Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-07-14 to 2012-xx-xx
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 421
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 421 reproduction/developmental toxicity screening test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear liquid
Details on test material:
- Name of test material (as cited in study report): Gaskamine 240
- Physical state: clear liquid
- Analytical purity: 97.73 % (based on the sum of 4 main peaks HPLC(ELSD) Area %)
- Lot/batch No.: 1R101
- Expiration date of the lot/batch: 30-Sep-2012
- Storage condition of test material: at room temperature (20±5 °C) in the dark, under nitrogen gas

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, BV, Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: (P) x 10 wks
- Weight at study initiation: (P) Males: 339-385 g; Females: 203-236 g; (F1)
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland; batch/lot no. 02105120301) with paper enrichment (Envirodri from Lillico, Biotechnology, Surrey / UK), batch/lot no. 100099). During the pre-pairing period, cages with males were interspersed amongst those
holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): for stability of the test item
- Concentration in vehicle: o, 1.25, 3.75 and 10.0 mg/mL/day
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): Charge 260156161
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analyzed by an HPLC method adapted at Harlan Laboratories. The samples (approximately 2 g each) were delivered to the analytical laboratory.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): no data
Duration of treatment / exposure:
males: 4 weeks, females: 7 to 9 weeks
Frequency of treatment:
once daily
Duration of test:
Males were sacrificed after they had been treated for at least 28 days, when no longer needed for the assessment of reproductive effects. Dams and pups were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on
day 25 post coitum.
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects but not death or severe suffering. A descending sequence of dose levels was selected with a view to demonstrating any dose related response. 2.67 to 3 fold intervals were chosen for the descending dose levels.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Recorded on the first day of dosing, once weekly thereafter and at termination. Females: Recorded on the first day of dosing, once weekly thereafter and on days 0, 7, 14 and 20 post coitum, on days 0, 1 and 4 post partum.
Ovaries and uterine content:
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively: in females (ovaries) that did not give birth and liver of all animals. In addition, microscopic examination of the reproductive organs of all infertile males was made.
Fetal examinations:
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: for any structural changes

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
no data
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Indices:
ERproductive indices:
Percentage mating = ( Females mated / Females paired) * 100
Fertility index = ( Females achieving a pregnancy / Females paired) * 100
Conception rate = ( Females achieving a pregnancy / Females mated) * 100
Gestation index = ( Number of females with living pups / Females pregnant) * 100

Offspring viability indices:
Viability index = (pups alive before culling on day 4 p.p. / pups born alive) * 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: LOAEL = 40 mg/kg bw/d

Details on maternal toxic effects:
Abnormal posture or gait, body weight loss and lower food consuption, clay- or tan-colored livers (for more details see RSS Braun 2012 in section 7.8.1 Toxicity to reproduction).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In an OECD 421 Reproduction / Developmental Toxicity Screening Study no teratogenic effects observed up to the highest dose level tested of 40 mg/kg bw/d (NOEL).
Executive summary:

In a Klimisch-1 -rated OECD 421 Reproduction / Developmental Toxicity Screening Study no teratogenic effects were being observed up to the highest dose level tested of 40 mg/kg bw/d (NOEL).

The NOAEL for systemic toxicity in liver has to be based on the gross lesions that were recorded only in a few high dose animals. A correlating increase in glycogen contents was noted in these few males and females although the body weights were decreased in high dose animals. Therefore, a relation to an effect of the test item cannot be excluded definitely at 40 mg/kg bw/day and the NOAEL was established at 15 mg/kg bw/day based on the livers with gross lesions.