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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization. Study according to OECD guideline no. 421 under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction / developmental Toxicity screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: white powder
- Analytical purity: 100%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Wistar Crl:WI
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation:10-11 weeks
- Weight at study initiation: (P) Males: 286.6-317.9 g; Females: 176.9-198.4 g

ENVIRONMENTAL CONDITIONS
- Housing: After an adequate acclimatisation period (at least five days) the animals were barrier maintained (full-barrier) in air conditioned rooms
under the following conditions:
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approx. 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)
- housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item for each dose concentration was taken separately and grounded in morter before dissolving in vehicle Polyethylene Glycol 300 (PEG 300). The formulation preparation was carried out on each administration day, immediately before the administration procedure by using ultrasonic bath and magnetic stirrer/vortex machine.
Homogeneity of the test item in the vehicle was maintained during the daily administration period by using a magnetic stirrer/vortex machine.

EXPOSURE:
Based on the available information from other toxicity studies following doses were selected:
- Control: 0 mg/kg bw
- Low Dose (LD): 100 mg/kg bw
- Mid Dose (MD): 300 mg/kg bw
- High Dose (HD): 1000 mg/kg bw

The highest dose level was chosen with the aim of inducing toxic effects but not death or severe suffering (it is also a limit dose). Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL. The animals in the control group were handled in an identical manner to the dose group subjects and received the vehicle in the volume same as treated groups.

The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-29 days. The maximum dose volume administered was 5 mL / kg body weight. For each animal the individual dosing volume was calculated on the basis of the most recently measured body weight.

VEHICLE:
- Justification for use and choice of vehicle (if other than water): proven to be an adequate vehicle in 28-day study
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 d
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear was referred to as day 0 of gestation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the each nominal dosing concentration was analyzed for nominal concentration. Stability and homogeneity of the test item in the vehicle was analyzed for the low and high dose concentrations.

Samples for the nominal concentration verification was taken in study week 1 (First week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation).

Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation in study week 1 and 5.

Samples for stability analysis were taken in first week of the study. Small portion of preparations (in triplicate) were stored (approximately 24°C) for up to 14 days post-preparation to determine the stability of the test item in the vehicle.

All concentration samples were stored frozen (approximately -20°C) till the analysis was performed.
Duration of treatment / exposure:
The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-29 days.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10 (randomly assigned)
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 (it is also a limit dose). Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
Positive control:
not required

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day except during weekend and holidays where observation was made only once, approximately at the same time
each day and considering the peak period of anticipated effects after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day except during weekend and holidays where observation was made only once, approximately at the same time
each day and considering the peak period of anticipated effects after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Rats were weighed at randomisation, males weekly during the entire study period and at termination. Females were weighed weekly during pre mating period, on gestation day 0, 7, 14, 20 and on PND 1 (PND = post natal day, within 24 hours of parturition) and 4 along with pups.

FOOD CONSUMPTION:
Food consumption was measured on corresponding day of body weight after beginning of the dose administration. Food consumption was not measured during mating period.
Estrous cyclicity (parental animals):
The subsequent morning and the next morning after pairing onwards the vaginal smear of female were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm was considered as day 0 of gestation. Cages were arranged in such a way that possible effects due to cage placement was minimised.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
- testis weight, epididymis
Litter observations:
The duration of gestation was recorded and is calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing actual numbers on the back with the help of permanent marker In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Males were sacrificed after the completion of mating period (total dosing of 28-29 days)
- Females were sacrificed on respective post natal day 4 along with pups by using high dose of sodium pentobarbital.

GROSS NECROPSY
At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.

The number of implantation sites and corpora lutea was recorded in all pregnant females.

The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin. Testes and epididymides were preserved in modified Davidson’s Solution.

ORGAN WEIGHTS
The testes and epididymides of all male adult animals and ovaries, uterus with cervix of all female adult animals were weighed along with liver, kidneys, adrenals, thymus, spleen, brain, heart. Paired organs were weighed separately. Organs of found dead animals were not weighed.

HISTOPATHOLOGY
Full histopathology was carried out on the reproductive organs and tissues of all animals in the control and high dose groups. These examinations were not extended to animals of all other dosage groups, as no treatment-related changes were observed in the high dose group as compared to controls.

For testis, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.

