Reaction product of D-Glucopyranoside, methyl; esterified with oleic acid, methyl ester

Endpoint summary

Description of key information

OECD Guideline 407, Repeated Dose 28-Day Oral Toxicity Study in Rats, NOAEL = 1000 mg/kg bw,

There were no test substance-related alterations in body weights, food consumption, clinical pathology parameters, or organ weights. In addition, there were no test substance-related macroscopic or microscopic findings noted at the scheduled necropsies. No test substance-related neurobehavioral findings (FOB or motor activity alterations) were noted at the end of the dosing and recovery periods.

Key value for chemical safety assessment

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:

2015-01-16 - 2016-06-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Male body weights were 186 g to 225 g. Relative humidity on day 40 was 25.1%. On study day 29 was no water available. Doses on day 7 were calculated based on the study day 0 body weights. No observation at day 35 and 42. No urine sample for female 1882.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

All animals were housed throughout acclimation and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71 ± 5°F (22 ± 3°C) and 50 ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 68.1°F to 71.9°F (20.1°C to 22.2°C) and mean daily relative humidity ranged from 25.1% to 48.3% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

The test substance and vehicle control formulations will be administered orally by gavage. Doses will be administered via syringes (of appropriate volume) equipped with flexible Teflon, steel ball-tipped dosing cannula. The dose volume will be 5 mL/kg.
Test substance and vehicle control formulations will be stirred continuously at room temperature for the duration of the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

An HPLC method using CAD for the determination of test item concentration in formulations containing peanut oil and test substance ranging in concentration from
5.00 to 250 mg/mL was validated in a previous study (Bystry, 2015). In the present study, test substance homogeneity and, following 8 days of refrigerated storage, resuspension homogeneity was assessed in formulations prepared at target concentrations of 6, 20, 50, and 200 mg/mL. In addition, formulations used for dose administration were analyzed to verify test substance concentration acceptability.

Duration of treatment / exposure:

28 consecutive days.

Frequency of treatment:

Once daily.

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals/sex/dose

Control and high-dose groups: 10 animals/sex/dose (5 animals were euthanized following a 14-day nondosing (recovery) period)

Control animals:

yes, concurrent vehicle

Details on study design:

The test substance, in the vehicle (peanut oil) was administered orally by gavage once daily for 28 consecutive days to 4 groups (Groups 2-5) of Crl:CD(SD) rats. Dosage levels were 30, 100, 250, and 1000 mg/kg/day for Groups 2, 3, 4, and 5, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Groups 1 and 5 each consisted of ten animals/sex, and Groups 2-4 each consisted of five animals/sex. Following 28 days of dose administration, 5 rats/sex/group were euthanized; the remaining five rats/sex in the control and high-dose groups were euthanized following a 14-day nondosing (recovery) period.
All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body and food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and locomotor activity data were recorded for 5 animals/sex/group during study week 3 and on all remaining animals during study week 5. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals scheduled for the primary (study day 28) and recovery (study day 42) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.

Observations and examinations performed and frequency:

NEUROBEHAVIOURAL EXAMINATION: yes
FOB assessments were recorded for five animals/sex/group during the final week of test substance administration (study week 3) and on all remaining animals during the final week of the recovery period (study week 5). Testing was performed by the same technicians, whenever possible, without knowledge of the animals’ group assignments, and was performed at approximately the same time each day. The FOB was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB.

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical examinations were performed at the time of dose administration and 1-2 hours following dose administration. During the recovery period, the animals were observed once daily. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals 1 week (± 2 days) prior to randomization, on the day of randomization, weekly (± 2 days) during the study period, and on the days of the scheduled necropsies. Due to social housing, some observations could not be attributed to a single animal. In these instances, the observation was recorded in a separate computer protocol for the social group.

BODY WEIGHT: Yes
Individual body weights were recorded 1 week (± 2 days) prior to randomization, on the day of randomization, on study day 0 (prior to dosing), weekly (± 2 days) during the study period, and on the day prior to the scheduled necropsies (nonfasted). Mean body weights and mean body weight changes were calculated for the corresponding intervals. In addition, individual body weights were recorded for male nos. 1775 and 1777 in the 1000 mg/kg/day group on study day 20. Final body weights (fasted) were recorded on the day of the scheduled necropsies.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Cage food weights were recorded once weekly (± 2 days) beginning following randomization and throughout the study period. Food consumption was calculated and normalized to the number of animals/cage and reported as g/animal/day for the corresponding body weight intervals.

