Lecithins, acetylated

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Study included FOB and motor activity endpoints.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (2000)

Deviations:

yes

Remarks:

Study included FOB and motor activity endpoints.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Lecithins, acetylated

EC Number:

293-316-5

EC Name:

Lecithins, acetylated

Cas Number:

91053-50-8

Molecular formula:

Not applicable- complex UVCB substance

IUPAC Name:

91053-50-8

Details on test material:

Purity: Unknown variable composition biological substance (UVCBS)
Composition of test material, percentage of components: Unknown variable composition biological substance (UVCBS)

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 65 days
- Weight at study initiation: 300-357 grams (males) and 215-260 grams (females)
- Housing: Solid-bottom cages contained bedding material, and a nestlet and nylabone for enrichment. Sexes were placed on separate racks (except during mating).
- Diet (e.g. ad libitum): ad libitum except when fasted
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Approximately 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): approximate 12-hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared by mixing the test substance into the vehicle control substance. Formulations were prepared daily. Formulations were stored at room temperature.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of mating was observed or until a period of 2 weeks had elapsed
- Proof of pregnancy: Copulation plug in situ or sperm in vaginal lavage sample referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually. Females that did not mate at the end of the 2-week period were transferred to individual cage housing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test Formulation Sampling: Near the beginning of the study, dose formulations were sampled and analyzed for verification of concentration, homogeneity, and stability. Near the middle and end of the study, dose formulations were sampled for an additional concentration verification check.

Method of Analysis: Analysis was performed by 31P NMR Spectroscopy. Samples were shipped on the day they were collected at ambient temperature for NMR analysis. At the time of analysis, the samples were diluted with an appropriate solvent and analyzed by 31P Nuclear Magnetic Resonance Spectroscopy.

Analyses results show that the test substance was homogeneously mixed at the targeted concentrations, and stable in the vehicle under the storage conditions for all dose levels for the study. Test substance was not detected in the control samples.

Duration of treatment / exposure:

All animals were dosed once daily by gavage for approximately 14 days prior to cohabitation and during the cohabitation period (up to 2 weeks). Male and female rats showing no evidence of copulation continued to be dosed after the end of the cohabitation period until sacrifice. Females showing evidence of copulation were dosed throughout gestation. Pregnant females in the process of delivery or showing signs of delivery were not dosed. Females were dosed after delivering litters, until day 3 postpartum. Females that did not deliver a litter continued to be dosed until the day before sacrifice.

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: 79 days
Female rats were observed at least twice daily for signs of delivery and offspring beginning GD 20. Each litter was examined as soon as possible after delivery was completed. The day when delivery was complete was designated LD 0 for dams and postnatal day (PND) 0 for pups. On PND 0 and 4 (day of litter sacrifice), live and dead pups in each litter were counted by sex and live pups were individually weighed. Each live pup was individually handled and examined for abnormal behaviour and an external examination conducted. Dead pups were examined externally to the extent possible and underwent a gross postmortem examination. Pups were euthanized by decapitation on PND 4. Carcasses were discarded without gross pathological examination. If litters died prior to PND 4, the female was sacrificed. If the female died prior to LD 4, the litter was examined externally and euthanized and discarded without gross pathological examination.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

12 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previous acute oral toxicity study (up and down procedure) in which the test substance was dosed neat, the LD50 value was determined to be > 5000 mg/kg in female rats. Based on this data, doses of 100, 300, and 1000 mg/kg/day were selected for the current study. These dose levels include the limit dose level of 1000 mg/kg/day as specified by the relevant testing guidelines. The low and intermediate dose levels were selected to enable detection of potential dose responses across a wide range of dose levels.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily, at least 2 hours post-dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to dosing on test day 0 (baseline) and weekly thereafter at approximately the same time of day (± 2 hours); mortality and moribundity check twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing on test day 0 and weekly during premating and postmating; gestation days (GD) 0, 7, 14, and 20 and lactation days (LD) 0 and 4 and at neurobehavioral evaluations and scheduled sacrifice

FOOD CONSUMPTION:
- Food consumption for each animal determined (g/food/animal/day): Yes; Weekly during premating GD 0, 7, 14, and 20 and LD 0 and 4. Food consumption was not measured during the postmating period for males, and females that did not mate.

FOOD EFFICIENCY:
- Calculated as g weight gained/g food consumed: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Test day 14
- Anaesthetic used for blood collection: Yes; Carbon dioxide or Isoflurane
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Test day 14
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters in table No. 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes (refer to Section 7.9.1, DI.K1.Neuro.D-18511-1422 for additional details).

