Registration Dossier

Administrative data

Endpoint:
developmental toxicity
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:
other: OECD 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
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
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 given at similar times each day
Analytical verification of doses or concentrations:
no
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
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
Duration of test:
up to 54 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 400, 1600 mg/kg bw./day
Basis:
actual ingested
No. of animals per sex per dose:
12
satellite group: (1600 mg/kg bw./day ):5 individuals (5 females and 5 males)
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

Examinations

Maternal 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

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 satellite 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 satellite groups and from the control and high dose group.

URINALYSIS: No
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
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 date 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 cas of repeated data measurement (body weight, food intake): Repeated measures ANOVA (procedure General Linear Model (GLM) for Repeated Measures in SPSS statistical software).
Indices:
implantation/fertility index, delivery index, live birth index, neonatal survival rate, copulation index, lactation status
Historical control data:
no

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
LOEL
Effect level:
1 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental 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.
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 testarticle related changes.

The organ weights showed some statistically significant differences, but these were not related to dosage or not affirmed by the dindings in other groups, for example by the results of the satelite 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 satellite 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 the results of the present study 400 mg ASC plus/kg bw. per day can be defined as "No Observed Adverse Effect Level" (NOAEL) for the developmental toxicity.