Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Remarks:
Study conducted on a structurally similar analogue
Adequacy of study:
key study
Study period:
17 July 2009 to 11 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental toxicity screening test
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Name of test material {as cited in study report):AD-1500
- Substance type:White powder
- Physical state: solid
-Expiration data of the lot/balch: 01 May 2011
-Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark
-Lot number: L-952191

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
-Source: Charles River Deutschland, Sulzfeld, Germany
-Age at study initiation: Approximately 12 weeks
-Weight at study in iation: males: 302-340 grams, females: 201-224 grams
- Fasting period before study: no
-Housing:
Pre-mating: During acclimatization, females were housed in groups of 5 animals/cage in Macrolon cages (MIV type, height 18 em). Mating: Females were caged together with males.
Post-coitum: Females were individually housed in Macrolon cages (Mill type, height 18 cm).
Lactation: Offspring was kept with the dam until termination in Macrolon cages (Mill type, height 18 cm). General: Sterilized sawdust as bedding material and paper as cage-enrichment was supplied.
-Diet (e.g. ad lib um): ad libitum
-Water (e.g. ad libitum): ad libitum
-Acclimation period: At least 5 days prior to start of treatment under laboratory cond ions

Accommodation
Pre-mating Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 em). Mating Females were
caged together with males on a one-to-one-basis in Macrolon cages (Mill type, height 18 ern). Post-mating Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (Mill type, height 18 cm).
Lactation Offspring were kept wh the dam until termination in Macrolon cages (Mill type, height 18 cm). General Sterilized sawdust as bedding material (L Iabo, S.P.P.S., Argentauil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
-Temperature ("C): 18.3--21.9
-Humidity(%): 42- 82
-Air changes (per hr): approx. 15
-Photoperiod (hrs dark I hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (wlw) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment
was made for specific gravity of the vehicle (factor 1.036).

VEHICLE
-Justification for use and choice of vehicle (W other than water): Based on trial formulations performed at NOTOX and based on a previous
28-day study with AD-1500 (NOTOX Project 472343, see section 7.5.1).
- Concentration in vehicle: 10, 30 and 200 mg/mL on Days 1 and 2, 5, 15 and 100 mg/mL from Day 3 onwards.
-Amount of vehicle (if gavage): 5 mUkg body weight on Days 1 and 2. The dose volume was increased to 10 mUkg from Day 3 of
premating onwards.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Char1es River supplied non-litter mates). Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 postcoitum. Once mating had occurred, the males and females were
separated. A maximum of 14 days was allowed for mating. After 14 days of mating, any females who had not shown evidence of mating were separated from their males.

The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Examination of maternal care revealed no deficiencies (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed according to a validated method {NOTOX Project 477023, see section 8), on formulations prepared on Days 1 and 7 {i.e. before and after adjusting the dose volume from 5 to 10 ml/kg).

Group 1-4 formulations were analysed for accuracy. Group 2 and 4 formulations were also analysed for stability {over 6 hours) and homogeneity.

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was s 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40 to 49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 3 days of lactation.
Frequency of treatment:
Once daily
Details on study schedule:
-Age at mating of the mated animals in the study: approximately 14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected on the basis of a 5-day dose range finding study. The animals were randomized prior to treatment by a computer-generated random algorithm according to body weight, with all animals within +/-20% of the sex mean
Positive control:
Not required

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
-Time schedule: twice daily (early morning/late afternoon)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from Day 0 post-coitum onwards
BODY WEIGHT: Yes
-Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females
were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-co um and during lactation on Days 1 and 4. FOOD CONSUMPTION: Yes
-Food consumption for each animal determined: Weekly, for males and females. Food consumption was not recorded during the breeding
period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-co um and during lactation on Days 1 and 4.
WATER CONSUMPTION: subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no
effect was expected.
REPRODUCTION PROCESSES: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded
Oestrous cyclicity (parental animals):
Not determined
Sperm parameters (parental animals):
Parameters examined in all male parental animals:
testis weight, epididymis weight, spermatogenesis staging, microscopic examination of testes and epididymides.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:


number of live and dead pups, sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnonnalities,
GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnonnalities; possible cause of death was not detennined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
-Male animals: All surviving animals after 28 days of exposure
-Maternal animals: All surviving animals after at least 3 days of lactation

GROSS NECROPSY
-Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
-The number of corpora lutea and former implantation sites were recorded. Nongravid uteri were stained using the Salewski technique.

Samples of the following tissues and organs were collected and fiXed in 10% buffered formalin:
Identification marks (not processed), Preputial gland, Cervix, Prostate gland, Clitoral gland, Seminal vesides, Coagulation gland, Testes*,
Epididymides*, Uterus, Ovaries, Vagina, Pituitary gland, All gross lesions
*Fixed in modified Davidson's solution and transferred to formalin after fiX8tion for at least 24 hours.