All gross lesions were examined microscopically.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY
Dead pups and pups killed at day 4 post-partumwere carefully examined for gross abnormalities.
Statistics:
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control- and test groups. These statistics were performed with GraphPad Prism V software (p<0.05 was considered as statistical significant). In the evaluation of laboratory parameters all values within a range of the mean value +/- the two fold standard deviation (x +/- 2s) are considered to be „normal“ values within a „normal“ population.
Reproductive indices:
- Fertility index (No. of pregnant females/No. of copulated females X 100)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

DAY OF SACRIFICE:
All survived male animals were sacrificed on day 29 and 30. Non pregnant females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating. Lactatating females along with pups were sacrificed on respective post natal day 4.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No test item related clinical observations and mortalities were observed in male and female animals during the entire period of the study. One male animal from mid dose (No. 25) was observed for respiratory sound on day 11 of treatment. Female No. 54 (low dose) was observed for bloody vagina on treatment day 16 and female No. 57 (low dose) was observed for vocalisation on day 3.

Male No. 12 (low dose), 33 (high dose) and female No. 57 (low dose) were found dead due to gavaging error which was confirmed by microscopic evaluations and therefore not attributed to the treatment. Female no.56 (low dose) was sacrificed interimly at the beginning of the mating period when observed for absent vaginal opening.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
In males, no effect on body weight and body weight change was observed throughout the study period in treated groups when compared with controls. In females, decreased body weight change was observed during entire gestion period (0-20) in high dose group when compared with controls. However, this change was not statistically significant. Therefore, no toxicological relevance can be attributed to this finding. Body weight and body weight change during premating and lactation period was comparable with that of controls.

No effect on food consumption was observed in males during premating and in females during premating period and lactation period. In females, statistical analysis of gestation period food consumption data revealed statistically significant decrease during gestation day 0-7 in mid dose, gestation day 7-14 in low and mid dose and during gestation day 14-20 in high dose group when compared with controls. However, due to lack of consistent and dose dependent pattern of effect on food consumption in treated groups indicates no toxicological relevance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males, there were no statistically significant differences in the absolute and relative organ weights of the treatment groups when compared with the controls. However, marginal increase in absolute and relative testes and epididymides weights were observed in high dose group when compared with controls. In females, statistically significant decrease in absolute spleen weights and increase in relative heart weights were observed in high dose when compared with controls. There was also a slight decrease in absolute and relative thymus weights in high dose group as compared to the controls but this change was withought statistical significance. This decrease or increase in female organ weights is marginal and therefore has no toxicological significance. Absolute and relative organ weights are summarised in the attached document "Organ_Weights_Summary.pdf".

GROSS PATHOLOGY (PARENTAL ANIMALS)
In males, various macroscopic findings observed were small sized thymus , bloody oesophagus and stomach with remains of the test item
and blood, slightly small sized thymus, small sized testes and right epididymis, stomach and lungs with remains of the testitem.

In females, various macroscopic findings observed were lungs - with remains of the test item and accessory liver lobe.

All other males and females from various treatment groups did not show any macroscopic findings at necropsy. Gross pathological observation of males and females at scheduled necropsy revealed no treatment related findings and were considered to be spontaneous in nature.

A summary of gross pathological findings is given in the attached document "Gross_Pathology_Summary.pdf".

HISTOPATHOLOGY (PARENTAL ANIMALS)
The histopathological evaluation of male and female reproductive organs did not reveal any histopathological lesions considered to be test item-related. Isolated testicular and epididymal changes in one control male and one male of the 100 mg/kg/day group were considered spontaneous. Female reproductive organs showed similar post-partum histomorphology in the control and treated groups. There was no indication of test item-related changes by histopathological evaluation of sporadic macroscopic findings in other organs.

FERTILITY:
Fertility index (No. of pregnant females/No. of copulated females X 100) in control was 70% and in low dose group was 66% as compared to mid (100%) and high dose group (90%).

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: NOAEL = highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
not examined
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)

LITTER WEIGHT DATA
Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4 when compared with controls.

PRECOITAL INTERVAL, DURATION OF GESTATION AND FERTILITY INDEX
No treatment related effect was observed on precoital interval, duration of gestation when compared with controls and values were comparable between the groups. All pregnancies resulted in normal births. Successful mating resulted in 10 and 9 pregnancies in the mid and high dose respectively. However, only 7 and 6 pregnancies occurred in control and low dose group.

Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in control (70%) and low dose group (66%) as compared to mid (100%) and high dose group (90%).

