complex mixture from biological origin

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data for the substance "Sophorolipids: fermentation products of glucose and fatty acids, C18 (unsaturated), glycerol esters with yeast Starmerella bombicola, hydrolysed" on toxicity to reproduction is available. Therefore, data from a suitable read-across partner, was used to assess the toxicitiy to reproduction for the target substance. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

No effects on reproductive toxicity were observed in an OECD 421. The No Observed Adverse Effects Level (NOAEL) of the source substance for reproductive and developmental toxicity including satellite group animals was considered to be 500 mg/kg bw/day (for active ingredient) for both parental animals and offspring, this being the highest dose investigated. Dose levels have been selected based on information from a previous repeated dose toxicity study (Rossiello 2018, OECD 407, LOAEL 1000 mg/kg/day).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

OECD 421

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

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.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

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.

Histopathological findings: neoplastic:

no effects observed

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:

test mat.

Remarks:

in terms of test item corrected for solid content (47.2%)

Sex:

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:

test mat.

Remarks:

in terms of test item corrected for solid content (47.2%)

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:

Under the experimental conditions of this study, 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 active ingredient) for both parental animals and offspring, this being the highest dose investigated.

Executive summary:

In a reproduction/developmental toxicity screening test according to OECD guideline 421, the test substance “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed” (47.2%) was orally administered to Sprague Dawley rats (10/sex/dose) at doses of 1, 100, 300 and 500 mg/kg bw/day. Dose levels have been selected based on information from a previous repeated dose toxicity study (Rossiello 2018, OECD 407)

Males of groups 1 to 4 were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 29 or 30 days. Females of groups 1 to 4 were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum. The following investigations were performed in all groups: mortality check, clinical signs, bodyweight, bodyweight gain, food consumption and mating performance and thyroid hormone determination only for parental males and pups at day 14 post partum (1 pup/sex/group), litter data, macroscopic observations, organ weights. Clinical signs, anogenital distance, external and internal examination of pups at necropsy were also recorded. The histopathological examination was performed only on control and high dose group animals and included identification of the stages of the spermatogenic cycle. In addition, 2 satellite groups (control and high dose) were included for specific histopathology investigations (spleen, thymus and bone marrow) and sacrificed after 4 weeks of treatment.

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 active ingredient) for both parental animals and offspring, this being the highest dose investigated.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

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

Effects on developmental toxicity

Description of key information

No data for the substance "Sophorolipids: fermentation products of glucose and fatty acids, C18 (unsaturated), glycerol esters with yeast Starmerella bombicola, hydrolysed" on developmental toxicity is available. Therefore, data from a suitable read-across partner was used to assess the developmental toxicity for the target substance. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Based on the absence of adverse maternal or fetal effects in an OECD 414 study, a dosage level of 725 mg/kg bw/day (for active ingredient) was considered to be the no‑observed‑adverse‑effect level (NOAEL) for maternal toxicity and embryo/fetal development when the source substance “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida bombicola, partially hydrolysed” (solid content: 46.6%) was administered orally by gavage to time-mated Crl:CD(SD) rats. Dose levels have been selected based on information from a previous repeated dose toxicity study (OECD 407) and an OECD 414 DRF study (both Rossiello, 2018).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

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:

test mat.

Remarks:

in terms of test item corrected for solid content (46.6%)

Remarks on result:

other: no adverse effects at highest dose

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:

test mat.

Remarks:

in terms of test item corrected for solid content (46.6%)

Sex:

male/female

Remarks on result:

other: no adverse effects at highest dose

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 (for active ingredient) was considered to be the no observed adverse effect level (NOAEL) for maternal toxicity and embryo/fetal development when 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" (46.6% solid content) 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 (based on solid content of 46.6%. Animals were dosed via oral gavage once daily during Gestation Days 6–20. The dose levels were selected based on a LOAEL of 1000 mg/kg bw/day form a GLP-compliant 28 -day study in Sprague Dawley CD rats (Rossiello, 2018) and an OECD 414 DRF study (Rossiello, 2018).

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 (for active ingredient) 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.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

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

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse effects on fertility or developmental toxicty were reported. The substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008.