Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

No effects on reproduction or development were observed. The No Observed Adverse Effects Level (NOAEL) for reproductive and developmental toxicity including satellite group animals was considered to be 500 mg/kg/day for both parental animals and offspring, this being the highest dose investigated.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
OECD 421
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2017 - Date of study director signature; end of experimental phase 06 March 2018 (last day of necropsy)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain. The oral route was selected as it is a possible route of exposure of the test item in man.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia, S.p.A., Calco (Lecco), Italy.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 200-225 g; Females: 175-200 g
- Fasting period before study: no
- Housing: up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese).
- Diet: commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water: Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 5 weeks was allowed before the start of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-20
- Photoperiod: artificial light for 12 hours each day
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
softened by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was dissolved in the vehicle. During the formulation procedure, particular attention was paid to avoid foaming production as much as possible. The formulations were prepared daily (concentrations of 10, 30 and 50mg/mL). Concentrations were calculated and expressed in terms of test item corrected for purity.
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: the female was paired with the same male until positive identification of copulation occurred
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): The females were transferred to individual solid bottomed cages measuring 42.5×26.6×18 cm (Tecniplast Gazzada S.a.r.l.) for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (RTC Study No. A2478). Samples of the formulations prepared during the current study (the first and the last week of treatment) were analysed to check the concentration. Chemical analysis was carried out by the Analytical Chemistry Department at RTC. The software used for this activity was Empower® 2 Build No. 2154.
Duration of treatment / exposure:
Main groups
Males
Animals of the main groups were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Day 15 or 16 of the mating phase), for a total of 30 or 31 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice. Female no. X0780053 which had total litter loss was dosed up to Day 2 post partum (the day before sacrifice). Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Satellite groups
Animals were dosed once a day, 7 days a week, for 4 consecutive weeks until the day of necropsy (Day 29 of the premating phase). Animals were dosed for a total of 28 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12-13 weeks
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Groups 1 (main) and 5 (satellite)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Groups 4 (main) and 6 (satellite)
No. of animals per sex per dose:
Main groups: 10 per sex per dose
Satellite groups: 5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels have been selected based on information from a previous repeated dose toxicity study
- Fasting period before blood sampling for clinical biochemistry: yes
Positive control:
none
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study
BODY WEIGHT: Yes / No / No data
- Time schedule for examinations:
Main groups: Males were weighed weekly from allocation to termination and on the day of necropsy. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1, 4, 7 and 13 post partum and on the day of necropsy.
Satellite groups: Animals were weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and on the day of necropsy.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Oestrous cyclicity (parental animals):
Vaginal smears and oestrous cycle (Main groups)
Stock females
Oestrous cycle was monitored by vaginal smears in all stock females for at least 2 weeks before dosing in order to exclude from the study females with irregular cycle.
Females allocated to groups
Vaginal smears were taken in the morning from Day 1 of dosing up to positive identification of mating. The vaginal smear data were examined to determine the following:
– anomalies of the oestrous cycle;
– pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also taken from all females, before despatch to necropsy.
Mating (Main groups)
Matings were monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plug found on the cage tray). Each cage tray was checked each morning for the presence of copulation plugs. The female was paired with the same male until positive identification of copulation occurred.

