Registration Dossier

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 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:
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
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
Sex:
male/female
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
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
:
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.
Duration of treatment / exposure:
males: 4 weeks, females: 7 to 9 weeks
Frequency of treatment:
once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
15 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
40 mg/kg/day
Basis:
actual ingested
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.
Positive control:
no

Examinations

Parental animals: Observations and 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.
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight, sperm staging
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after they had been treated for at least 28 days
- Maternal animals: All surviving animals 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.

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 males (testes and epididymides, prostate, seminal vesicles with coagulating gland, liver); 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.
Postmortem examinations (offspring):
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.
Reproductive 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: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
abnormal posture or gait, body weight loss and lower food consuption at 40 mg/kg/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males, the mean daily food consumption values at 40 mg/kg/day were significantly lower than the control males during pre-pairing period and in females, slightly lower during pre-pairing period and significantly lower during the gestation and lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males, the mean daily food consumption values at 40 mg/kg/day were significantly lower than the control males during pre-pairing period and in females, slightly lower during pre-pairing period and significantly lower during the gestation and lactation.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 40 mg/kg/day, three males had clay-colored livers and three females had clay or tan-colored livers.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
An incerase in hepatocellular glycogen deposits was observed in the livers with gross lesions in six high dose animals.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence in the mean post-implantation loss, postnatal loss, and lower viability index were observed at the high dose level.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Clinical signs of abnormal posture or gait were observed in males and females from the pairing period in males and from the gestation period in the females. Body weight loss and lower food consumption of males and females were observed at 40 mg/kg/day.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In males, the mean body weights at 40 mg/kg/day were significantly lower (generally p<0.01) than the control males from day 12 of the pre-pairing and pairing periods. Body weight gain was significantly lower (p<0.01) than the control males from day 2 of the pre-pairing period and from day 4 of the pairing period. In females, the mean body weights at 40 mg/kg/day were generally significantly lower (p<0.05) than the control females from day 12 of the pre-pairing period and during the pairing period. During the gestation period female mean body weights at 40 mg/kg/day were significantly lower (p<0.01) than the control females. Body weight gain was significantly lower (p<0.05 or p<0.01) than the control females from day 11 during the pre-pairing period and significantly lower (generally p<0.01) than the control females during the gestation and lactation periods.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
not examinated - gavage

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
no effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
An increased incidence in the mean post-implantation loss, postnatal loss, and lower viability index were observed at the high dose level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
no effects

GROSS PATHOLOGY (PARENTAL ANIMALS)
At 40 mg/kg/day, three males had clay-colored livers and three females had clay or tan-colored livers.

HISTOPATHOLOGY (PARENTAL ANIMALS)
An incerase in hepatocellular glycogen deposits was observed in the livers with gross lesions in six high dose animals.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
for reproductive toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Higest dose tested. No adverse effects observed
Dose descriptor:
NOEL
Remarks:
for reproductive toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Dose descriptor:
NOAEL
Remarks:
and NOEL for systemic toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Remarks:
for systemic toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: abnormal posture or gait, body weight loss and lower food consumption, clay- or tan-coloured livers

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Pup data at high dose group showed a high pup death in one litter (9 pups) or missing pups in other litter (12 missing and 2 dead pups).
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
lower viability index were observed at the high dose level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the remaining pups (out of 9 death in this litter) were lower on day 4 of lactation when compared with day 1.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
One pup from the low dose and two pups from the mid dose had sores on the tail tip.
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
Lower viability index were observed at the high dose level.

CLINICAL SIGNS (OFFSPRING)
Pup data at high dose group showed a high pup death in one litter (9 pups) or dead and missing pups in other litter (12 missing and 2 dead pups).

BODY WEIGHT (OFFSPRING)
The body weights of the remaining pups (out of 9 death in this litter) were lower on day 4 of lactation when compared with day 1.

SEXUAL MATURATION (OFFSPRING)
not examinated

ORGAN WEIGHTS (OFFSPRING)
no data

GROSS PATHOLOGY (OFFSPRING)
generally no effects, but one pup from the low dose and two pups from the mid dose had sores on the tail tip.

HISTOPATHOLOGY (OFFSPRING)
no data

Effect levels (F1)

Remarks on result:
other: Not determined

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was considered to be at 40 mg/kg body weight/day since no histological evidence of a direct toxicological property in the reproductive organs was detected. The No Observed Effect Level (NOEL) for reproductive toxicity was established at 15 mg/kg body weight/day. The NOAEL and NOEL for systemic toxicity were established at 15 mg/kg body weight/day. The findings at 40 mg/kg/day including the clinical signs, moribundity, lower food consumption and decrease in body weight as well as the liver gross lesions correlating to an increase in glycogen contents.
Executive summary:

The purpose of this study was to generate preliminary information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it will provide information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

This study should provide information to assess the need to conduct further investigations and may provide guidance in the design of subsequent studies. Eighty eight rats were assigned to 4 groups each containing 11 males and 11 females. The control, low, intermediate, and high dose group animals (groups 1 to 4) were treated with 0, 5, 15 or 40 mg/kg bw/day, respectively. At the end of the study period, all surviving parent males were sacrificed on study day 29, except high dose male no. 38 which was sacrificed on day 35. All surviving parent females were sacrificed on day 4 post partum (study days 40 to 46). All animals were necropsied and examined post mortem. Histological examinations were performed in all parent males and females of groups 1 and 4 as well as in parent male nos. 13, 14, 21, and 33 (groups 2 and 3) and parent female nos. 57, 58, 65, and 77 (groups 2 and 3) because no pregnancy or delivery occurred after mating. Under the conditions of this experiment, the test item “Gaskamine” did induce the following direct or indirect test item-related findings:

• One high dose male and two high dose females were sacrificed in moribund conditions. Only in one female the remaining fetuses in the uterus horns without delivery could be considered as cause of moribundity. In the male and the other female, the causes of death

were not evident from the organs examined.

• In three high dose males and three females, the livers were recorded as clay-colored or with a tan discoloration. All other gross lesions were considered to be background findindings.

• Possibly test item-related microscopical findings such as increased hepatocellular glycogen deposits were observed in the six livers with gross lesions although the body weights were decreased in high dose animals.

• No test item-related microscopical findings were noted in testes and epididymides; the minimally to slightly reduced secretory activity in prostate, coagulating glands, and/or seminal vesicles in some high dose males was considered to be consequent to the lower

body weights and not to be a direct effect of the test item. No histological evidence of a direct toxicological property in the reproductive organs was detected. Therefore the No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was

considered to be at 40 mg/kg bw/day. The No Observed Effect Level (NOEL) for reproductive toxicity was established at 15 mg/kg bw/day.

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.