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

Endpoint summary

Description of key information

In an OECD 422 and GLP study, no systemic toxicity was observed by oral route, NOAEL was the highest dose tested (500 mg/kg bw/d).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

Route of administration:

oral: gavage

Details on route of administration:

The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days. Females were dosed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until 1 day prior to scheduled euthanasia. Females with no evidence of mating were dosed through the day prior to euthanasia. All animals were dosed at approximately the same time each day.
The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Vehicle:

water

Details on oral exposure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

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

Observations and examinations performed and frequency:

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, neurobehavior, motor activity, thyroid hormones, clinical pathology, gross necropsy findings, organ weights, and histopathologic examinations

Other examinations:

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

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

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

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

Endocrine findings:

no effects observed

Description (incidence and severity):

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

Urinalysis findings:

no effects observed

Description (incidence and severity):

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

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were
unaffected by test substance administration at all dosage levels

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

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 rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of Sophorolipids.
The mean numbers of implantation sites and post-implantation loss in the 100, 250, and 500 mg/kg/day groups were similar to the control group values, with the following exception. Mean post-implantation loss in the 500 mg/kg/day group was statistically significantly higher than the control group; however, the difference was slight and the mean number of live newborn pups in the 500 mg/kg/day group was higher than the control group. Therefore, this was not considered test substance-related.

Neuropathological findings:

no effects observed

Description (incidence and severity):

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

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

ophthalmological examination

organ weights and organ / body weight ratios

serum/plasma hormone analyses

urinalysis

Key result

Critical effects observed:

no

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD 422 and GLP study.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Given the absence of systemic toxicity in the OECD 422 study, no classification for STOT RE is warranted for the substance.