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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:
2020-11-08 to 2021-06-01 (draft final study report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 2001
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed
EC Number:
941-809-7
IUPAC Name:
Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed
Test material form:
liquid

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, United States
- Age at study initiation: 11–13 weeks
- Weight at study initiation: between 219 and 301 g
- Housing: Single/Individual. Solid-bottom cages containing appropriate bedding material (Bed-O-Cobs or other suitable material). Housing set-up was as specified in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). For enrichment, animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.
- Diet: ad libitum. PMI Nutrition International, LLC Certified Rodent LabDiet® 5002. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: ad libitum. Municipal tap water, treated by reverse osmosis and ultraviolet irradiation. Automatic watering system. Water bottles were provided, if required. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: yes, duration not reported
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 25
- Humidity (%): 30 to 70
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: Completion of In-life: 2020-11-25

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion. Frequency of preparation: approximately weekly. Dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dose formulations were stirred continuously at room temperature during dosing.
VEHICLE
- Concentration in vehicle: 0, 14.5, 43.5, 145 mg/mL
- Amount of vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose analysis results were verified prior to dose/diet administration at each sampling interval. If results were deemed unacceptable, the formulations were prepared again and analysed. All samples to be analyzed were transferred to the Analytical Chemistry Department at the Testing Facility for same day analysis, where possible or stored for analysis within known formulation stability period.
Analytical Method
Analyses described below were performed by high performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) using a validated analytical procedure.
Concentration Analysis
Storage Conditions: Temperature set to maintain a target of 5°C.
Acceptance Criteria: Mean sample concentration within 100% ± 10% of theoretical concentration. Individual sample concentration of ± 15%.
Stability Analysis
Test substance formulations have been previously shown to be stable over the range of concentrations used on this study for at least 8 days (refrigerated). Therefore, stability of test substance formulations was not assessed on this study.
Details on mating procedure:
Time-mated female Crl:CD(SD) rats were received from Charles River Laboratories, Inc., Raleigh, NC on Gestation Day 2, 3, or 4.
Duration of treatment / exposure:
The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 6 through 20.
Frequency of treatment:
Daily, at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
72.5 mg/kg bw/day (nominal)
Dose / conc.:
217.5 mg/kg bw/day (nominal)
Dose / conc.:
725 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on information provided by the Sponsor based on preceding GLP-compliant studies in Sprague Dawley CD rats, including a 4-week oral toxicity study with mortality at 1000 mg/kg. The NOAEL was set at 500 mg/kg. For further information on dose selection rationale, please see section "any other information on materials and methods".
The dose levels chosen in this study were expected to produce graded responses to the test substance. It was anticipated that the high-dose level would show drug-specific effects but not produce an incidence of fatalities that would prevent a meaningful evaluation. The lower dose levels were selected at intervals that were predicted to be narrow enough to reveal any dose related trends.

- Time of day for (rat) dam blood sampling: prior to noon on each day of collection, around the same time each day, and within a 2-hour window on each collection day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (morning and afternoon), beginning upon arrival through termination/release.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning with the day of animal arrival and continuing through (and including) the day of euthanasia.
BODY WEIGHT: Yes
- Time schedule for examinations: Gestation Days 0 (by supplier) and 5–21 (daily).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined: thyroid gland, kidney, liver, muscle, diaphragm, placenta, spleen, ovary, uterus
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
Blood sampling:
- Plasma: No
- Serum: Yes
- Volume collected: approximately 150 µL
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
- Anogenital distance of all live rodent pups: yes
Statistics:
Yes. For details please refer to the document in section "Attached background material"
Indices:
Pre-Implantation Loss = (No. of corpora lutea – no. of implants x 100) / No. of corpora lutea.

Post-Implantation Loss = (No. of implants – no. of live fetuses x 100) / No. of implants.

Sex Ratio (% males) = (No. male fetuses x 100) / Total no. of fetuses.