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals males were killed after the mating of all females.
- Maternal animals:
Females with live pups were killed on Day 14 post partum.
Female no X0780053 with total litter loss on Day 0 post partum was killed on Day 3 post partum.
Female no. X0780019 which did not give birth was killed on Day 26 post coitum.
GROSS NECROPSY
- Gross necropsy consisted of a detailed post mortem examination including examination of the external surface and orifices. Changes were noted, the requisite organs weighed and the required tissue samples processed for histopathological examination.
All females were examined also for the following:
– external and internal abnormalities
– number of visible implantation sites (pregnant animals)
– number of corpora lutea (pregnant animals)
HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues which were prepared for microscopic examination and weighed, respectively, are summarized in section “any other details on results incl. tables”.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at Day 4 post partum (culled pups) and at Day 14 post partum
- These animals were subjected to postmortem examinations as follows:
GROSS NECROPSY
All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrificed at Day 14 post partum were examined for external abnormalities. Sex was determined by internal gonads inspection.
HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid was weighed from one male and one female from each litter (pups bled for thyroid hormone determination) and preserved. The thyroid weight (expressed in mg) was determined after fixation.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The criteria for statistical significance was p<0.05 and p<0.001.
Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test.
Reproductive indices:
Reproductive indices
1) Males:
- Copulation Index (%) = no. of animals mated x 100/ no. of animals paired
- Fertility Index (%) = no. of males which induced pregnancy x 100/ no. of animals paired
2) Females:
- Copulatory Index (%) = no. of animals mated x 100/ no. of animals paired
- Fertility Index (%) = no. of pregnant females x 100/ no. of females paired was calculated as a percentage from the formula:
3) Males and females:
- Copulatory Interval = Mean number of days between pairing and mating

Offspring viability indices:
Pre-implantation loss was calculated as a percentage from the formula:
[(No. of corpora lutea – No. of implantation) / No. of corpora lutea] * 100
Pre-natal loss was calculated as a percentage from the formula:
[(No. of visible implantations – live litter size at birth) / No. of visible implantations] * 100
Pup loss at Day 0 post partum was calculated as a percentage from the formula:
[(Total litter size – live litter size) / total litter size] * 100
Post natal loss at Day 4 post partum (before culling) was calculated as a percentage from the formula:
[(live litter size at birth – live litter size at day 4 before culling) / live litter size at birth] * 100
Post natal loss at Day 13 post partum (after culling) was calculated as a percentage from the formula:
[(live litter size at Day 4 after culling – live litter size at day 13) / live litter size at Day 4 after culling] * 100
Sex ratios were calculated at birth, on Day 4 and on Day 14 post partum and were presented as the percentage of males per litter.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No relevance was attributed to the hairloss and/or staining observed in one lowdose female, one mid-dose female of the main group or in one high dose male and two high dose females of the satellite groups. These signs were considered incidental. Since the hairloss was observed at fore- and hindlimbs this was caused by licking.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Parental males
Thyroxine showed a statistically significant increase in a number of males dosed at 500 mg/kg/day (mean group value was 12 % above controls). Due to the minimal severity and the absence of other related changes, this finding was considered of no toxicological relevance.
Offspring - Day 14 post partum
A decrease of Thyroid Stimulating Hormone was recorded in some male pups of all treated groups. Compared with controls, changes were 21 % to 31 %, with no dose-relation. Due to the absence of dose-relation and of other related changes, this finding was not considered to be treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Parental animals
No treatment-related changes were noted, following histopathology evaluation. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. As regular layering in the germinal epithelium was noted, there was no treatment-related effect on the spermatogenic cycle. All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated
Sprague Dawley SD rats of the same age.

Satellite groups
No treatment-related changeswere noted, following histopathology evaluation of bone marrow, spleen and thymus. All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Parental females
No mortality occurred during the study. One control female (no. X0780015) was proved not pregnant at necropsy and the mid-dose female no. X0780053 had total litter loss on Day 0 post partum.
The number of females with live pups on Day 14 post partum was: 9 in the control, 10 in the low dose (100 mg/kg/day), 9 in the mid-dose (300 mg/kg/day) and 10 in the high dose group (500 mg/kg/day).
No signs were recorded in males of the main groups during the study. No relevance was attributed to the hairloss and/or staining observed in one low dose female, one mid-dose female of the main group or in one high dose male and two high dose females of the satellite groups. These signs were considered incidental.
No differences of toxicological relevance were observed in body weight or body weight gain between the control and treated animals.
The food consumption was comparable between groups.

