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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 18-MAR-2010 to 19-AUG-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study performed according to OECD guideline and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Vegeflux soy
- Substance type: UVCB
- Physical state: liquid
- Stability under test conditions: data not available
- Storage condition of test material: at room temperature, keep away from heat and dampness

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Velaz Praha, Czech Republic
- Age at study initiation: adult
- Weight at study initiation: the mean body weight of animals at the start of experiment was in the males 237.71g, in the females 198.96g
- Fasting period before study: no
- Housing: all animals were kept in cages with the bedding in experimental animal house with the standard conditions.
The keeping of the animals was organised to the time table:
1. 4 animals in the cage (males and females separately)
2. 1 male and 1 female in the cage (mating procedure)
3. 1 pregnant female individually
4. 1 female and offspring individually
After the mating procedure males were returned to the original cages
- Diet: conventional laboratory diet
- Water: ad libitum
- Acclimation period: for 6 days before the study in the condition identical to the condition during the experiment

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 45 to 65%
- Air changes: data not available
- Photoperiod: 12-hour light / 12-hour dark cycle

IN-LIFE DATES: from 18.3.2010 to 14.5.2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the test substance was dissolved in the olive oil every day, before the application.

VEHICLE
- Justification for use and choice of vehicle: Vegeflux Soy is oil; therefore olive oil was used as the vehicle.
- Concentration in vehicle: data not available
- Amount of vehicle (if gavage): fixed application volume of 0.5mL/100g body weight
Details on mating procedure:
Normally, 1:1 (1 male to 1 female) mating was used in this study. The female was placed with the same male until the pregnancy occurs or 14 days have elapsed. Every morning during the mating period the females were examined for the presence of sperms or vaginal plugs. The day 0 of pregnancy was defined as the day a vaginal plug or sperm are found. Pregnant female was keeping individually up to 4th day post-partum. In case of pairing was unsuccessful, re-mating of females with the successful males of the same group was considered.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Application period:
- Males: 24.3.2010 – 4.5.2010 (42 days, beginning 14 days before mating)
- Females: 24.3.2010 – 13.5.2010 (51 days, beginning 14 days before mating)

Daily dosing of both sexes began 14 days before the mating procedure. The dosing was continued in the both sexes during the mating period (max. 14 days – 2 complete estrus cycles). Daily dosing of males were continued 14 days after the mating process still (42 days), and then were humanely killed. The dosing of females was continued throughout the pregnancy and up to 4th day after the birth. Females showing no-evidence of copulation were dosing 24 days after the last day of the mating period. They were humanely killed, too. Females with offspring were sacrificed on day 4th post-partum.
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 500 a,d 1000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Twelve males and twelve females were used in control and in all dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Selection of the doses was estimated from the available information about the test article.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day 1 hour after the test article application.
- Cage side observations: health condition of all animals, reaction of animals to applied substance, and their condition were monitored every day and recorded. Number of pregnant females was recorded.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: once a week
Oestrous cyclicity (parental animals):
The uterus and ovaries were weighed during the necropsy. The samples from these organs with prostate and glands vesicles were processed for the microscopic examination.
At time of the death the animals were examined macroscopically for any abnormalities or pathological changes. The special attention was given to the organs of reproductive system and to the liver and kidneys as the target organs. In females the number of corpora lutea and count of implantation sites were monitored.
The detailed histological examination was performed on uterus, ovaries, liver and kidneys (target organs) in all females of the control (olive oil) and all dose groups (1000mg.kg-1 Vegeflux Soy).
Sperm parameters (parental animals):
Sperm samples from left epididymis of control and high dose group males were obtained.
Motility of sperms was measured. Two hundred sperms per animal were evaluated for head and flagellar defects.
The testes and epididymis were weighed during the necropsy. The samples from these organs with prostate and glands vesicles were processed for the microscopic examination.
At time of the death the animals were examined macroscopically for any abnormalities or pathological changes. The special attention was given to the organs of reproductive system and to the liver and kidneys as the target organs. In males of control and highest dose groups left of epididymis for examination of sperm parameters were used. Sperm comparative concentration, motility and morphology were monitored.
The detailed histological examination was performed on testes, epididymes, prostate, liver and kidneys (target organs) in all males of the control (olive oil) and all dose groups (1000mg.kg-1 Vegeflux Soy).
Litter observations:
Number of live and death pups, weight of litters and sex of pups were observed.
Live litters was weighting within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum.
Offspring were sexed on 4th day post-partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals (after 14 days of the mating procedure)
- Maternal animals: All surviving animals (pregnant females after 4 day post-partum, and non-pregnant females 24th day after the mating procedure)

GROSS NECROPSY
At time of the death the animals were examined macroscopically for any abnormalities or pathological changes. The special attention was given to the organs of reproductive system and to the liver and kidneys as the target organs. In females the number of corpora lutea and count of implantation sites were monitored. In males of control and highest dose groups left of epididymis for examination of sperm parameters were used (3, 15). Sperm comparative concentration, motility and morphology were monitored.

