Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No significant adverse effects of the registration substance were revealed when tested in a reproductive toxicity screening tests according to OECD TG 421 in rats at doses up to 1000 mg/kg body weight. Based on the results tthe NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was established at 1000 mg/kg/day for both males and females.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline-conform study under GLP without deviations
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, S.r.l. Calco, (Lecco), Italy.
- Age at study initiation: 8-9 wks
- Weight at study initiation: (P) Males: 255.13-257.18 g; Females: 193.39-193.90 g
- Fasting period before study: no
- Housing: 5 per cage (polysulfone solid bottomed cages)
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: approximately 2 wks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): approximately 15-20 changes
- Photoperiod (hrs dark / hrs light): 12 hrs

IN-LIFE DATES: 20 June - 12 August 2012
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The required amount of Hostapon TPHC was suspended in purified water at the final concentrations of 10, 30 and 100 mg/mL. The formulations
were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.
Details on mating procedure:
- M/F ratio per cage: one to one
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): single
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 91450).
The validation of the formulation procedure for Hostapon TPHC was performed at 10, 30 and 100 mg/mL. The results were obtained in RTC Study
No. 91450 and all levels were within the acceptability limits for concentration and homogeneity.
The stability was found to be 24 hours at room temperature in the concentration range of 10 to 100 mg/mL in the same study.
Samples of the formulations prepared on Weeks 1 and 6 (when all females were present) of the present study were also analysed to check the concentration and homogeneity.
The overall results of the analyses were within the limits of acceptance stated in RTC’s SOPs for concentration (90-110%) and homogeneity (CV <10%).
The software used for this activity was Analyst 1.5.2 (AB Sciex).
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, with the exception of female no. 91480067 which was dosed for 15 consecutive days, and thereafter during pairing, post coitum and post partum periods until Day 3 post partum. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
Once a day
Details on study schedule:
Males were treated for a total of 33 days including 2 weeks prior to pairing and continuously thereafter up to the day before necropsy.
Females were dosed throughout the study including 2 weeks before pairing, thereafter during pairing, gestation and lactation periods until Day 3
post partum.
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg body weight
Basis:
nominal in water
No. of animals per sex per dose:
10 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dosages of 100, 300 and 1000 mg/kg/day were selected in consultation with the Sponsor based on preliminary dose-range-finder. The oral route was selected as it is a possible route of exposure of the test item in man.

Parental animals: Observations and examinations:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs
All clinical signs were recorded for individual animals.
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Body weight
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

Food consumption
Food consumption was recorded at weekly intervals by each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 1 post partum).
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Sperm parameters (parental animals):
Parameters examined in all control and high dose parental male generations:
Epididymides, testis, prostate gland and seminal vesicles weights, morphological evaluation of the seminiferous epithelium (staging of the
spermatogenic cycle)

Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies. Live pups were individually weighed on Days 1 and 4
post partum. Observation was performed once daily for all litters.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were killed after the mating of all females (i.e. after 33 days of treatment).
- Maternal animals: The females with live pups were killed on Day 4 post partum

GROSS NECROPSY
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted
(including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples
preserved in fixative and processed for histopathological examination.

All females were examined also for the following:
a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).

The uterus of female no. 91480063 with no visible implantations was immersed in a 20% solution of ammonium sulphide to reveal evidence of
implantation.

HISTOPATHOLOGY / ORGAN WEIGHTS
From all animals completing the scheduled test period the organs were dissected free of fat and weighed (Annex 1 of the study protocol). The ratios of organ weight to terminal body weight
were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed in the Annex 1 of the study protocol were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid) from all animals.

Histopathological examination and staging of spermatogenic cycle
The tissues required for histopathological examination are listed in Annex 1 of the study protocol. After dehydration and embedding in paraffin wax,
sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted as detailed below:

a) Tissues from all animals in the control and high dose groups killed at term.
b) Tissues from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.



Postmortem examinations (offspring):
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Thiopenthal.