Parental males
Thyroxine showed a statistically significant increase in a number of males dosed at 500 mg/kg/day (mean group value was 12% above controls). Due to the minimal severity and the absence of other related changes, this finding was considered of no toxicological relevance.
Pups - Day 14 post partum.
A decrease of Thyroid Stimulating Hormone was recorded in some male pups of all treated groups. Compared with controls, changes were 21 % to 31 %, with no dose-relation. Due to the absence of dose-relation and of other related changes, this finding was not considered to be treatment-related.
Oestrous cycle, reproductive parameters, pairing combination and mating performance
Oestrous cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups. All females were pregnant except for one control female (animal no. X0780015).
Implantation sites, pre-implantation loss data, pre-natal loss data and gestation length of females
Gestation periods were similar between treated and control group females. All pregnant females gave birth on Day 22 post coitum (mean value). Corpora lutea, implantation sites, total litter size, pre-implantation loss and pre-natal loss (percentage) were similar in control and treated groups.
Litter data at birth, on Day 1 and on Day 4 post partum (before culling), on Day 13 (after culling) post partum and sex ratio: No differences in litter data were seen between control and treated groups.
Sex ratios (calculated as a percentage of males) at birth and on Days 4 and 14 post partum did not show differences between groups.
Anogenital distance
Anogenital distance (normalised for the cube root of the pup weight collected on Day 1 post partum) was unaffected by treatment.
Clinical signs of pups and nipple observations
Pup no. 12 of dam X0780031 of the low dose group was found moribund and with no milk in the stomach on Day 4 post partum. This pup was selected for culling. No abnormalities were found at necropsy. No signs related to treatment were seen in the remaining pups.
No nipples were present in any male pup, when observed on Day 13 post partum. Necropsy findings in decedent pups, culled pups and in pups sacrificed on Day 14 post partum.
No treatment-related findings were described. Pups thyroid weight on Day 14 post partum. Thyroid weight in treated pups was comparable to controls.
Terminal body weight of treated animals of the three main groups (Groups 2, 3 and 4), as well as the high dose satellite group (Group 6) was comparable to the concurrent control group (Group 1 or 5).
No relevant changes were observed in absolute and relative organ weight in all treatment groups of both sexes, when compared to the concurrent control data. All organ weight variations between control and treated animals were considered to be within the physiological range of Sprague Dawley SD rats of this age.
Macroscopic observations
No treatment-related changes were noted, following gross pathology examination in either main or satellite groups. All observed changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Microscopic observations
Parental animals
No treatment-related changes were noted, following histopathology evaluation. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. As regular layering in the germinal epithelium was noted, there was no treatment-related effect on the spermatogenic cycle.
All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Satellite groups
No treatment-related changes were noted, following histopathology evaluation of bone marrow, spleen and thymus. All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at highest dose level
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pup no.12 of dam X0780031 of the low dose group was found moribund and with no milk in the stomach on Day 4 post partum. This pup was selected for culling. No abnormalities were found at necropsy.
No signs related to treatment were seen in the remaining pups.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A decrease of Thyroid Stimulating Hormone was recorded in some male pups of all treated groups. Compared with controls, changes were 21 % to 31 %, with no dose-relation. Due to the absence of dose-relation and of other related histopathological changes, this finding was not considered to be treatment-related.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid weight in treated pups was comparable to controls.
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
other: Treatment of parental generation
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at highest dose level
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Tissues which were prepared for microscopic examination and weighed:

Organs / tissues

Weight

Fixation / Preservation

Microscopic examination

Abnormalities

-

Yes

Yes

Adrenal glands

-

Yes

Yes

Bone marrow (from sternum), only satellite groups

-

Yes

Yes

Brain (cerebrum, cerebellum,

medulla/pons)