Litter % of Fetuses with Abnormalities = (No. of fetuses in litter with a given finding x 100) / No. of fetuses in litter examined.
Historical control data:
The test facility has provided historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat.
- Embryo-Fetal Developmental Historical Control Data
- Modal Distribution of Fetal Body Weights
- Historical Control Summary of Clinical Pathology Values

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related increased incidences of abnormal breathing sounds were observed in 8 females at approximately 2 hours post-dose in the 725 mg/kg/day group and persisted to the daily examinations in 3 females only during Gestation Days 7–21. These findings were considered non-adverse due to the animals being in otherwise general good health throughout the treatment period. Other clinical observations noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean maternal body weights, body weight gains, adjusted body weights, adjusted body weight gains in the 72.5, 217.5, and 725 mg/kg/day groups were unaffected by test substance administration. Any statistically significant differences from the control group were transient, did not occur in a dose-responsive manner, and/or did not impact mean absolute body weights.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 725 mg/kg/day group, test substance-related statistically significantly lower mean food consumption was noted during Gestation Days 6–9 compared to the control group. Mean food consumption in this group was comparable to the control group for the remainder of the dosing period (Gestation Days 9–21) and when the entire dosing period (Gestation Days 6–21) was evaluated. There were no corresponding effects on mean body weights or body weight gains, and therefore, the effects on mean food consumption in the 750 mg/kg/day group were considered non-adverse.
Mean food consumption in the 72.5 and 217.5 mg/kg/day were unaffected by test substance administration throughout the study. Differences from the control group were slight and not statistically significant.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were test substance-related effects on thyroid hormone values (TSH, T3, and T4) at any dose level. Differences from the control group were not statistically significant, observed in a manner that was not dose-related, and were within the ranges of the Historical Control Data.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Gravid uterine weights in the 72.5, 217.5, and 725 mg/kg/day groups were unaffected by test substance administration.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
725 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
behaviour (functional findings)
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
endocrine findings
food consumption and compound intake
gross pathology
histopathology: neoplastic
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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related external malformations were noted in fetuses in this study. A single fetus in the 217.5 mg/kg/day group (No. 3518-09) was observed with proboscis and absent eye bulges, mouth, and lower jaw. These findings were noted in a single mid-dose fetus, and therefore were not considered test substance related. No other external malformations or external developmental variations were observed in fetuses on this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were noted for 0(0), 1(1), 2(2), and 6(3) fetuses (litters) in the control, 72.5, 217.5, and 725 mg/kg/day groups, respectively; incidences in the test substance-treated groups were not statistically significantly higher than the concurrent control group. In the 725 mg/kg/day group, different types of vertebral malformations were noted in 3 individual litters: Fetus Nos. 4504 05, 4504-16, and 4504-17 were observed with absent lumbar vertebra, Fetus Nos. 4513-01 was observed with an absent rib and thoracic hemivertebra; this fetus was also noted with a lower body weight (3.87 g) compared to the group mean value (5.92 g), and Fetus Nos. 4521-04 and 4521-10 were observed with supernumerary lumbar vertebra. Because the skeletal malformations in the 725 mg/kg/day group were of specific types to fetuses in individual litters, they were considered familial and not related to the test substance administration. In the 217.5 mg/kg/day group, Fetus No. 3503 10 was observed with fused ribs, cervical centrum, cervical arches, and thoracic arches and absent cervical centrum and Fetus No. 3518 09 was noted with multiple malformations in the skull (absent mandible and jugal bones, misshapen nasal, premaxilla, maxilla, squamosal, frontal, parietal, and interparietal bones, small premaxilla, nasal, maxilla, squamosal, tympanic annulus, frontal, parietal, and interparietal bones, large supraoccipital bone, and unossified parietal bone), corresponding to the external malformations noted in this fetus. In the 72.5 mg/kg/day group, Fetus No. 2501-03 was observed with an absent lumbar vertebra. Skeletal malformations in the 50 and 150 mg/kg/day groups were limited to single fetuses, and therefore were not considered test substance related.
Higher incidences (statistically significant) of skeletal developmental variations were noted in the 72.5 and 725 mg/kg/day groups. However, the individual findings were not observed in a dose related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the historical control data (version 2017.03). Therefore, the skeletal developmental variations were not considered test substance-related.
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
725 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Dose Formulation Analyses


The analyzed dosing formulations contained 92.4% to 101% of the test substance which was within the protocol-specified range of target concentrations for solutions (90% to 110%). The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1). Results of the analyses of dosing formulations are summarized below.
Results of Concentration Analyses (mean concentration, mg/mL % of target))
- Date of preparation: 2020-11-05. Group 2 (10 mg/mL) 9.49 mg/mL, Group 3 (30 mg/mL) 28.9 mg/mL, Group 4 (100 mg/mL) 95.1 mg/mL.
- Date of preparation: 2020-11-19. Group 2 (10 mg/mL) 9.24 mg/mL, Group 3 (30 mg/mL) 30.4 mg/mL, Group 4 (100 mg/mL) 93.6 mg/mL.