PRE AND POST NATAL DATA
Group mean number of corpora lutea, No. of implantation sites, number of live pups born on PND 0, percent preimplantation loss and post implantation loss remained unaffected due to treatment when compared with controls. Although there was a slight increase in preimplantation loss in high dose as compared to controls, there was no statistical significance and percent post implantation loss was decreased in high dose group as compared to controls therefore this effect can not be attributed to the treatment.

LITTER DATA
No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and total No. of live pups and sex ratio on PND 4.

PUP SURVIVAL DATA
Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups.

One pup (Pup No. 2) from female No. 49 of control group was found dead on PND 4. Gross necropsy findings of found dead pup revealed no specific findings.

Relevant data concerning LITTER WEIGHT, PRECOITAL INTERVAL, DURATION OF GESTATION AND FERTILITY INDEX, PRE AND POST NATAL, LITTER, and PUP SURVIVAL is summarised in the attached document "Offspring_data.pdf".

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: NOAEL = highest dose tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, the repeated dose administration of the test item in PEG 300 to the male (28-29 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight revealed no major toxicological findings.

Based on the data generated from this reproduction/ developmental toxicity screening test, the no observed adverse effect level (NOAEL) was determined to be 1000 mg/kg bodyweight for reproduction/ developmental toxicity screening in males and females.

The study was performed acc. to OECD test guideline 421 under GLP conditions. Based on these results, the test item does not have to be classified according to DSD (Directive 67/548/EEC) or GHS/CLP (Regulation (EC) No. 1272/2008).
Executive summary:

The aim of this study was to assess the possible effect of the test item on male, female fertility and embryofetal development in Wistar rats.

In this study, four groups comprised of 10 adult males and 10 non pregnant nulliparous female rats (Wistar RjHan:WI) were dosed daily by oral gavage with 100, 300 and 1000 mg/kg body weight per day test item at dose volume of 5 mL/kg body weight. The test item was formulated in PEG 300 with administration volume of 5 mL/kg body weight. Control animals were handled identically as treated groups and received PEG 300 in similar volume as treated groups.

The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-29 days. Dose volumes were adjusted weekly based on the recent body weight measurement.

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except food consumption was not measured during the mating period.

After 14 days of treatment to both male and female, animals were paired (1:1) for maximum 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

Males and females were sacrificed on treatment day 29-30, post natal day 4 respectively and subjected to necropsy. Non pregnant females were sacrificed on their respective day 26 after the evidence of mating.

No test item related clinical observations and mortalities were observed in male and female animals during the entire period of the study.

Two males from low and high dose group and one female from low dose groups were found dead due to gavaging error which was confirmed by microscopic evaluations. Female from low dose was sacrificed interimly at the beginning of the mating period when observed for the absent vaginal opening.

In males, no effect on body weight and body weight change was observed through out the study period in treated groups when compared with controls. In females, decread body weight change was observed during entire gestion period (0-20) in high dose group when compared with controls. However, this change was not statistically significant. Body weight and body weight change during premating and lactation period was comparable with that of controls.

No effect on food consumption was observed in males during premating and in females during premating period and lactation period.

In females, statistical analysis of gestation period food consumption data revealed statistically significant decrease during gestation day 0-7 in mid dose, gestation day 7-14 in low dose and mid dose and during gestation day 14-20 in high dose group when compared with controls.

Statistical analysis of reproduction and litter data revealed no treatment related effect on group mean litter weight, number of males, number of females, total litter weight, male and female litter weights on PND 0 and PND 4, number of live pups born on PND 0, percent preimplantation loss, post implantation loss, total number of pups born, sex ratio, live pups, still birth and runt on PND 0, total No. of live pups and sex ratio on PND 4, precoital interval, duration of gestation, No. of corpora lutea, and No. of implantation sites when compared with controls. Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups.

No treatment related gross pathological findings were observed in males, females and pups at necropsy or death during the study.

In males, there were no statistically significant differences in the absolute and relative organ weights of the treatment groups when compared with the controls. However, marginal increase in absolute and relative testes and epididymides weights were observed in high dose group when compared with controls. In females, statistically significant decrease in absolute spleen weights and increase in relative heart weights were observed in high dose group when compared with controls. There was also a slight decrease in absolute and relative thymus weights in high dose group as compared to the controls but this change was without statistical significance.

The histopathological evaluation of male and female reproductive organs did not reveal any histopathological lesions considered to be treatment related.