-

Yes

-

Clitoral gland

-

Yes

-

Epididymides

Yes

Yes

Yes

Kidneys

-

Yes

Yes

Liver

-

Yes

Yes

Mammary gland - Females

-

Yes

Yes

Mammary gland - males

-

Yes

Yes

Ovaries with oviducts

Yes

Yes

Yes

Parathyroid glands, weighed and preserved with thyroid gland

Yes

Yes

-

Pituitary gland

-

Yes

-

Penis

-

Yes

-

Prostate gland (dorsolateral and ventral)

Yes

Yes

-

Sciatic nerve

-

Yes

Yes

Seminal vesicles with coagulating glands

Yes

Yes

Yes

Spleen, only satellite groups

Yes

Yes

Yes

Testes

Yes

Yes

Yes

Thymus, where present, only in satellite groups

Yes

Yes

Yes

Thyroid gland

Yes

Yes

Yes

Uterus – cervix

Yes

Yes

Yes

vagina

-

Yes

Yes

Conclusions:
The toxicity, as well as any possible effects of the test item on male and female reproductive performance were investigated in this study (main groups). Two satellite groups (control and high dose) sacrificed after 4 weeks of treatment were included for specific histopathology investigations of spleen, thymus and bone marrow.
The dosages used were 100, 300 and 500 mg/kg/day. The study was conducted according to OECD Test Guideline No. 421 adopted on 29 July 2016.
For parental main groups animals, no adverse effects were found on oestrous cycle, copulation, fertility, delivery or lactation gestation length, number of corpora lutea or implantation sites.
No adverse effects were found in anogenital distance, pup sex ratios, pup weight, pup viability and no treatment related findings were noted at necropsy. Thyroid weights of the parental animals and of pups on Day 14 post partum were comparable to the control group.
Macroscopic observation and organ weight were unaffected by treatment. No treatment related changes were described following histopathology evaluation in main and satellite groups, including spermatogenic cycle.
Slight changes noted in thyroid hormone levels were considered not related to treatment.
The histopathological examination performed in satellite group animals did not show any changes related to treatment.
The No Observed Adverse Effects Level (NOAEL) for reproductive and developmental toxicity including satellite group animals was considered to be 500 mg/kg/day for both parental animals and offspring, this being the highest dose investigated.
Executive summary:

In this study according to OECD guideline 421, GLP, the toxicity as well as any possible effects of “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed” on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptus and parturition, were investigated (main groups). Two satellite groups (control and high dose) sacrificed after 4 weeks of treatment were included for specific histopathology investigations of spleen, thymus and bone marrow.

The dosages used were 100, 300 and 500 mg/kg/day. For parental main groups animals, no adverse effects were found on oestrous cycle, copulation, fertility, delivery or lactation gestation length, number of corpora lutea or implantation sites.

No adverse effects were found in anogenital distance, pup sex ratios, pup weight, pup viability and no treatment related findings were noted at necropsy. Thyroid weights of the parental animals and of pups on Day 14 post partum were comparable to the control group.

Macroscopic observation and organ weight were unaffected by treatment. No treatment related changes were described following histopathology evaluation in main and satellite groups, including spermatogenic cycle.

Slight changes noted in thyroid hormone levels were considered not related to treatment.

The histopathological examination performed in satellite group animals did not show any changes related to treatment.

