; n: Number of dams

TABLE 14.           SUMMARY OF MEAN FETAL ANOGENITAL DISTANCE (AGD) RATIO RECORD

Group & Dose
 (mg/kg body weight)

Mean Fetal AGD Ratio

Mean Male Fetal AGD Ratio

Mean Female Fetal AGD Ratio

G1 & 0

Mean

3.02

1.40

±SD

0.18

0.29

n

21

21

G2 & 50

Mean

2.98

1.41

±SD

0.19

0.17

n

20

20

G3 & 130

Mean

3.02

1.39

±SD

0.16

0.19

n

21

21

G4 & 350

Mean

3.06

1.43

±SD

0.16

0.15

n

21

21

SD: Standard Deviation; n: Number of dams

TABLE 15.           SUMMARY OF EXTERNAL EXAMINATION OF FETUSES

Parameters

Group

G1

G2

G3

G4

Dose

(mg/kg body weight)

0

50

130

350

Number of Dams

21

20

21

21

Number of fetuses for External Examination

263

244

251

254

Number of fetuses observed with No Abnormality Detected

262

244

250

251

Number of fetuses observed with Malformations

0

0

0

0

Number of fetuses observed with Variations

1

0

1

3

VARIATIONS

Haemorrhagic spot - Hind limb unilateral

No.

1 (1)

0 (0)

0 (0)

1 (1)

%

0.38

0.00

0.00

0.39

Haemorrhagic spot - Ventral side of trunk

No.

0 (0)

0 (0)

1 (1)

0 (0)

%

0.00

0.00

0.40

0.00

Haemorrhagic spot – head region

No.

0 (0)

0 (0)

0 (0)

1 (1)

%

0.00

0.00

0.00

0.39

Haemorrhagic spot -
Fore limb unilateral

No.

0 (0)

0 (0)

0 (0)

1 (1)

%

0.00

0.00

0.00

0.39

Number of dams in parentheses

 

 

No. of fetuses with abnormality

% of Abnormality

:

------------------------------------------ X 100

 

 

 Total No. of fetuses examined

TABLE 16. SUMMARY RECORD OF VISCERAL EXAMINATION OF FETUSES

Parameters

Group

G1

G2

G3

G4

Dose

(mg/kg body weight)

0

50

130

350

Number of Dams

21

20

21

21

Number of fetuses for visceral examination

125

118

118

123

Number of fetuses observed with No Abnormality Detected

117

111

111

116

Number of fetuses observed with Malformations

0

1

1

0

Number of fetuses observed with Variations

8

6

6

7

MALFORMATIONS

 

Liver Lobes Fused

No.

0 (0)

0 (0)

1 (1)

0 (0)

%

0.00

0.00

0.85

0.00

Adrenals Smaller (unilateral)

No.

0 (0)

1 (1)

0 (0)

0 (0)

%

0.00

0.85

0.00

0.00

VARIATIONS

Kidney - Renal pelvis Dilation – Unilateral (right)

No.

2(2)

1(1)

1(1)

2(2)

%

1.60

0.85

0.85

1.63

Kidney – Pale colored

No.

0 (0)

0 (0)

0 (0)

1(1)

%

0.00

0.00

0.00

0.81

Kidney – Malpositioned

No.

4(4)

4(3)

4(3)

3(3)

%

3.20

3.39

3.39

2.44

Microopthalmia

No.

2(2)

1(1)

0 (0)

0 (0)

%

1.60

0.85

0.00

0.00

Number of dams in parentheses

 

 

No. of fetuses with abnormality

% of Abnormality

:

-----------------------------------------  X 100

 

 

 Total No. of fetuses examined

TABLE 17.           SUMMARY RECORD OF SKELETAL EXAMINATION OF FETUSES

Parameters

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

50

130

350

Number of Dams

21

20

21

21

Number of fetuses for skeletal examination

138

126

133

131

Number of fetuses observed with No Abnormality Detected

100

92

92

95

Number of fetuses observed with Malformations

18

16

17

16

MALFORMATIONS

SKULL

Skull – Interparietal - Misshapen

No.

