Sophorolipids: fermentation products of glucose and rapeseed-oil fatty acids methyl esters with yeast Candida Bombicola

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / 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

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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sophorolipids: fermentation products of glucose and rapeseed-oil fatty acids methyl esters with yeast Candida Bombicola

EC Number:

700-786-1

Cas Number:

1194682-64-8

IUPAC Name:

Sophorolipids: fermentation products of glucose and rapeseed-oil fatty acids methyl esters with yeast Candida Bombicola

Test material form:

solid

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group. Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycles (e.g., EDDDE), during the pretest period, were excluded from the study; therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, 10 females per group was an appropriate number of animals to obtain a sample size of 8 females per group at termination.

Environmental conditions: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.
Target temperatures of 68°F to 78°F (20°C to 26°C) with a relative target humidity of 30% to 70% were maintained. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The route of administration was oral (gavage) because this is a potential route of exposure to humans. Historically, this route has been used extensively for studies of this nature.
Dosage levels were determined from results of previous studies and were provided by the Sponsor Representative. These studies were based on very similar Sophorolipids; ECHA profile : fermentation products of glucose and fatty acids, C18 (unsaturated), glycerol esters with yeast Candida Bombicola, partially hydrolysed. EC / List no. 941-809-7. In a previous 28-day repeat-dose oral toxicity study in rats (OECD 407), Sophorolipids was administered daily via oral gavage to 10 males and 10 females per group at dosage levels of 0, 100, 300, and 1000 mg/kg bw/day. Due to unexpected mortality and overall adverse effects during the first 2 weeks of treatment, the high-dosage level was reduced from 1000 to 500 mg/kg/day. Following the reduction in dosage level, no adverse effects were noted. Therefore, 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for a subsequent oral gavage reproduction/developmental toxicity screening study (OECD 421) in rats. In the subsequent OECD 421 study, Sophorolipids was administered daily via oral gavage to 10 males and 10 females per group at doses of 100, 300, and 500 mg/kg/day. In the absence of adverse effects at all dosage levels, 500 mg/kg/day was considered to be the NOAEL for both parental animals and offspring. Based on these results, dosage levels of 0, 100, 250, and 500 mg/kg/day were selected for the current OECD 422 study.

Details on mating procedure:

After a minimum of 14 days of dosing, the animals were paired on a 1:1 basis within each group. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses described below were performed by a high performance liquid chromatography (HPLC) method with ultraviolet light (UV) absorbance detection, using a validated analytical procedure (Akalkotkar, 2020, 00810018).
Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration, with each individual sample concentration within ± 20% of the target concentration. After acceptance of the analytical results, backup samples were discarded.
Test substance formulations (solutions) have been previously shown to be stable and uniform over the range of concentrations used on this study for at least 24 hours at room temperature (18ºC to 24ºC) and up to 8 days refrigerated (target of 5°C), protected from light, purged with nitrogen (Akalkotkar, 2020, 00810018). Therefore, stability and uniformity of test substance formulations (solutions) were not assessed on this study.

Duration of treatment / exposure:

Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days (Study Days 0-27). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were dosed via oral gavage once daily. Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days (Study Days 0-27). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13.
The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, neurobehavior, motor activity, thyroid hormones, clinical pathology, gross necropsy findings, organ weights, and histopathologic examinations.
No test substance-related effects were noted on F0 survival, body weights, food consumption, estrous cyclicity, reproductive performance, parturition, neurobehavior, motor activity, thyroid hormones (males only), clinical pathology, macroscopic findings, organ weights, or microscopic findings. No test substance-related effects were noted on F1 litter viability and survival, clinical observations, body weights, anogenital distance, areolae/nipple anlagen retention, thyroid hormones, macroscopic findings, and organ weights.
A clinical observation of abnormal breathing sounds was noted at the daily examinations and/or postdosing observations for F0 males and females in the 100, 250, and 500 mg/kg/day groups during the respective treatment periods. Due to the absence of this observation in control group animals, it was considered test substance-related. However, because this observation was noted sporadically during the treatment period, occurred in a manner that was not clearly dose related, and/or did not impact the overall health and well-being of the affected animals, it was not considered adverse, but likely a secondary effect of dosing procedures. No other test substance related clinical observations were noted for F0 males and females at any dosage level.