The No Observed Adverse Effects Level (NOAEL) for reproductive and developmental toxicity including satellite group animals was considered to be 500 mg/kg/day for both parental animals and offspring, this being the highest dose investigated.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017-10-24 to 2017-11-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Thyroid hormone analysis was not conducted. No blood samples were collected. Not all developmental endpoints were addressed. Oestrus cyclicity was not examined.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 9 weeks (females), 11 weeks (males)
- Weight at study initiation: 221-241 g (females), at least 342-363 g (males)
- Fasting period before study: not reported
- Housing: clear polysulphone cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material (Scobis 0 Mucedola) was provided inside suitable bedding bags as necessary and changed at least 2 times a week.
- Diet: commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water: Drinking water was supplied ad libitum.
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 2 °C
- Humidity: 55 % ± 15 %
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was dissolved in the vehicle. During formulation procedure, particular attention was paid to avoid foaming production as much as possible. The formulation was prepared daily or, based on stability data (8-day stability at + 5 °C ± 3 °C - RTC Study No. A2478), for more days up to 5 (concentrations of 10, 30 and 50 mg/mL). Concentrations were calculated and expressed in terms of test item corrected for purity (47.2 %).
VEHICLE
- Amount of vehicle: dose volume was 10 mL/kg bw.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: females were paired with the males in the home cage of the male.
- M/F ratio per cage: 1/1
- Length of cohabitation: one night
- Verification of same strain and source of both sexes: no; as provided by animal supplier
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
All animals were dosed once a day from Day 6 through Day 19 post coitum.
Frequency of treatment:
Once a day
Details on study schedule:
From day 0 of pregnancy till day 20 post coitum. All animals were dosed once a day from Day 6 through Day 19 post coitum.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
6 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose selection was based on a previous developmental screening study.
- Rationale for animal assignment: animals were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: early in each working day and again in the afternoon. At weekends and public holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15 and 20 post coitum.
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
- Other: number of intra-uterine deaths; gross evaluation of placentae, number, sex and weight of all live foetuses.
Postmortem examinations (parental animals):
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: ovaries and uteri
Postmortem examinations (offspring):
- External examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- other: malformations in general, anomalies, variants
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Reproductive indices:
Pre-implantation loss % = (no. of corpora lutea − no. of implantations) / no. of corpora lutea X 100

Post-implantation loss % = (no. of implantations − no. of live foetuses) / no. of implantations X 100

Total implantation loss % = (no. of corpora lutea − no. of live foetuses) / no. of corpora lutea X 100

Sex ratios of the foetuses were calculated as the percentage of males per litter.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were seen. Hairloss and/or scab on the head were seen in three females receiving 300 mg/kg/day and in one female receiving 500 mg/kg/day. This last female also showed abrasion and scab on the ear. The above mentioned signs were not considered related to the test item administration.
All mated females were pregnant with live foetuses on Day 20 post coitum.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on body weight and body weight gain were seen in treated females, compared to controls.
Organ weight findings including organ / body weight ratios:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
All mated females were pregnant with live foetuses on Day 20 post coitum.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormalities were seen in foetuses examined, with the exception of malrotated hind-limbs observed in one foetus each in one low (100 mg/kg/day) and mid-dose (300 mg/kg/day) female and in three foetuses in two high dose (500 mg/kg/day) females.
Rotation of limbs is also present in the Historical Control Data obtained by the Supplier, although with a lower incidence, due to the high number of foetuses examined.
Reproductive effects observed:
no
Conclusions:
No significant treatment-related changes were seen in the in vivo and post mortem data in treated females. The test item was well tolerated up to the dose of 500 mg/kg/day.
Malrotation of hindlimbs was noted, at the external examination, in single foetuses in all treated groups. Further examination could be suggested in order to evaluate the concern of this finding.
Executive summary:

In this developmental toxicity screening study similar to OECD guideline 421, the effects of “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed” were investigated, after oral administration at dose levels of 100, 300 and 500 mg/kg/day, in each 6 Sprague Dawley female rats during pregnancy and on embryofoetal development.

All animals were administered during the gestation period, starting from Day 6 through Day 19 post coitum at the dose volume of 10 mL/kg. Body weight and daily clinical signs were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late intrauterine deaths, live foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities.

No mortality occurred.

All females were pregnant and with live foetuses on Day 20 post coitum.

No treatment-related clinical signs were seen during the study.

No effects on body weight and body weight gain were seen.

No intergroup differences in terminal body weight, uterus weight and absolute weight gain were seen.

Litter data and sex ratio were not affected by treatment.

No treatment-related macroscopic observations in females were recorded.

