Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance to OECD guideline
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Physical state: solid, white crystalline powder
- Batch No.: 110526S
- Expiry date: 26.05.2013
- Purity: 99.24% (taken as 100%)
- Instructions for storage: Room temperature, tighly closed container, cool, dry, only in original container
- Colour: white
- Odour: odourless

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany, D-97633 Sulzfeld
- Age at purchase: 11 weeks
- weight rage at time of grouping: males, 175-200 g
- Fasting period before study: no
- Housing: 2 per cage,
- Cages: TECHNIPLAST filter top cages type 2145 F with an G-Temp (PSU) durable filter cover, 480x265x210 mm³, floor area 940 cm²,
- Source: Techniplast Company, Italy
- Diet: ad libitum, M3, BONAGRO Ltd., reg. CZ 10174, Czech Republic
- Water: ad libitum, tap water
- Acclimation period: 9 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE: suspension in water
- Amount of vehicle: 10 ml/kg at similar times each day
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1 (probably) (male animals: see Endpoint study record in section 7.5.1 "Repeated dose Toxicity")
- Length of cohabitation: until copulation/ up to 14 days
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- M/F ratio per cage: 1/1
- Length of cohabitation: Each morning the females were examined for the presence of sperm in vaginal lavages
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
males: 54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating period)
females: 14 days pre-mating period and until mating, through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period)
Frequency of treatment:
daily by stomach tube
Details on study schedule:
- Age at mating of the mated animals in the study: 14 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
actual ingested doses
Basis:
actual ingested
0, 100, 400, 1600 mg/kg bw./day
No. of animals per sex per dose:
12
Control animals:
yes
Details on study design:
- Control groups: drinking water
- Dose selection rationale: Results of an acute toxicity study with oral administration to male and female rats
- Result: no effects up to and including 2000 mg/kg bw.
- Rationale for animal assignment: randomly grouped
- Section schedule rationale: all animals were sacrificed
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on administration days 1, 8, 15, 22, 29, 36, 42, 50 and on day of autopsy, pups within 24 hours of parturition and on day 4 post-partum

FOOD CONSUMPTION : 2 weeks before mating, after 2 week mating period and weekly until the end of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 14 (before mating) and prior to autopsy from the satelite groups and from the control and high dose group.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:day 14 (before mating) and prior to autopsy from the satelite groups and from the control and high dose group.

URINALYSIS: No
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight, histopathology of testis and epidymidis
Litter observations:
PARAMETERS EXAMINED
numbers of pups born, delivery index, number of pups alive, birth index, live birth index, pup weight on day 0 of lactation, sex ratio, number of pups alive on day 4 of lactation, pup weight on day 4 of lactation, general status of pups, observation of external deformities, corpora lutea, implantations, visible resorptions, early resorptions


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities
Postmortem examinations (parental animals):
POST-MORTEM EXAMINATIONS: Yes

SACRIFICE
- Male animals after 54 days: All surviving animals
- Female animals: Sacrifice on lactation day 4 (starting with day 0),

ORGAN WEIGHTS
- Organ weights: brain, heart, thymus, spleen, liver, testis, epididymis, kidney, adrenal gland

HISTOPATHOLOGY: Yes: high dose and control animals
- Organ: medulla oblongata, brain, heart, pancreas + lymphnode, spleen, liver, lung, small intestine, stomach, kidneys, adrenal gland, testes, prostrate, urinary bladder, bone + bone marrow, thymus, trachea, white + brown fat, muscle, pituitary gland
Postmortem examinations (offspring):
Gross pathology: observation of external deformities
Statistics:
Statistical evaluation was operated using the software SPPS version 16.0. Group data were represented by mean, standard deviation and median. Statistical analysis in case of data measured once during the study (organ weight, haematology, clinical chemistry) : Mann-Whitney U test for pairwise comparison between control and individual experimental groups on significance level alpha = 0.05.
Statistical analysis in case of repeated data measurement (body weight, food intake): Repeated measures ANOVA (procedure General Linear Model (GLM) for Repeated Measures in SPSS statistical software).
Reproductive indices:
number of mated pairs, number of copulated pairs, copulation index, number of pregnant animals, fertility index, pairing days until copulation, frequency of vaginal estrus, number of pregnant females, number of pregnant females with pups alive, gestation index, gestation lenth in days, number of corpora lutea, number of imlantation sites, implantation index, number of pups born, delivery index
Offspring viability indices:
number of pups alive, birth index, live birth index, pup weight on day 0 of lactation, sex ratio, number of pups alive on day 4 of lactation, viability index, pup weight on day 4 of lactation, general status of pups, observation of external deformities