0(0)

0(0)

1(1)

1(1)

%

0.00

0.00

0.75

0.76

STERNUM

Sternum No. 5 - Absent

No.

3(3)

4(2)

3(3)

5(5)

%

2.17

3.17

2.26

3.82

Sternum No. 5 & 6 - Absent

No.

3(3)

0(0)

0(0)

0(0)

%

2.17

0.00

0.00

0.00

RIBS

Rib No.13 short (unilateral)

No.

1(1)

0(0)

0(0)

1(1)

%

0.72

0.00

0.00

0.76

THORACIC VERTEBRAE

Centra No.10 Split

No.

0(0)

0(0)

0(0)

1(1)

%

0.00

0.00

0.00

0.76

Centra No.11 Split

No.

0(0)

1(1)

0(0)

2(2)

%

0.00

0.79

0.00

1.53

Centra No.12 Split

No.

1(1)

1(1)

2(2)

1(1)

%

0.72

0.79

1.50

0.76

Centra No.13 Split

No.

1(1)

3(2)

0(0)

0(0)

%

0.72

2.38

0.00

0.00

Centra No.11 & 12 Split

No.

0(0)

0(0)

1(1)

0(0)

%

0.00

0.00

0.75

0.00

LUMBAR VERTEBRAE

Centra No. 1 & 2

No.

1(1)

0(0)

0(0)

0(0)

%

0.72

0.00

0.00

0.00

FORE LIMB

Metacarpal No.5 Absent (Bilateral)

No.

2(2)

2(2)

1(1)

1(1)

%

1.45

1.59

0.75

0.76

Proximal phalanges No. 3 and 4 - Absent (Bilateral)

No.

6(4)

5(5)

9(9)

4(4)

%

4.35

3.97

6.77

3.05

Number of dams in parentheses

 

 

No. of fetuses with abnormality

% of Abnormality

:

------------------------------------------ X 100

 

 

 Total No. of fetuses examined

TABLE 17 (Contd...). SUMMARY OF SKELETAL EXAMINATION OF FETUSES


Parameters

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

50

130

350

Number

of Dams

21

20

21

21

Number of fetuses for skeletal examination

138

126

133

131

Number of fetuses observed with
No Abnormality Detected

100

92

92

95

Number of fetuses observed withVariations

20

18

24

20

VARIATIONS

SKULL

Intra parietal bones - Poor Ossification

No.

1(1)

0(0)

0(0)

0(0)

%

0.72

0.00

0.00

0.00

Intra parietal bones - Incomplete Ossification

No.

0(0)

0(0)

1(1)

0(0)

%

0.00

0.00

0.75

0.00

Supraoccipital bones - Poor Ossification

No.

0(0)

1(1)

0(0)

0(0)

%

0.00

0.79

0.00

0.00

Intra parietal bones & Supraoccipital bones - Incomplete Ossification

No.

0(0)

0(0)

0(0)

1(1)

%

0.00

0.00

0.00

0.76

STERNUM

Sternum No. 5 - Poor Ossification

No.

1(1)

0(0)

0(0)

0(0)

%

0.72

0.00

0.00

0.00

Sternum No. 5 - Incomplete Ossification

No.

1(1)

1(1)

4(3)

8(8)

%

0.72

0.79

3.01

6.11

Sternum No. 5 - Bipartite Ossification

No.

0(0)

0(0)

2(2)

0(0)

%

0.00

0.00

1.50

0.00

Sternum No. 6 - Poor Ossification

No.

4(3)

6(4)

2(2)

0(0)

%

2.90

4.76

1.50

0.00

Sternum No. 6 - Incomplete Ossification

No.

0(0)

0(0)

4(4)

0(0)

%

0.00

0.00

3.00

0.00

Sternum No. 5 and 6 - Poor Ossification

No.