No abnormalities were seen at the external examination of foetuses, except for malrotated hindlimbs observed in one foetus each in one low (100 mg/kg/day) and mid-dose (300 mg/kg/day) female and in three foetuses in two high dose (500 mg/kg/day) females. Malrotation of hindlimbs could be confirmed by a skeletal evaluation. Rotation of limbs is also present in the Historical Control Data obtained by the Supplier although with a lower incidence, due to the high number of foetuses examined. Further examination could be suggested in order to evaluate the concern of this finding.

In conclusion, no significant treatment-related changes were seen in the in vivo and post mortem data in treated females. The test item was well tolerated up to the dose of 500 mg/kg/day in female rats.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available key study was conducted according to OECD guideline 421 and is of high quality.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a study according to OECD guideline 421, GLP, the toxicity as well as any possible effects of “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed” on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptus and parturition, were investigated (main groups). Two satellite groups (control and high dose) sacrificed after 4 weeks of treatment were included for specific histopathology investigations of spleen, thymus and bone marrow.


The dosages used were 100, 300 and 500 mg/kg/day. For parental main groups animals, no adverse effects were found on oestrous cycle, copulation, fertility, delivery or lactation gestation length, number of corpora lutea or implantation sites.


No adverse effects were found in anogenital distance, pup sex ratios, pup weight, pup viability and no treatment related findings were noted at necropsy. Thyroid weights of the parental animals and of pups on Day 14 post partum were comparable to the control group.


Macroscopic observation and organ weight were unaffected by treatment. No treatment related changes were described following histopathology evaluation in main and satellite groups, including spermatogenic cycle.


Slight changes noted in thyroid hormone levels were considered not related to treatment.


The histopathological examination performed in satellite group animals did not show any changes related to treatment.


The No Observed Adverse Effects Level (NOAEL) for reproductive and developmental toxicity including satellite group animals was considered to be 500 mg/kg/day for both parental animals and offspring, this being the highest dose investigated.


 


In a supporting study, the effects of “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed” were investigated, after oral administration at dose levels of 100, 300 and 500 mg/kg/day, in each 6 Sprague Dawley female rats during pregnancy and on embryofoetal development.


All animals were administered during the gestation period, starting from Day 6 through Day 19 post coitum at the dose volume of 10 mL/kg. Body weight and daily clinical signs were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late intrauterine deaths, live foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities.


No mortality occurred.


All females were pregnant and with live foetuses on Day 20 post coitum.


No treatment-related clinical signs were seen during the study.


No effects on body weight and body weight gain were seen.


No intergroup differences in terminal body weight, uterus weight and absolute weight gain were seen.


Litter data and sex ratio were not affected by treatment.


No treatment-related macroscopic observations in females were recorded.


No abnormalities were seen at the external examination of foetuses, except for malrotated hindlimbs observed in one foetus each in one low (100 mg/kg/day) and mid-dose (300 mg/kg/day) female and in three foetuses in two high dose (500 mg/kg/day) females. Malrotation of hindlimbs could be confirmed by a skeletal evaluation. Rotation of limbs is also present in the Historical Control Data obtained by the Supplier although with a lower incidence, due to the high number of foetuses examined. Further examination could be suggested in order to evaluate the concern of this finding.


In conclusion, no significant treatment-related changes were seen in the in vivo and post mortem data in treated females. The test item was well tolerated up to the dose of 500 mg/kg/day in female rats.


 


Studies on prenatal developmental toxicity via the inhalation or dermal route with the test item are not available.

Effects on developmental toxicity

Description of key information

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 PC-2020-913 was administered orally by gavage to time-mated Crl:CD(SD) rats.

Link to relevant study records
Reference
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
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)
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
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.
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.
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
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
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
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
Key result
Abnormalities:
no effects observed
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
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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

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.

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
725 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available study is a guideline study and is of high quality.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a 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.


Studies on developmental toxicity via the inhalation or dermal route with the test item are not available.

Justification for classification or non-classification

Reliable animal studies report that the test item did not show reproductive toxicity and is thus not classified according to GHS.

Additional information