ORGAN WEIGHTS Epididymides Testes

HISTOPATHOLOGY:
The following slides were examined by a pathologist:
-The ovaries and epididymides of the animals of Groups 1 and 4 and testes of all groups.
- The addijional slides of the testes of the males of Groups 1 and 4 to examine staging of spennatogenesis.
-The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal
vesicles, testis, uterus, and vagina) of all animals that failed to mate, conceive, sire or deliver healthy offspring.
-The preserved organs and tissues of the animals of all dose groups which died spontaneously.
-All gross lesions of all animals (all dose groups).
Postmortem examinations (offspring):
SACRIFICE
-Animals were subjected to external postmortem examinations.
Statistics:
The following statistical methods were used to analyze the data:
-lithe variables could be assumed to follow a nonnal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled
variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data oould not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.
-The corpora lutes and implantation sites were subjected to the Kruskai-Wallis onparametric N>IOVA test (Kruskal, 1952) to determine intergroup difference. As no results of the ANOVA were significant {p<0.05), the Wilcoxon test (Wilcoxon, 1945) was not applied to the
data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.
Reproductive indices:
For each group the following calculations were perfonned:
Percentage mating males = (Number of females mated/Number of males paired) x 100
Percentage mating females= (Number of females mated/Number of females paired) x 100
Fertility index males= (Number of males generating a pregnancy/Number of males paired) x 100
Fertility index females = (Number of pregnant females/Number of females paired) x 100
Conception rate = (Number of pregnant females/Number of females mated)x 100
Gestation index= (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confinnation of mating and the beginning of parturition
Offspring viability indices:
For each dose group pup parameters were expressed in two ways:
-As a mean (with standard deviation) of the number observed for each litter
- Relative to a second parameter and calculated on a total group basis

Percentage live males at First Litter Check= (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x
100
Percentage live females at first litter check= (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x
100
Percentage of postnatal loss Days 0-4 of lactation = (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check)
X 100
Viability index= (Number of live pups on Day 4 of lactation/Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

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):
No substance related mortality occurred. Slight to moderate salivation was transiently noted for 8 males and 4 females of Groups 2 and 3 and 5 males and 4 females of Group 4. This finding was transient and noted in absence of a dose response relationship. Salivation for these animals was likely due to the taste of the test substance and is not considered to be a sign of treatment-related toxicity.

BODY WEIGHT AND FOOD CONSUMPTION {PARENTAL ANIMALS: No effects.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
ORGAN WEIGHTS (PARENTAL ANIMALS)
In males, testes and epididymides weights and organ to body weight ratios remained unaffected wijh treatment up to 1000 mglkg of AD-
1500. At 50 mg/kg, absolute testes weights and testes:body weight ratios were significanUy higher than controls. Testes weights were
similar to controls for all other treatment Groups. In absence of a dose response relationship, this findings was considered to be fortuitous.

GROSS PATHOLOGY (PARENTAL ANIMALS):
There were no macroscopic findings that were considered to be related to treatment with AD-1500 up to 1000 mglkg. Incidental findings seen for control and treated animals at macroscopic examination included alopecia of several body parts, reddish focus/foci and tan soft nodules on the right clitoral glands, tan or yellowish soft nodules on the right epididymide (head or body), reddish soft or yellowish hard nodules on the uterine adipose tissue, a flaccid smaller left testis and left epididymides reduced in size. At the limited incidence and in the absence of a dose-response relationship, these were not considered to be toxicologically-relevant. Uterus contains fluid was noted for a single female at 150 mg/kg (female no. 68, Group 3); this finding correlates with a particular stage in the estrous cycle and is not indicative of an effect related to treatment wijh AD-1500.

HISTOPATHOLOGY (PARENTAL ANIMALS):
There were no treatment related microscopic findings. For the seven males and seven females failed to mate, conceive, sire or deliver
hea hy offspring, there were no morphological alterations to account for any lack of fertility. Spennatogenic staging profiles were nonnal for all Group 1 and Group 4 males. All microscopic findings recorded were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

OTHER FINDINGS (PARENTAL ANIMALS):
The number of corpora lutea, implantation sites, implantation index, number of pups born, live and dead pups at first litter check, delivery index, and duration of gestation were unaffected by treatment of AD-1500 up to 1000 mglkg. There were no treatment-related effects on
the duration of gestation, number of litters, numbers of living or dead pups at first litter check, postnatal loss, or on the viability index, number of corpora lutea or the number of implantation sites up to 1000 mg/kg.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No treatment-related mortality occurred. Twelve pups of the control group, three pups of the low dose group, fiVe pups of the mid dose group and three pups of high dose group were found dead or missing during the first days of lactation: No relationship with treatment was established for these deaths.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at highest dose level tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with AD-1500 by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 1000 mg/kg body weight/day revealed no parental, reproduction, breeding and developmental toxicity up to 1000 mg/kg body weight/day.

The parental, reproduction, breeding and developmental NOAEL was established as being at least 1000 mg/kg body weight/day.