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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-21 - 2016-03-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

946-364-2

Molecular formula:

C43H78O8

IUPAC Name:

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

Test material form:

liquid

Details on test material:

- State of aggregation: yellow liquid
-Others: storage at room temperature, in the dark

Specific details on test material used for the study:

see "Test material information"

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Remarks:

Sprague-Dawley

Details on species / strain selection:

This species and strain of animal is recognized as appropriate for reproductive toxicity studies. WIL Research has reproductive historical control data in the Crl:CD(SD) rat. 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:

Controls will be set to maintain temperature at 71 ± 5°F (22 ± 3°C) and relative humidity at 50 ± 20%. Temperature and relative humidity will be monitored continuously. Data for these two parameters will be scheduled for automatic collection on an hourly basis. Fluorescent lighting controlled by light timers will provide illumination for a 12 hour light/dark photoperiod. The ventilation rate will be set at a minimum of 10 room air changes per hour, 100% fresh air.

Reverse osmosis-purified water will be available ad libitum.

PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 will be offered ad libitum during the study except during fasting of males and recovery phase females prior to blood collection for clinical chemistry as described in section 7.9.6.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily. The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated, protected from light. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. On each dosing day, aliquots were stirred at room temperature for at least 30 minutes prior to dispensation for dosing. The first test item dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

VEHICLE
The vehicle used in preparation of the test item formulations and for administration to the control group was arachis (peanut) oil, NF (lot nos. 2EA0347 and 2EA0218, exp. date: 31-Jan-2016). The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored refrigerated (2°C to 8°C), protected from light. The vehicle was mixed throughout the sampling and dose administration procedures. On each dosing day, aliquots were stirred at room temperature for at least 30 minutes prior to dispensation for dosing.

Details on mating procedure:

The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was cohabitated with 1 male in a solid-bottom cage containing bedding material. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages. For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the test formulations will be performed by the Analytical Chemistry Department at WIL Research using a validated method (Bystry, WIL-168232, Draft). In this study, the test substance was determined to be stable in the vehicle under conditions utilized on this study at a concentration range of 5 to 230 mg/mL. In addition, resuspension homogeneity was assessed at the same concentrations in a previous 28-Day Toxicity study (Haas, WIL-168233, Draft). For the current study, samples will be taken from the top, middle and bottom strata of the first and last control and test substance formulations prepared during the in-life phase of the study (middle samples only will be collected from the control group formulations). Samples will be analyzed to verify the homogeneity and concentration of the test substance in the formulations; any remaining samples will be stored refrigerated as backup samples. Any backup samples kept at WIL Research will be discarded following acceptance of the analytical results by the Study Director.

Duration of treatment / exposure:

The males selected for pairing were dosed during study days 0-28 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 29 doses. The females selected for pairing were dosed during study days 0 through the day prior to
euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-43 doses. Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses. The extra 5 rats/sex in the control and
high-dose groups were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 29 doses for males and 39 doses for females, these animals remained on study for a 14-day recovery period. The dosage volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.

Frequency of treatment:

The males selected for pairing were dosed during study days 0-28 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 29 doses. The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-43 doses. Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses. The extra 5 rats/sex in the control and high-dose groups were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 29 doses for males and 39 doses for females, these animals remained on study for a 14-day recovery period. The dosage volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Dose (mg/kg/day):
0 (15 males and 15 females)*
30 (10 males and 10 females)
100 (10 males and 10 females)
250 (10 males and 10 females)
1000 (15 males and 15 females)*

*5 animals/sex in Groups 1 and 5 were necropsied following a 14-day recovery period. Recovery animals were not evaluated for reproductive performance.

Control animals:

yes, concurrent vehicle

Details on study design:

Dosage levels were selected based on the results of a previous 28-day study in rats (Haas, 2015, WIL-168233). In that study, the test item was administered to male and female rats for 28 consecutive days at dosage levels of 30, 100, 250, and 1000 mg/kg/day. There were no significant clinical signs noted at any dosage level. In addition, body weights and food consumption were unaffected by treatment.

Examinations

Litter observations:

LITTER VIABILITY AND DEATHS, CLINICAL OBSERVATIONS, BODY WEIGHTS, SEX DETERMINATION

Postmortem examinations (parental animals):

Complete postmortem examinations were performed on all F0 parental animals euthanized at the scheduled necropsies. Animals were euthanized by carbon dioxide inhalation and exsanguinated. At the time of necropsy, the following tissues and organs were collected and placed in 10% neutral-buffered formalin fixative unless otherwise noted:
Adrenals (2)
All gross lesions (all groups)
Aorta
Bone with marrow
(sternebrae)
Brain
Coagulating glands (2)
Eyes with optic nerves (2)
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys (2)
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed
by inflation with fixative)
Lymph nodes
Axillary (2)
Mandibular (2)
Mesenteric
Ovaries with oviducts (2)
Pancreas
Peripheral nerve (sciatic)
Pituitary
Prostate
Salivary glands [mandibular (2)]
Seminal vesicles (2)
Skeletal muscle (rectus femoris)
Skin with mammary gland
Spinal cord (cervical)
Spleen
Testes with epididymides (2)
and vas deferens
Thymus
Thyroid with parathyroids (2)
Trachea
Urinary bladder
Uteruse with cervix and vagina