CLINICAL PATHOLOGY: Yes
Blood and urine samples for clinical pathology evaluations (hematology, coagulation, serum chemistry, and urinalysis) were collected at the scheduled necropsies (study days 28 and 42) from animals scheduled for necropsy. The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected at the time of euthanasia via the inferior vena cava of animals anesthetized by
inhalation of isoflurane. Blood was collected into tubes containing potassium (K2) EDTA (hematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

Sacrifice and pathology:

A complete necropsy was conducted on all animals. Animals were anesthetized by isoflurane inhalation and euthanized by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Body weight, body weight change, food consumption, continuous FOB, clinical pathology, and organ weight data were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test. All repeated measures analysis of variance (RANOVA) statistical analyses for total and ambulatory locomotor activity counts were conducted by BioSTAT Consultants, Inc., Portage, MI, using SAS version 9.2 software.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related clinical observations were noted for the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females during the dosing period. Test substance-related increased incidences of red material around the nose were noted for the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females during the dosing period. In addition, test substance-related clear material around the mouth was noted for the 1000 mg/kg/day group males and females, and clear material around the ventral neck was noted for the 1000 mg/kg/day group females. These clinical findings were primarily noted at the time of dosing and/or 1-2 hours post-dosing and generally did not persist to the recovery period. All other clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights were unaffected by test substance administration. There were no statistically significant differences when the control and test substance-treated groups were compared, with the exception of a single statistically significantly lower mean body weight gain noted for the 250 mg/kg/day group males during study days 14-21 compared to the control group; this change was transient, did not occur in a dose-related manner,
and was not considered test substance-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by test substance administration. A statistically significantly lower mean food consumption value was noted for the 1000 mg/kg/day group females during study days 35-41 (recovery period) when compared to the control group; however, the difference from the control group was slight and no test substance-related effects on food consumption were noted during the dosing period. Therefore, this change was not considered test substance-related. There were no other statistically significant differences when the control and test substance-treated groups were compared.

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 or coagulation parameters. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Mean absolute neutrophil and monocyte counts were higher in the 1000 mg/kg/day group males at the recovery necropsy, but these values were within the range of mean values in the WIL Research historical control database. Additionally, there were no alterations in these parameters in the test substance-treated groups at the primary necropsy; thus, these alterations were considered to be due to biologic variability. Statistically significant findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for
interpretative purposes.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on serum chemistry parameters. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Mean aspartate aminotransferase was higher in the 250 mg/kg/day group males at the primary necropsy, but this level was within the range of mean values in the WIL Research historical control database. Additionally, there was no alteration in this parameter in the 1000 mg/kg/day group at either the primary or recovery necropsies; thus, this alteration was considered to be due to biologic variability. The mean glucose level was lower in the 1000 mg/kg/day females at the recovery necropsy; however, the level was within the WIL Research historical control database. Additionally, there was no alteration in this parameter in the primary test substance-treated groups; thus, this alteration was considered to be due to biologic variability.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test substance-related alterations in urinalysis parameters.

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on organ weights. However, the following statistically significant differences were observed when the control and test substance-treated groups were compared. The mean liver/brain weight ratio was higher in the 1000 mg/kg/day group females at the primary necropsy compared to the control group; however, the ratio was within the range of the WIL Research historical control database. Therefore, this apparent weight ratio variation was considered to be due to biologic variation. The mean epididymides/body weight ratio was higher in the 1000 mg/kg/day group males at the recovery necropsy compared to the control group; however, the ratio was within the range of the WIL Research historical control database, there were no notable histologic lesions in the treated animals, and no changes in the primary treated animals. Therefore, this apparent weight ratio variation was considered to be due to biologic variation.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No effects observed

Remarks on result:

other: Highest dosage level tested.

Critical effects observed:

no

Conclusions:

The NOAEL was considered to be 1000 mg/kg/day (the highest dosage level tested).

Executive summary:

A study in accordance with OECD Guideline 407 was conducted to evaluate the potential toxic effects of the test

substance, when administered via gavage to rats for 28 consecutive days, as well as to evaluate the recovery, persistence, or progression of any effects following a 14 day recovery period. This included evaluation of potential neurotoxicity by functional observational battery and motor activity assessment.

The test substance, in the vehicle (peanut oil) was administered orally by gavage once daily for 28 consecutive days to four groups (Groups 2-5) of Crl:CD(SD) rats. Dosage levels were 30, 100, 250, and 1000 mg/kg/day for Groups 2, 3, 4, and 5, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Groups 1 and 5 each consisted of ten animals/sex, and Groups 2-4 each consisted of five animals/sex. Following 28 days of dose administration, five rats/sex/group were euthanized; the remaining five rats/sex in the control and high-dose groups were euthanized following a 14-day nondosing (recovery) period.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body and food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and locomotor activity data were recorded for five animals/sex/group during study week 3 and on all remaining animals during study week 5. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals scheduled for the primary (study day 28) and recovery (study day 42) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.

All animals survived to the scheduled necropsy.

Test substance-related clinical observations were noted during the dosing period and consisted of red material around the nose for the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females, clear material around the mouth for the 1000 mg/kg/day group males and females, and clear material around the ventral neck for the 1000 mg/kg/day group females. Recovery from these clinical observations was evident during the recovery period.

There were no test substance-related alterations in body weights, food consumption, clinical pathology parameters, or organ weights. In addition, there were no test substance-related macroscopic or microscopic findings noted at the scheduled necropsies. No test substance-related neurobehavioral findings (FOB or motor activity alterations) were noted at the end of the dosing and recovery periods.