1(1)

1(1)

3(3)

0(0)

%

0.72

0.79

2.26

0.00

Number of dams in parentheses

 

 

No. of fetuses with abnormality

% of Abnormality

:

------------------------------------------ X 100

 

 

 Total No. of fetuses examined

TABLE 17 (Contd…). SUMMARY OF SKELETAL EXAMINATION OF FETUSES


Parameters

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

50

130

350

Number of Dams

21

20

21

21

Number of fetuses for skeletal examination

138

126

133

131

Number of fetuses observed with
No Abnormality Detected

100

92

92

95

Number of fetuses observed withVariations

20

18

24

20

VARIATIONS

Sternum No. 5 and 6 - Incomplete Ossification

No.

0(0)

1(1)

2(2)

0(0)

%

0.00

0.79

1.50

0.00

Sternum No. 4 - Misshapened

No.

0(0)

3(3)

1(1)

0(0)

%

0.00

2.38

0.75

0.00

Sternum No. 5 - Misshapened

No.

0(0)

2(2)

2(2)

1(1)

%

0.00

1.59

1.50

0.76

RIBS

Rib No. 13 - Wavy

No.

2(1)

0(0)

0(0)

0(0)

%

1.45

0.00

0.00

0.00

Rib No.12 and 13 – Wavy (bilateral)

No.

1(1)

0(0)

0(0)

0(0)

%

0.72

0.00

0.00

0.00

Rib No.11,12 and 13 – Wavy (bilateral)

No.

0(0)

0(0)

1(1)

0(0)

%

0.00

0.00

0.75

0.00

Rib No.11 and 12 – Wavy (unilateral)

No.

0(0)

0(0)

0(0)

1(1)

%

0.00

0.00

0.00

0.76

Rudimentary Rib

No.

0(0)

0(0)

0(0)

2(1)

%

0.00

0.00

0.00

1.53

Rib No.10 – Wavy (unilateral)

No.

0(0)

0(0)

0(0)

1(1)

%

0.00

0.00

0.00

0.76

THORACIC VERTEBRAE

Centrum No. 10 – Dumbbell shaped

No.

0(0)

0(0)

0(0)

1(1)

%

0.00

0.00

0.00

0.76

Centrum No. 11 – Assymetrical bilobed

No.

1(1)

0(0)

0(0)

0(0)

%

0.72

0.00

0.00

0.00

Number of dams in parentheses

 

 

No. of fetuses with abnormality

% of Abnormality

:

------------------------------------------ X 100

 

 

 Total No. of fetuses examined

TABLE 17 (Contd...). SUMMARY OF SKELETAL EXAMINATION OF FETUSES

Parameters

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

50

130

350

Number of Dams

21

20

21

21

Number of fetuses for skeletal examination

138

126

133

131

Number of fetuses observed with

100

92

92

95

No Abnormality Detected

Number of fetuses observed with Variations

20

18

24

20

VARIATIONS

Centrum No. 11 - Dumbbell shaped

No.

1(1)

0(0)

0(0)

0(0)

%

0.72

0

0

0

Centrum No. 12 - Dumbbell shaped

No.

0(0)

2(2)

2(2)

0(0)

%

0

1.59

1.5

0

Centrum No. 11 & 13 - Dumbbell shaped

No.

0(0)

0(0)

0(0)

1(1)

%

0

0

0

0.76

Centrum No. 10, 11, 12 & 13 - Dumbbell shaped

No.

0(0)

0(0)

0(0)

1(1)

%

0

0

0

0.76

Centrum No. 10 & 12 - Dumbbell shaped

No.

0(0)

0(0)

0(0)

1(1)

%

0

0

0

0.76

LUMBAR VERTEBRAE

Arch No. 4 & 5 - Incomplete Ossification

No.

0(0)

1 (1)

0(0)

0(0)

%

0

0.79

0

0

Arch No. 5 - Incomplete Ossification

No.

1 (1)

0(0)

0(0)

0(0)

%

0.79

0

0

0

Arch No. 1: Extra ossification site (Bilateral)

No.

1 (1)

0(0)

0(0)

0(0)

%

0.79

0

0

0

Arch No.2: Extra Ossification Site (Unilateral)

No.