HISTOPATHOLOGY / ORGAN WEIGHTS
The detailed histological examination was performed on uterus, ovaries, testes, epididymes, prostate, liver and kidneys (target organs) in all males and females of the control (olive oil) and all dose groups (1000mg.kg-1 Vegeflux Soy). The histopathological examination was made by the standard operation procedures.
The liver, kidneys (target organs), testes, epididymis, uterus, and ovaries were weighed during the necropsy.
Statistics:
Results obtained during the study were statistically evaluated by the statistical program Statgraphics. Statistical evaluated was made separately for the males, females, and litters. For the identification homogeneity the groups were used the Bartlett’s test. In the case of homogeneity One-Way Analysis of variance with consecutive Multi Rangers tests was accomplished. In the case non homogeneity Kruskal-Wallis One-Way Analysis by Ranks was applied. For the sperm parameters Student’s t-test were used.
Reproductive indices:
Fertility Index = (Number of females with implants / Number of females mating) x 100
Gestation (Pregnancy) Index = (Number of females delivering live young / Number of females with evidence of pregnancy) x 100
Offspring viability indices:
Live Birth Index = (Number of live offspring / Number of offspring delivered) x 100
Viability (Survival) Index = (Number of live offspring at lactation day 4 / Number of live offspring delivered) x 100

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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
During the study any deaths loss of animals were observed. All animals lived through observation without important visible signs of intoxication. There were registered the temporary occurrence of alopecia (3 females in dose group 200mg.kg-1, 2 females in dose group 500mg.kg-1) and sporadic of smooth stool (1 male in control group, 2 males in dose group 200mg.kg-1, 1 male and 1 female in dose group 500mg.kg-1, 3 males and 1 female in dose group 1000mg.kg-1). The temporary lethargy in three males was registered too. These incidences were not being indicated relation to the test article.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
The average of the body weight at the beginning of study was 256.88g in males, 198.96g in females. During study the body weight of all animals was increased. After the statistical evaluation of the body weight significant differences between control group and all dose groups of both sexes were not detected.
A normally laboratory diet was served according to recommended daily dose. Intake of drinking water was unlimited. The food intake in the males and females of all dose groups were similar with the control groups.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS):
The pregnancy of 22 days was established in 4 females of control group, 6 females on low dose group, 7 females of medium dose group, and 9 females of highest dose group. The pregnancy of 23 days was established in 6 females of control group, 5 females of low dose group, 3 females of medium dose group, and 1 female of highest dose group. The length of pregnancy 24 days was observed in 1 female of dose group 500mg.kg-1.
All indices of reproductive parameters in female control group and all dose groups were stable.
The number of corpora lutea and implantations sites was monitored and any statistical differences in these parameters were observed.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS):
There were not significant differences between control and highest dose group regarding the following sperm parameters: comparative concentration, motility, and pathology.
Pathological changes of sperms were determined by morphology analysis. Changes of smears indicated any differences between control and highest dose group, too.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
The copulation of majority females control and all dose groups were established during the first five days.

ORGAN WEIGHTS (PARENTAL ANIMALS):
Significant increase of liver relative weight in males high dose group against control group was observed.

GROSS PATHOLOGY (PARENTAL ANIMALS):
Macroscopical lesions were observed during necropsy of rats. Hypoplasia of testes was found in 1 male at 200mg.kg-1. Histopathological examination was not confirmed any changes in the spermiogenesis. Lighter color of the liver was observed in 1 female at 1000mg.kg-1.