OTHER: Coagulation
- Time schedule for collection: at sacrifice
- How many animals: 5/sex/group
- Anaesthetic used: Carbon dioxide

Oestrous cyclicity (parental animals):

Not evaluated

Sperm parameters (parental animals):

Not evaluated

Litter observations:

Each litter was examined as soon as possible after delivery was completed. The day when delivery was complete was designated lactation day (LD) 0 for dams and postnatal day (PND) 0 for pups. On LD 0 and 4 (day of litter sacrifice) live and dead pups in each litter were counted by sex and live pups were individually weighed. Each live pup was individually handled and examined for abnormal behaviour and an external examination was conducted.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 3)

Gross examinations were performed on all adult rats. Final body and organ weights were recorded for all rats euthanized at the scheduled sacrifice. Microscopic examination of all reproductive organs was conducted for all rats in the control (0 mg/kg/day) and high-treatment (1000 mg/kg/day) groups and for all reproductive failures (male and female pairs). One mated pair (300 mg/kg/day) required a caesarean section and was also evaluated microscopically. Microscopic examination of all remaining tissues collected was limited to the first 5 surviving rats in the males and female high-treatment and control groups. All collected tissues were also examined microscopically for the 2 decedents. Most gross lesions recorded at necropsy were evaluated microscopically. Gross lesions which were unlikely to have a microscopic correlate (e.g., wet, stain) were not examined.

Postmortem examinations (offspring):

Dead or moribund pups were examined externally to the extent possible and underwent a gross post-mortem examination. Pups surviving to scheduled sacrifice on postnatal day (PND) 4 were discarded without gross pathological examination.

Statistics:

Statistical analysis was conducted such that data from each group were compared to the vehicle control group using an appropriate pairwise analysis (see Table 4). Male and female data were evaluated separately. For litter parameters, the proportion of affected pups per litter or the litter mean were used as the experimental unit for statistical evaluation. The level of significance selected was p <0.05. For each parameter analyzed with a trend test, the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected.

Reproductive indices:

Refer to Table 5 for reproductive indices calculated.

Offspring viability indices:

Refer to Table 5 for offspring viability indices calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): Refer to Section 7.5.1, DI.K1.28Day.Gav.Screen.RD/REPRO/DEV.R.D-18511-1422.KD for details.

HAEMATOLOGY: Refer to Section 7.5.1, DI.K1.28Day.Gav.Screen.RD/REPRO/DEV.R.D-18511-1422.KD for details.

CLINICAL CHEMISTRY: Refer to Section 7.5.1, DI.K1.28Day.Gav.Screen.RD/REPRO/DEV.R.D-18511-1422.KD for details.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose tested.

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

not measured/tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Remarks on result:

other: no effects observed in measured endpoints at the highest dose tested

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL = 1000 mg/kg/bw/day

Executive summary:

The objective of this study was to evaluate the potential subchronic and reproductive/developmental toxicity of the test substance when administered by oral gavage to male and female rats during premating, cohabitation, gestation, until postnatal day (PND) 3. Four groups of parental (P₀) male and female Crl:CD(SD) rats (12/sex/group) were administered formulations of the test substance via once daily oral gavage for 14 consecutive days prior to mating, throughout mating, and then continuing through one day prior to the day of euthanasia. The dose levels administered were 0, 100, 300, or 1000 mg/kg/day. Samples of the dose formulations were analyzed and confirmed that the formulations were at targeted concentrations, homogeneous, and stable under the conditions of use.  During the in-life phase of the study, all animals were observed twice daily for appearance and behaviour. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for P₀ males throughout the study and for P₀ females prior to mating and during gestation and lactation. Neurobehavioral examinations consisting of an abbreviated functional observational battery (FOB) and motor activity (MA) were performed for all P₀ animals once prior to dosing to provide baseline data and once more near the end of the premating period. In addition, clinical pathology data consisting of haematology, coagulation, and clinical chemistry data were collected prior to the end of the premating period. All P₀ females were allowed to deliver and rear their pups until weaning on PND 4. Each P₀ parental animal received a complete detailed gross necropsy and selected organs were weighed and/or retained for histopathologic examination.

 

There was no evidence of test substance-related toxicity at any dose level tested. The in-life data, including body weight and food consumption parameters, clinical observations, neurobehavioural endpoints (FOB and MA), clinical pathology, as well as reproductive performance data including mating and fertility indices, gestation length, offspring viability, litter sizes, litter sex ratio, and pup weights, were comparable across all groups throughout the study. In addition, there were no test substance-related effects on either gross or microscopic histopathology or on organ weights. 

 

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for reproductive toxicity was 1000 mg/kg/day. This conclusion is based on the lack of any evidence of any adverse and/or test substance-related effects at any dose level tested.