0(0)

0(0)

0(0)

1(1)

%

0

0

0

0.76

SACRAL VERTEBRAE

Arch No.4 : Extra Ossification Site – (Bilateral)

No.

2(2)

0(0)

0(0)

0(0)

%

1.45

0

0

0

Number of dams in parentheses

 

 

No. of fetuses with abnormality

% of Abnormality

:

------------------------------------------ X 100

 

 

 Total No. of fetuses examined

TABLE 17 (Contd...). SUMMARY OF SKELETAL EXAMINATION OF FETUSES

 

 

Parameters

 

 

 

Group

G1

G2

G3

G4

Dose (mg/kg body weight)

0

50

130

350

Number of Dams

21

20

21

21

Number of fetuses for skeletal examination

138

126

133

131

Number of fetuses observed with No Abnormality Detected

100

92

92

95

Number of fetuses observed with Variations

20

18

24

20

VARIATIONS

CAUDAL VERTEBRAE

Arch No.2: Incomplete Ossification

No.

2 (2)

0 (0)

0 (0)

0 (0)

%

1.45

0

0

0

Arch No.1 & 2: Incomplete Ossification

No.

0 (0)

0 (0)

0 (0)

1 (1)

%

0

0

0

0.76

FORE LIMB

Proximal Phalanx No. 3 & 4 – Incomplete Ossification

No.

1 (1)

0 (0)

0 (0)

0 (0)

%

0.72

0

0

0

Number of dams in parentheses

 

 

No. of fetuses with abnormality

% of Abnormality

:

------------------------------------------ X 100

 

 

 Total No. of fetuses examined

Applicant's summary and conclusion

Conclusions:
The Test item, Hordaphos MDGB was administered via oral route at a dose volume of 10 mL/kg body weight to mated female rats from gestation day 5 (GD5) through gestation day 19 (GD19). The dose levels were 0 mg/kg, 50 mg/kg, 130 mg/kg and 350 mg/kg for the control (G1), low (G2), mid (G3) and high (G4) groups, respectively. The results of the experiment support the conclusion that the NOAEL [No Observed Adverse Effect Level] for the test item, Hordaphos MDGB for maternal and developmental toxicity is 350 mg/kg, the high dose, as there were no effects noted on maternal parameters as well as fetal parameters at all the tested dose groups.
Executive summary:

The objective of this study was to assess the effects of prenatal exposure of Hordaphos MDGB on pregnant rats and on the developing fetus, including assessment of maternal effects as well as death, structural abnormalities, or altered growth in the fetus.

The test item,Hordaphos MDGB, was administered by the oral route at 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19. For dams the end points of assessment were maternal death, maternal body weight and clinical signs of maternal toxicity. In the fetuses the end points of assessment were fetal death, structural variations and malformations or altered growth.

A total of 100 mated female Sprague Dawley rats were allocated to four groups. Each group consisted of 25 mated presumed-pregnant female Sprague Dawley rats (Dams).Hordaphos MDGBwas administered by the oral route in graduated doses at three dose levels:

Group G1: 0 mg/kg (Vehicle Control)

Group G2: 50 mg/kg (Low Dose)

Group G3: 130 mg/kg (Mid Dose)

Group G4: 350 mg/kg (High Dose)

All the animals were observed for clinical signs of toxicity once daily, mortalities twice daily during the experimental period, the body weight and feed consumption were recorded on 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section). On the day of caesarean section, all the dams were observed for gravid uterus weight, uteri observations (no. of corpora lutea, no. of implantations, no. of resorptions and no. of live and foetuses). The sex ratio, pre/post implantation loss per dam, percentage of male and female foetuses was calculated based on the uteri observations. The weight of thyroid along with the parathyroid was recorded post-fixation for all the group animals.

All the animals were euthanized on gestation day 20 by exposing to CO2and subjected to detailed gross pathological examination. The histopathology of thyroid along with parathyroid conducted for all the tested dose group animals.

A total number of 21, 20, 21 and 21 mated females were confirmed pregnant across groups yielding pregnancy rates of 84%, 80%, 84% and 84% at the time of caesarean section for the G1 (0 mg/kg), G2 (50 mg/kg), G3 (130 mg/kg) and G4 (350 mg/kg), respectively.