HISTOPATHOLOGY (PARENTAL ANIMALS):
In all animals the kidney and liver were evaluated as the possible target organs, too. In females of control and highest dose group pathological changes related to the effect of test article were not observed. In males significant increase of liver relative weight in highest dose group, and degenerative (but not necrotic) changes (nephropathy) of kidneys in all dose groups against control group were observed.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for reproduction/developmental parameters of males and females

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:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Alive and dead pups were counted on day 1 and 4 post-partum. In control group 105 live-born and 11 dead-born pups were observed. In dose group 200mg.kg-1 112 alive and 4 dead pups were observed after the birth. In dose group 500mg.kg-1 were observed 93 alive and 14 dead pups after the birth. In dose group 1000mg.kg-1 102 alive and 12 dead pups after the birth were found. Up to 4th day from the delivery died in control group 15 (No 1 – 9 pups, No 5 – 6 pups), in low and medium dose groups 1 (No 21 in low dose group, No 25 in medium dose group), and in high dose group 2 pups (No 38, and 41).
After the 4th day post-partum the mean count of live pups in one female of control and dose group 500mg.kg-1 8, in dose group 200 and 1000mg.kg-1 10 was observed.
The litters were weighted on day 1 and day 4th post-partum. The mean weight of litter after the birth was 65.50g in control group, 62.27g in low dose group, 51.82g in medium dose group, and 62.50g in highest dose group. The mean weight of litter determined on day 4th post-partum was 83.00g in control group, 90.91g in low dose group, 76.36g in medium dose group, and 85.50g in high dose group.
Offspring were sexed on 4th day post-partum. The mean sex ratio was 5 males/3 females in control group, 5 males/5 females in dose groups 200 and 1000mg.kg-1, and 4 males/4 females in dose group 500mg.kg-1.
The health condition of all pups was standard.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL (No-Observed-Adverse-Effect Level) for reproduction/developmental parameters of males and females determined in this study is > 1000mg.kg-1 bw/day, excluding the findings of male rat nephropathy.
Executive summary:

This study was conduced to provide initial information concerning possible effects of Vegeflux Soy on reproduction and/or developmental when administered orally, by gavage, to male and female rats using an OECD 421 protocol (according to GLP).

The Wistar rats, 48 adult males and 48 adult females were divided to four groups.Vegeflux Soywas administered at doses of 200, 500 and 1000 mg.kg-1in olive oil;the control group of rats receivedthe vehicle at an equivalent volume. The administered dose was calculated on the current body weight. Fixed application volume 0.5mL/100g body weight was administered.

The males were treated 42 days: 14 days before the mating procedure, 14 days during the mating procedure, and 14 days after the mating procedure. The females were exposed to effect of Vegeflux Soy 41 – 51 days: 14 days before the mating procedure, at most 14 days during the mating process, 22 - 24 days the gestation, and 4 days during the lactation.

All animals were weighted weekly. The litters were weighted 24 hours after the birth and on day 4 post-parturition. All animals were monitored for the signs of toxicity during the application of the test substance. During study clinical observation and food consumption were recorded. After finishing of the test all animals were sacrificed and moved to the histological evaluation. The results were elaborated in form of tables.

 

There was no test article-related mortality of animals during the study. There were no clinical signs of toxicity in all animals during observation period. The temporary occurrence of alopecia and smooth stool in animals were registered. The lethargy in three males (one male in each tested group except control group) was registered, too. These incidences were not being indicated relation to the test article treatment.

The body weight of all animals moderately increased during the study. There were not significant differences between of all tested groups and the control groups. The food consumption in all animals was adequate.

After 8th day of beginning mating procedure two males were replaced (one in dose group 500mg.kg-1, and one in dose group 1000mg.kg-1). The length of pregnancy in all females was 22 – 23 days (in one female of dose group 500mg.kg-1 - 24 days).

Pup body weigh, external observation and necropsy data were comparable among the groups.  In litters of all dose levels were not found any abnormal pups. 

Copulation and fertility indices, precoital intervals, gestation lengths and pregnancy rates were comparable among the groups and no signs of prolonged delivery or unusual nesting behaviours were noted.

The indices of reproductive parameters in all dose groups against control group and number of corpora lutea and implantation sites in all females were stable.

At termination of study in males of control and highest dose groups left of epididymis for examination of sperm parameters were used. Sperm comparative concentration, motility and morphology were monitored. These parameters in highest dose group against control group were comparable.

Detailed histological examination was performed on reproduction organs of the males and females control and highest dose groups. We have not seen pathological changes related to the effect of Vegeflux Soy.  

In all animals the kidney and liver were evaluated as the possible target organs, too. In females of control and highest dose group pathological changes related to the effect of test article were not observed. In males significant increase of liver relative weight in highest dose group, and degenerative (but not necrotic) changes (nephropathy) of kidneys in all dose groups against control group were observed.

The  NOAEL (No-Observed-Adverse-Effect Level) for reproduction/developmental parameters of males and females determined in this study is > 1000mg.kg-1 bw/day, excluding the findings of male rat nephropathy.