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
f
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS:
- 100, 400 and 1600 mg/kg bw/day:
no differences to the control animals observable

MORTALITY: no

BODY WEIGHT GAIN: 1600 mg/kg bw: reduced

FOOD CONSUMPTION: no statistical differences

HAEMATOLOGY/ CLINICAL CHEMISTRY:
Haematology and clinical chemistry reveales some statistically significant differences, but these were not related to dosage or not confirmed by the findings in other groups, for example by the results of the satellite groups, or the effects are of biological low relevance i.e..

URINALYSIS: not examined

NEUROBEHAVIOUR: not examined

ORGAN WEIGHTS: no statistical differences

GROSS PATHOLOGY: no dosage related effects

HISTOPATHOLOGY: NON-NEOPLASTIC: no statistical differences

HISTOPATHOLOGY: NEOPLASTIC: no statistical differences

HISTORICAL CONTROL DATA: not given

Effect levels (P0)

Dose descriptor:
NOEL
Remarks:
fertility
Effect level:
>= 1 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: P and F1 (migrated information)

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:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

CLINICAL SIGNS (OFFSPRING): no abnormalities in the general status

BODY WEIGHT (OFFSPRING): no significant differences between the control group and the various dosage groups werde observed in males or females in body weights on day 0

On lactation day 4 a higher number of dead pups was counted in the high-dose level group. Moreover, there was a stastitically significant but very low difference in the body weight between the control group and the 400 and the 1600 mg/kg bw dose groups. However, this difference is considered to be of low biological relevance.

GROSS PATHOLOGY (OFFSPRING): no external deformities were observed in the surviving pups on the day of birth. In autopsies of pups on lactation day 4, no abnormalities were observed in the control, nor were any abnormalities in the autopsy of the dead pup.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male and female rats. Treatment of males: 54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating period), treatment of females: 14 days pre-mating period and until mating, through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period) for 54 days.
Additional satelite groups of 5 male and 5 female were treated with 0 and 1600 mg ASC plus/kg bw. and used for observation of reversibility, persistence or delayed occurence of systemic toxic effects for at least 14 days post treatment.
The test-article was formulated in drinking water and administered in 10 ml/kg bw..
At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOAEL).
1600 mg ASC plus/kg bw. may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth.
NOAEL: 400 mg/kg bw. However NOAEL (Fertility) = 1600 mg/kg bw.
Executive summary:

Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male rats for 54 days. The test-article was formulated in drinking water and administered in 10 ml/kg bw..

Histopathology and gross necropsy did not reveal any test article related changes.

The organ weights showed some statistically significant differences, but these were not related to dosage or not affirmed by the findings in other groups, for example by the results of the satellite groups.

The same is true for the statistical significant differences of the haematological parameters and the result of the clinical chemistry, especially decreased activities of the liver enzymes in the blood. These are merely statistical effects without biological relevance.

Two effects with a possible relation to the oral administration of ASC plus were observed:

- At birth no differences were observed between the groups, but on day 4 after birth a higher number of dead pups was counted in the high-dose level group.

- With male rats a dose dependent decrease in body weight is observed. This effect is in the highest dose group statistically significant and confirmed by the satelite group but not confirmed by the body weight development of the females and not confirmed by the food consumption of males and females.

Although an influence of an infection with paracites seems possible and may explain both effects, a non-specific influence of ASC plus cannot be excluded. The daily administration of a suspension of ASC plus in 10 ml/ kg bw. may have led to a reduced appetite of the animals. This should be especially true for the highest dosage group, as here the suspension was relatively dense. Imaginable is also an osmotic effect of non-resorbed ASC plus in the intestine. At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOEL).

1600 mg ASC plus/kg bw. may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth.

With regard to effects on fertility of the present study 1600 mg ASC plus/kg bw. per day can be defined as "No Observed Adverse Effect Level" (NOAEL).