The following organs were weighed from all animals at the scheduled necropsies:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries (with oviducts)
Spleen
Testes
Thymus
Thyroid with parathyroids

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 item-treated group to the control group by sex.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males: Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day group males were unaffected by test item administration throughout the study. A significantly (p<0.05) lower mean body weight gain was noted for males in the 250 mg/kg/day group during study days 13-20 and significantly (p<0.05) higher mean body weight gains were noted for males in the 100 and 1000 mg/kg/day groups for the pre-mating period (study days 0-13). However, these results did not occur in a dose-related manner and did not affect mean absolute body weights. No other differences from the control group were statistically significant.
Females (Pre-mating): Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating period (females selected for breeding) or the entire study (females selected for recovery phase). Significantly (p<0.05) higher mean body weight gains were noted for females in the 1000 mg/kg/day group during study days 34-38 and 46-51. However, these results were transient and did not affect mean absolute body weights. No other differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in hematology and coagulation parameters at the scheduled necropsies. However, some statistically significant (p<0.05) differences were observed when the control and test substance-treated groups were compared. Lower absolute basophil values were noted for females in the 100 and 1000 mg/kg/day groups at the primary necropsy. There was no dose-response relationship, values were of minimal magnitude difference, and were similar to the control group at the recovery necropsy; the differences were attributed to biological variability. Higher prothrombin time (PT) values were noted in the 1000 mg/kg/day group males at the recovery necropsy. Values were of minimal magnitude change and were similar to control and 1000 mg/kg/day group values at the primary necropsy; the finding was attributed to biological variability and not related to administration of test item.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in serum chemistry parameters at the scheduled necropsies. However, some statistically significant (p<0.01 or p<0.05) differences were observed when the control and test item-treated groups were compared. Lower serum chloride values were noted in the 1000 mg/kg/day group males at the primary necropsy. Other serum electrolyte values were similar to the control group, the difference was of minimal magnitude, and there were no histologic correlates; the finding was attributed to biological variability. Higher serum urea nitrogen values were noted in the 1000 mg/kg/day group males and higher serum triglyceride values were noted in the 1000 mg/kg/day group females at the recovery necropsy. Both values were similar to the control group at the primary necropsy and were of minimal magnitude difference; the findings were attributed to biological variability and not related to administration of test item. Other statistically significant differences lacked a dose-response relationship, and were attributed to biological variability.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed (highest dose tested).

Results: F1 generation

General toxicity (F1)

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

NECROPSIES OF PUPS FOUND DEAD: The numbers of pups (litters) found dead during PND 0-4 numbered 1(1), 0(0), 3(2), 3(2), and 0(0) in the control, 30, 100, 250, and 1000 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.
GENERAL PHYSICAL CONDITION: The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental administration of the test item. Pups that were found dead numbered 1, 0, 3, 3, and 0 in the control, 30, 100, 250, and 1000 mg/kg/day groups, respectively. One, 1, 0, 1, and 3 pups in the same respective groups was missing and presumed to have been cannibalized.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level (highest dose tested).

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL for neonatal and reproductive toxicity was 1000 mg/kg/day (the highest dosage tested).

Executive summary:

A Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity

Screening Test in Rats, with Recovery, has been conducted in accordance with OECD Guideline 422 and GLP compliance to

evaluate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.

The test item, in the vehicle (arachis [peanut] oil) was administered orally by gavage once daily to four groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of fifteen rats/sex. Dosage levels were 30, 100, 250, and 1000 mg/kg/day. A concurrent control group of fifteen rats/sex received the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups. Males and females were approximately ten weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to

euthanasia for a total of 29 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-43 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 29 doses for males and 39 doses for females, these animals were assigned to a 14-day non-dosing recovery

period.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for five males/group following approximately 28 days of dose administration and for five females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on five F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period or 14-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 14-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups at the primary necropsy.

All F0 males and females in the control, 30, 100, 250, and 1000 mg/kg/day groups survived to the scheduled necropsies. No test item-related clinical findings were noted for males or females at the detailed physical examinations or approximately 1 hour following dose administration at any dosage level.

Mean F0 male and female body weights, body weight gains, and food consumption in the test item groups were unaffected by test item administration throughout the study.

There were no test item-related effects noted during the FOB or motor activity evaluations for F0 animals at any dosage level.

Mean male and female mating, fertility, copulation, and conception indices in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration. The mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration.

There were no test item-related gross necropsy observations, microscopic findings, or effects on organ weights noted for F0 males and females at any dosage level during the dosing and recovery periods. No test item-related alterations in clinical pathology parameters (hematology, coagulation, and serum chemistry) were noted for F0 males and females in the 30, 100, 250, and 1000 mg/kg/day groups during the dosing and recovery evaluations.

Mean numbers of former implantation sites and F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, and pup body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration.

Under the conditions of this screening study, no test item-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level.

Therefore, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 30, 100, 250, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day

based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.