Mortality and Observations
There were no test substance-related effects on survival. All animals survived to scheduled euthanasia and were gravid.
Test substance-related increased incidences of abnormal breathing sounds were observed in 8 females at approximately 2 hours postdose in the 725 mg/kg/day group and persisted to the daily examinations in 3 females only during Gestation Days 7–21. These findings were considered non-adverse due to the animals being in otherwise general good health throughout the treatment period. Other clinical observations noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.


Body Weights and Gravid Uterine Weights
Mean maternal body weights, body weight gains, adjusted body weights, adjusted body weight gains, and gravid uterine weights in the 72.5, 217.5, and 725 mg/kg/day groups were unaffected by test substance administration. Any statistically significant differences from the control group were transient, did not occur in a dose-responsive manner, and/or did not impact mean absolute body weights.


Food Consumption
In the 725 mg/kg/day group, test substance-related statistically significantly lower mean food consumption was noted during Gestation Days 6–9 compared to the control group. Mean food consumption in this group was comparable to the control group for the remainder of the dosing period (Gestation Days 9–21) and when the entire dosing period (Gestation Days 6–21) was evaluated. There were no corresponding effects on mean body weights or body weight gains, and therefore, the effects on mean food consumption in the 750 mg/kg/day group were considered non-adverse.
Mean food consumption in the 72.5 and 217.5 mg/kg/day were unaffected by test substance administration throughout the study. Differences from the control group were slight and not statistically significant.


Thyroid Hormone Analyses
There were test substance-related effects on thyroid hormone values (TSH, T3, and T4) at any dose level. Differences from the control group were not statistically significant, observed in a manner that was not dose-related, and were within the ranges of the Historical Control Data.


Macroscopic Pathology
No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to test substance administration.


Organ Weights
No test substance-related organ weight changes were noted at any dose level.


Microscopic Evaluations
No test substance-related microscopic findings in the thyroid gland or liver were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to test substance administration.


Ovarian and Uterine Examinations
Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 72.5, 217.5, and 725 mg/kg/day. Parameters evaluated included mean litter proportions of post implantation loss, mean number of live fetuses, mean fetal body weights, mean anogenital distance (absolute and relative to cube root of body weight), and fetal sex ratios. Differences from the control group were slight, not statistically significant, and/or noted in a manner that was not dose-related.
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups.


Fetal Morphological Data
The numbers of fetuses (litters) available for morphological evaluation were 317(25), 318(25), 301(25), and 333(25) in the control, 72.5, 217.5, and 725 mg/kg/day groups, respectively. Malformations were observed in 0(0), 1(1), 2(2), and 6(3) fetuses (litters) in these same respective dose groups.


External Malformations and Variations
No test substance-related external malformations were noted in fetuses in this study. A single fetus in the 217.5 mg/kg/day group (No. 3518-09) was observed with proboscis and absent eye bulges, mouth, and lower jaw. These findings were noted in a single mid-dose fetus, and therefore were not considered test substance related. No other external malformations or external developmental variations were observed in fetuses on this study.


Visceral Malformations and Variations
No visceral malformations were noted in fetuses in this study.
No test substance-related visceral developmental variations were noted. Findings observed in the test substance treated groups were noted similarly in the control group.