Examinations

Parental animals: Observations and examinations:

All F0 animals on the control, 100, 250, and 500 mg/kg/day groups survived to the scheduled necropsy. A clinical observation of abnormal breathing sounds was noted at the daily examinations and/or postdosing observations for F0 males and females in the 100, 250, and 500 mg/kg/day groups during the respective treatment periods. Due to the absence of this observation in control group animals, it was considered test substance-related. However, because this observation was noted sporadically during the treatment period, occurred in a manner that was not clearly dose related, and/or did not impact the overall health and well-being of the affected animals, it was not considered adverse, but likely a secondary effect of dosing procedures. No other test substance-related clinical observations were noted for F0 males and females at any dosage level.

Oestrous cyclicity (parental animals):

For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed or until the end of the mating period. The slides were microscopically examined to determine the stage of the estrous cycle. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] for 14 consecutive days before cohabitation and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Litter observations:

Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Postmortem examinations (parental animals):

Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).

Postmortem examinations (offspring):

No internal findings were noted at the necropsy of pups euthanized on PND 13.

Statistics:

Data collected during the predose period were not tabulated, summarized, or statistically analyzed, unless applicable to analyses in the proceeding sections. All statistical analyses were performed within the respective study phase, unless otherwise noted. Clinical and necropsy observations data were summarized but no inferential statistical analysis was performed. Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion or by litter. Values may also be expressed as a percentage of pretreatment period or control values, or fold change of control values, when deemed appropriate. Calculated values on Provantis tables may not be reproducible from the individual values presented because all calculations were conducted using non-rounded values.

Reproductive indices:

The mean number of pups born, live litter size and the percentage of males at birth in the 100, 250, and 500 mg/kg/day groups were similar to the control group values. Postnatal survival in the 100, 250, and 500 mg/kg/day groups were unaffected by test substance administration.

Offspring viability indices:

Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All F0 animals on the control, 100, 250, and 500 mg/kg/day groups survived to the scheduled necropsy. A clinical observation of abnormal breathing sounds was noted at the daily examinations and/or postdosing observations for F0 males and females in the 100, 250, and 500 mg/kg/day groups during the respective treatment periods. Due to the absence of this observation in control group animals, it was considered test substance-related. However, because this observation was noted sporadically during the treatment period, occurred in a manner that was not clearly dose related, and/or did not impact the overall health and well-being of the affected animals, it was not considered adverse, but likely a secondary effect of dosing procedures. No other test substance-related clinical observations were noted for F0 males and females at any dosage level.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males:Mean body weights and body weight gains in the 100, 250, and 500 mg/kg/day group males were unaffected by test substance administration throughout the study. None of the differences from the control group were statistically significant.
Females: Mean body weights and body weight gains in the 100, 250, and 500 mg/kg/day group females were unaffected by test substance administration during the premating period. A statistically significantly higher mean body weight gain was noted for females in the 500 mg/kg/day group during Study Days 9-13 compared to the control group; however, this difference was transient and did not affect mean body weights or the cumulative mean body weight gain, and therefore was not considered test substance-related. No other differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