Dose formulation analysis for dose concentration verification was performed during first week and last week of the treatment. The analysis results were within the limits and formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is <10%.

No clinical signs of toxicity and no mortalities were noted during the experimental period at all the tested dose group animals. No treatment related effects were observed on gestational body weight, body weight gain and corrected body weight across the dose groups when compared with controls. There were no treatment related differences in feed consumption across the dose groups during gestation period.

No treatment related changes were noted in gravid uterus weight, uteri observations (no. of corpora lutea, no. of implantations, no. of resorptions and no of live and foetuses), sex ratio, pre/post implantation loss per dam, percentage of male and female foetuses and weight of thyroid along with the parathyroid wat all the tested dose groups.

No gross pathological findings were reported in all the tested dose groups during conductance of the necropsy.

The histopathology of thyroid along with parathyroid conducted for all the dose group dams did not reveal any treatment related histopathological findings during conduct of the histopathological examination. Presence ofhemorrhage in thyroid of one animal from G1 group, MNC infiltration in thyroid of one animal from G2, ultimobranchial cyst in one animal from G1 and 3 animals each from G2 and G3, ectopic thymus in        2 animals from G3 and 1 animal from G4 were considered as background lesions.

For the maternal toxicity assessment, the results of the experiment support that there were no treatment related effects on maternal body weight, feed consumption, for the parameters which assess gestational integrity end points, e.g. mean gravid uterus weight, number of corpora lutea, number of implantations, litter size, number of live fetuses, number of dead fetuses, mean number of early resorptions per dam and percentage of implantation losses per dam across all the groups.

The gravid uterus was collected by hysterectomy and fetuses were removed by caesarean section. The fetuses were observed for fetal weights, crown rump length, ano-genital distance, observation of reproductive tract, comparison of external fetal sex with internal gonads. Examination of fetuses for external, soft tissue, and skeletal anomalies was performed.

No treatment related effects on fetal weight, crown-rump length, fetal ano-genital distance ratio and comparison of external fetal sex with internal gonads were noted at all the tested dose groups.

No treatment related gross external abnormalities were noted within any group after external examination of fetuses. The noted findings of haemorrhagic spots are common findings for fetuses of this species and strain.

No treatment-related abnormalities were observed during visceral examinations of fetuses at any dose. The noted findings of renal pelvic dilation, pale colored kidneys, malposition kidneys, fused liver lobes and smaller adrenal size unilateral are anatomical variations which are common findings for fetuses of this species and strain. The observations were not dose dependent, nor were the severity of the anomaly increased with dose. This result supports the conclusion that the findings are incidental and that the test item did not produce an adverse effect on the soft tissues during fetal development.

No treatment-related abnormalities were observed during skeletal examinations of fetuses at any dose. Misshapen interparietal bones of skull, absence of sternum no. 5 and 6, short ribs, Split thoracic/lumbar vertebral centrum; absence of proximal phalanges no. 3 and 4 and metacarpal bone no. 5 were recorded as malformations. Poor/incomplete ossification ofIntra parietal/supraoccipital bones of skull,Poor/incomplete ossification and misshapen of sternum no. 5 and 6, wavy ribs, dumbbell shaped thoracic vertebrae, extra ossification site/incomplete ossification of lumbar/sacral vertebrae centrum/arch, incomplete ossification of caudal vertebrae centrum/arch and incomplete ossification of proximal phalanges no. 3 and 4 were recorded as variations. The noted anomalies are common findings for fetuses of this species and strain. They did not occur in a dose-dependent pattern, nor was the severity of the anomalies increased with dose. This result supports the conclusion that the findings are incidental and that the test item, did not produce an adverse effect on the skeletal tissues during fetal development. 

For the developmental toxicity assessment, the results of the experiment support that there were no treatment related effects on the parameters which assess fetal development, i.e. fetal weight, crown-rump length, fetal ano-genital distance ratio and incidences of external, soft tissue or skeletal anomalies.