Skeletal Malformations and Variations
Skeletal malformations were noted for 0(0), 1(1), 2(2), and 6(3) fetuses (litters) in the control, 72.5, 217.5, and 725 mg/kg/day groups, respectively; incidences in the test substance-treated groups were not statistically significantly higher than the concurrent control group. In the 725 mg/kg/day group, different types of vertebral malformations were noted in 3 individual litters: Fetus Nos. 4504 05, 4504-16, and 4504-17 were observed with absent lumbar vertebra, Fetus Nos. 4513-01 was observed with an absent rib and thoracic hemivertebra; this fetus was also noted with a lower body weight (3.87 g) compared to the group mean value (5.92 g), and Fetus Nos. 4521-04 and 4521-10 were observed with supernumerary lumbar vertebra. Because the skeletal malformations in the 725 mg/kg/day group were of specific types to fetuses in individual litters, they were considered familial and not related to the test substance administration. In the 217.5 mg/kg/day group, Fetus No. 3503 10 was observed with fused ribs, cervical centrum, cervical arches, and thoracic arches and absent cervical centrum and Fetus No. 3518 09 was noted with multiple malformations in the skull (absent mandible and jugal bones, misshapen nasal, premaxilla, maxilla, squamosal, frontal, parietal, and interparietal bones, small premaxilla, nasal, maxilla, squamosal, tympanic annulus, frontal, parietal, and interparietal bones, large supraoccipital bone, and unossified parietal bone), corresponding to the external malformations noted in this fetus. In the 72.5 mg/kg/day group, Fetus No. 2501-03 was observed with an absent lumbar vertebra. Skeletal malformations in the 50 and 150 mg/kg/day groups were limited to single fetuses, and therefore were not considered test substance related.
Higher incidences (statistically significant) of skeletal developmental variations were noted in the 72.5 and 725 mg/kg/day groups. However, the individual findings were not observed in a dose related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data (version 2017.03). Therefore, the skeletal developmental variations were not considered test substance-related.


Summary of External, Visceral, and Skeletal Examinations
The numbers of fetuses (litters) available for morphological evaluation were 317(25), 318(25), 301(25), and 333(25) in the control, 72.5, 217.5, and 725 mg/kg/day groups, respectively. Malformations were observed in 0(0), 1(1), 2(2), and 6(3) fetuses (litters) in these same respective dose groups.
When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted, with the following exceptions. The mean litter proportion of fetuses with any skeletal developmental variations was statistically significantly higher in the 72.5 and 725 mg/kg/day groups compared to the control group; when individual developmental variations were evaluated, only the mean litter proportion of fetuses with incomplete ossification of the thoracic centrum was statistically significant in the 72.5 mg/kg/day group. Fetal malformations and developmental variations, when observed in the test article treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test article.

Applicant's summary and conclusion

Conclusions:
Based on the absence of adverse maternal or fetal effects, a dosage level of 725 mg/kg/day (the highest dose level tested) was considered to be the no observed adverse effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test substance "Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed" was administered orally by gavage to time-mated Crl:CD(SD) rats.
Executive summary:

In this developmental toxicity study according to OECD guideline 414 and OPPTS 870.3700, the test substance "Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed" was administered to 25 time-mated female Crl:CD(SD) rats/dose at dose levels of 0, 72.5, 217.5 and 725 mg/kg bw/day. Animals were dosed via oral gavage once daily during Gestation Days 6–20.


The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormone parameters (T3, T4, and TSH), organ weights, macroscopic and microscopic examinations, intrauterine growth and survival, and fetal morphology.


All females survived to the scheduled necropsy on Gestation Day 21. Test substance-related clinical observations noted at the daily examinations or approximately 2 hours postdosing were limited to abnormal breathing sounds in the 725 mg/kg/day group. The animals were otherwise in good health throughout the treatment period, so these effects were not considered adverse.


There were no test substance-related effects on mean maternal body weights, body weight gains, adjusted body weights, adjusted body weight gains, or gravid uterine weights at any dose level.


Test substance-related lower mean maternal food consumption was noted in the 725 mg/kg/day group during Gestation Days 6–9 but was comparable to the control group thereafter. Based on the transient nature of these effects and the lack of corresponding body weight effects, these effects on food consumption were considered non-adverse. No test substance-related effects were observed on food consumption in the 72.5 or 217.5 mg/kg/day groups.


There were no test substance-related effects on maternal thyroid hormone values at any dose level.


There were no test substance-related effects on maternal macroscopic pathology, organ weights, or microscopic evaluations at any dose level.


There were no test substance-related effects on intrauterine growth or survival at any dose level.


There were no test substance-related effects on external, visceral, or skeletal fetal morphology at any dose level.


Based on the absence of adverse maternal or fetal effects, a dosage level of 725 mg/kg/day (the highest dose level tested) was considered to be the no‑observed‑adverse‑effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test substance "Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed" was administered orally by gavage to time-mated Crl:CD(SD) rats.

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