MAles: Mean food consumption, evaluated as g/animal/day, in the 100, 250, and 500 mg/kg/day group males was similar to that in the control group throughout the study, with the following exceptions. Statistically significantly lower mean food consumption was noted for males in the 250 mg/kg/day group during Study Days 3-6 and for males in the 500 mg/kg/day group during Study Days 9-13; however, these results were transient, did not affect mean body weight gains, and/or were not noted in a dose-related manner. No other statistically significant differences were observed.
Females:Mean food consumption, evaluated as g/animal/day, in the 100, 250, and 500 mg/kg/day group females was unaffected by test substance administration during the premating period. Statistically significantly higher mean food consumption was noted for females in the 100 mg/kg/day group during Study Days 3-6 and statistically significantly lower mean food consumption was noted for females in the 100 and 500 mg/kg/day groups during Study Days 6-9; however, these changes were discordant in direction of change, did not occur in a dose-related manner, and did not affect mean body weights at any dosage level. No other differences from the control group were statistically significant.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on hematology. The following statistically significant differences were noted when compared to the control group: lower mean absolute neutrophil concentration for males in the 250 mg/kg/day group, higher mean hemoglobin concentration for females in the 100 mg/kg/day group, and higher mean hematocrit for females in the 100 and 500 mg/kg/day groups. However, these changes did not occur in a dose-related manner and were attributed to normal biological variation. Other differences from the control group were slight and not statistically significant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on serum chemistry. The only statistically significant difference from the control group was higher mean aspartate aminotransferase (AST) concentration for males in the 250 mg/kg/day group; however, this did not occur in a dose-related manner and was attributed to normal biological variation. Other differences from the control group were slight and not statistically significant.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F0 males at any dosage level. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

only males: There were no test substance-related effects on urinalysis for males at any dosage level. Differences from the control group were slight and not statistically significant.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance administration at all dosage levels when evaluated on Study Day 26 (males) or on Lactation Day 13 (females). Values obtained from the 6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50, and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data. Differences from the control group were slight, not statistically significant, within the Charles River Ashland historical control data ranges and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the F0 animals were evaluated at on Study Day 26 (males) or on Lactation Day 13 (females).

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Neurobehavioral assessments were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group on Study Day 26 (males) or on Lactation Day 13 (females).

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified in Text Table 21 for microscopic examination were evaluated from 5 animals/sex in the control and high-dose groups. Gross lesions were examined from all groups.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No test substance-related microscopic findings 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 administration of Sophorolipids.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed or until the end of the mating period. The slides were microscopically examined to determine the stage of the estrous cycle. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] for 14 consecutive days before cohabitation and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. One male in the 500 mg/kg/day group did not sire a litter. One female in the 500 mg/kg/day group was determined to be nongravid.
The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food efficiency

ophthalmological examination

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

neuropathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance exposure. One (1), 5(4), 4(2), and 3(2) pups (litters) in the control, 100, 250, and 500 mg/kg/day groups, respectively, were found dead. Two (2) pups (litters) in the 250 mg/kg/day group were missing and presumed to have been cannibalized.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One (1), 5(4), 4(2), and 3(2) pups (litters) in the control, 100, 250, and 500 mg/kg/day groups, respectively, were found dead. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on thyroid/parathyroid weights for F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 100, 250, and 500 mg/kg/day groups were generally similar to the control group values when evaluated on PND 1 and 4. Differences from the control group were not statistically significant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Numbers of areolae/nipple anlagen in the F1 male pups were unaffected by parental administration of the test substance when evaluated on PND 13. No retained nipples were noted for male pups at any dosage level.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on thyroid/parathyroid weights for F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic Pathology
Unscheduled Deaths
One (1), 5(4), 4(2), and 3(2) pups (litters) in the control, 100, 250, and 500 mg/kg/day groups, respectively, were found dead. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Scheduled Necropsy
No internal findings were noted at the necropsy of pups euthanized on PND 13.

Histopathological findings:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis : There were no test substance-related effects on thyroid hormone values in the F1 males and females at any dosage level on PND 4 or 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

other: anogenital distance, nipple retention, thyroid hormone analysis

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this screening study, based on the lack of adverse effects at any dosage level, a dosage level of 500 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic and reproductive toxicity of Sophorolipids when administered orally by gavage to Crl:CD(SD) rats. The NOAEL for F1 neonatal toxicity was 500 mg/kg/day based on the lack of effects at any dosage level.