Based on the results of this study, administration of test item via oral gavage to Sprague Dawley rats for 28 consecutive days was well tolerated at dosage levels of 30, 100, 250, and 1000 mg/kg/day and resulted in nonadverse clinical observations in the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day (the highest dosage level tested).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

good quality

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

Additional information

Repeated Dose - Oral Toxicity

The key study (Haas, 2015) in accordance with OECD Guideline 407 was conducted to evaluate the potential toxic effects of the test substance, when administered via gavage to rats for 28 consecutive days, as well as to evaluate the recovery, persistence, or progression of any effects following a 14 day recovery period. This included evaluation of potential neurotoxicity by functional observational battery and motor activity assessment.

The test substance, in the vehicle (peanut oil) was administered orally by gavage once daily for 28 consecutive days to four groups (Groups 2-5) of Crl:CD(SD) rats. Dosage levels were 30, 100, 250, and 1000 mg/kg/day for Groups 2, 3, 4, and 5, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Groups 1 and 5 each consisted of ten animals/sex, and Groups 2-4 each consisted of five animals/sex. Following 28 days of dose administration, five rats/sex/group were euthanized; the remaining five rats/sex in the control and high-dose groups were euthanized following a 14-day nondosing (recovery) period.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body and food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and locomotor activity data were recorded for 5 animals/sex/group during study week 3 and on all remaining animals during study week 5. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals scheduled for the primary (study day 28) and recovery (study day 42) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.

All animals survived to the scheduled necropsy.

Test substance-related clinical observations were noted during the dosing period and consisted of red material around the nose for the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females, clear material around the mouth for the 1000 mg/kg/day group males and females, and clear material around the ventral neck for the 1000 mg/kg/day group females. Recovery from these clinical observations was evident during the recovery period.

There were no test substance-related alterations in body weights, food consumption, clinical pathology parameters, or organ weights. In addition, there were no test substance-related macroscopic or microscopic findings noted at the scheduled necropsies. No test substance-related neurobehavioral findings (FOB or motor activity alterations) were noted at the end of the dosing and recovery periods.

Based on the results of this study, administration of test item via oral gavage to Sprague Dawley rats for 28 consecutive days was well tolerated at dosage levels of 30, 100, 250, and 1000 mg/kg/day and resulted in nonadverse clinical observations in the 250 and 1000 mg/kg/day group males and the 1000 mg/kg/day group females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day (the highest dosage level tested).

Repeated Dose - Inhalation Toxicity

According to Annex VIII,  testing by the inhalation route is justified if exposure of humans via inhalation is likely. In this case testing by the inhalation route is not justified since the test substance has low vapour pressure (0.00627 Pa at 25 °C), so the potential for the generation of inhalable forms is low, also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be unlikely to occur. For the hazard assessment purpose (DNEL derivation), a NOAEL from the 28 -days Repeated Dose Toxicity study in rats (Haas, 2015),  with application of an appropriate assessment factor, allows extrapolation towards repeated inhalation route administration.

Repeated Dose - Dermal Toxicity

Testing by dermal route is not justified for the test item. The physico-chemical and toxicological properties suggest low potential for significant rate of absorption through the skin (logPow = 6.79 - > 10.0, MW of 458.63 - 1260 g/mol, low water solubility (< 7.68  mg/L at 20°C)). Furthermore the results of laboratory animal studies show low acute dermal and oral toxicity. LD50 values are reported to be greater than 2000 mg/kg bw for the test item (Weinberg, 2015 (A); Weinberg, 2015 (B). In the 28-days repeated dose oral toxicity study in rats (Haas, 2015) no systemic toxicity effects were reported and a NOAEL of 1000 mg/kg bw was derived which suggest bioavailability is low, thereby there is low toxicity potential. The test item was not a skin irritant in a skin irritation study in rabbits (Sanders, 2015 (A)) nor an irritant to the eyes in rabbits (Sanders, 2015 (B)). Based on these results, a dermal repeated dose toxicity study for the test item is considered superfluous. For the hazard assessment purpose (DNEL derivation), a NOAEL from the oral repeated dose 28-days toxicity study in rats (Haas, 2015), with application of an appropriate assessment factor, allows extrapolation towards repeated dermal route administration.

Justification for classification or non-classification

No classification and labelling for repeated dose effects is required for the test item as there were no effects observed in the oral repeated dose toxicity study (Haas, 2015). There were no test substance-related alterations in body weights, food consumption, clinical pathology parameters, or organ weights. In addition, there were no test substance-related macroscopic or microscopic findings noted at the scheduled necropsies. No test substance-related neurobehavioral findings (FOB or motor activity alterations) were noted at the end of the dosing and recovery periods.

Concerning possible systemic effects by dermal route of exposure, they could be ruled out because no significant dermal absorption is expected for the test item due to its physico-chemical properties.

The inhalation route is also a not relevant route of exposure for the test item due to its physico-chemical properties.

Based on these data and in accordance with Regulation (EC) No 1272/2008 (CLP Regulation), classification and labelling is not required for systemic organ toxicity after repeated exposure (STOT-RE) for the test item.