All pups found dead in the cage were examined for external and internal abnormalities. Sex was also confirmed by gonadal inspection.
All live pups were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
The criterion for statistical significance was p<0.05.
Reproductive indices:
Males
Copulatory Index (%) =no. of animals mated/ no. of animals paired x 100

Fertility Index (%)=no. of males which induced pregnancy/no. of males paired x 100

Females
Copulatory Index (%) =no. of animals mated/no. of animals paired x 100

Fertility Index (%) =no. of pregnant females/no. of females paired x 100

Males and females
Copulatory Interval = Mean number of days between pairing and mating

Offspring viability indices:
Pre-implantation loss was calculated as a percentage from the formula:

(No. of corpora lutea - No. of implantations)/ No. of corpora lutea x 100

Pre-birth loss was calculated as a percentage from the formula:

(No. of visible implantations - total litter size at birth )/ No. of visible implantations x 100

Pup loss at birth was calculated as a percentage from the formula:

(Total litter size - live litter size)/ Total litter size x 100

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:

(Total litter size at birth - live litter size at Day 4)/Total litter size at birth x 100

Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two high dose animals, one male and one female, were found dead on Day 8 of the study and on Day 0 post coitum, respectively. The cause of death for these two animals was attributed to a mis-dosing.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Mortality
Two high dose animals were found dead during the study: one male (no. 91480068) on Day 8 of the study and one female (no. 91480063) on Day 0 post coitum. The cause of death was attributed to a mis-dosing on the basis of the macroscopic and histopathological findings.
All females were found pregnant at necropsy with the exception of the found dead animal no. 91480063 (Group 4).
The number of females with live pups on Day 4 post partum was: 10 in each of the control, low and mid- dose groups and 9 in the high dose group.

Clinical signs and observation of cage tray

Salivation was observed in the majority of high dose animals of both sexes throughout the study and occasionally in the low and mid-dose groups. This sign appeared early after dosing.
In one occasion, difficulty in breathing (dyspnoea) was observed in two high dose males (animal nos. 91480078 and 91480080).
No other relevant signs were recorded.
In several occasions, soft faeces and red/brown staining in the cage tray were noted in treated groups during the mating phase.
These data were not tabulated but retained as study raw data.

Body weight and body weight gain
Body weight and body weight gain were comparable between the groups throughout the study for both males and females.

Food consumption
Measurement of food consumption did not reveal relevant differences between the groups.

Oestrus cycle, reproductive parameters, pairing combination and mating performance
Measurements of oestrus cycle, pre-coital intervals, copulatory and fertility indices did not show differences between treated and control groups.

Implantation, pre-birth loss data and gestation length of females
No significant differences were found in the number of implantations, corpora lutea, total litter size, pre-implantation and pre-birth loss between control and treated groups.
Gestation length was comparable between groups.

Terminal body weight and organ weights
No differences were found in terminal body weight nor in organ weights between the groups.

Macroscopic observations

Unscheduled deaths
A high dose male (no. 91480068) and female (no. 91480063) died during the experimental phase.
At post mortem examination, the most relevant changes detected in the male or female rat were: red or brown staining in the muzzle, incomplete collapse, red colour or multiple dark areas in the lungs or red fluid in the thoracic cavity or red colour in the cervical lymph nodes or in the thymus.
Such changes, also confirmed by histopathological examination, were considered related to a possible misdosing and as consequence the factor contributory to death.

Final sacrifice
No relevant changes were detected at post mortem examination in treated animals, when compared with controls.

Microscopic observations
No treatment-related changes were seen in selected organs/tissues evaluated in males or females receiving Hostapon TPHC, sacrificed at the end of
treatment period, nor in the abnormalities detected in all groups at post mortem examination.
The sporadic lesions reported in single treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity could be considered 1000 mg/kg/day for both males and females.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups
Mean litter and pup weights were comparable between groups on Days 1 and 4 post partum.
No differences were found in sex ratio.

Clinical signs of pups
No differences were noted in the clinical signs observed in pups between groups.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum
No milk in stomach and autolysed organs in the abdomen were observed at necropsy in the decedent pups of control and treated groups.
No abnormalities were found in pups sacrificed on Day 4 post partum. Only one high dose pup had no milk in stomach.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Reproductive effects observed:
not specified
Conclusions:
On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity
could be considered 1000 mg/kg/day for both males and females.
Executive summary:

The registration substance was investigated according to OECD TG 421. The purpose of the study was to evaluate the reproduction/developmental toxicity of the test item in Sprague Dawley rats up to Day 4 post partum. The test item was administered orally, by gavage, at the dosages of 100, 300 and 1000 mg/kg/day. The treatment schedule included 2 weeks before pairing, during pairing, post coitum and post partum periods up to Day 3 post partum. Animals were administered for approximately 5 and 7 weeks for males and females, respectively. The parental animals were monitored for daily clinical signs, body weight, food consumption, oestrous cycle and mating performance. The dams were allowed to give birth and rear their offspring until Day 4 post partum. Macroscopic observation and histopathological examination were also performed. In parental animals, no relevant findings were found in life phase or at post mortem evaluation. The animals of the high dose group showed salivation early after dosing as major clinical sign. Body weight, body weight gain and food consumption were unaffected by treatment. No treatment-related findings were found at macroscopic and microscopic examinations. No abnormalities were found at the evaluation of the spermatogenic cycle. Likewise, no differences were noted in the reproductive performance including gonadal function, mating behaviour, conception, development of conceptus and parturition. The females had comparable length of gestation and gave births. Litter and mean pup weights were also comparable between groups and no relevant findings were observed in pups during the lactation period nor at post mortem examination. On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity could be considered 1000 mg/kg/day for both males and females.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Based on findings in a guideline conform reproductive screening study according to OECD TG 421 with the registration substance, neither effects with relevance to parental / maternal toxicity nor with regard to reproductive / developmental toxicity were observed. The NOAEL was established at 1000 mg/kg body weight per day for all endpoints. Additionally, a test proposal for an OECD TG 414 developmental toxicity study is included in this dossier.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
The available data base is considered sufficient for hazard / risk characterization. There are no indications of adverse effects on reproductive organs or tissues from the available data.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
The available data base is considered sufficient for hazard / risk characterization.
Additional information

The registration substance was investigated according to OECD TG 421 for reproduction/developmental toxicity in Sprague Dawley rats up to Day 4 post partum. The test item was administered orally, by gavage, at the dosages of 100, 300 and 1000 mg/kg/day. The treatment schedule included 2 weeks before pairing, during pairing, post coitum and post partum periods up to Day 3 post partum. Animals were administered for approximately 5 and 7 weeks for males and females, respectively. The parental animals were monitored for daily clinical signs, body weight, food consumption, oestrous cycle and mating performance. The dams were allowed to give birth and rear their offspring until Day 4 post partum. Macroscopic observation and histopathological examination were also performed. In parental animals, no relevant findings were found in life phase or at post mortem evaluation. The animals of the high dose group showed salivation early after dosing as major clinical sign. Body weight, body weight gain and food consumption were unaffected by treatment. No treatment-related findings were found at macroscopic and microscopic examinations. No abnormalities were found at the evaluation of the spermatogenic cycle. Likewise, no differences were noted in the reproductive performance including gonadal function, mating behaviour, conception, development of conceptus and parturition. The females had comparable length of gestation and gave births. Litter and mean pup weights were also comparable between groups and no relevant findings were observed in pups during the lactation period nor at post mortem examination. On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity could be considered 1000 mg/kg/day for both males and females.


Short description of key information:
The registration substance was tested in a guideline and GLP conform reproductive toxicity screening study according to OECD 421. No significant unspecific toxicity and mortality and no toxicologically relevant findings were noted for reproductive and developmental parameters in any dose group. The NOAEL for reproductive / developmental toxicity was considered to be 1000 mg/kg body weight. Additionally, a test proposal for an OECD TG 414 developmental toxicity study is included in this dossier.

Justification for selection of Effect on fertility via oral route:
Guideline study according to GLP. No derivations and/or confounders. Klimisch rating 1 representing reliability without restrictions. Information is valid and meet data requirements.

Effects on developmental toxicity

Description of key information
In the available GLP conform OECD TG 421 reproductive/developmental toxicity screening study, a NOAEL of 1000 mg/kg body weight per day was derived for male and female rats. Additionally, in a guideline conform OECD 407 repeated oral dose toxicity study in rats, there were no microscopic abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations, no systemic toxic effects,  and/or specific target organs identified. The NOAEL for repeated oral toxicity was also placed at 1000 mg/kg body weight per day.  From the available data, no significant concern with regard to potential fertility effects of the registration substance is deducible. However, based on the general REACH information requirements, an additional testing proposal for an OECD 414 prenatal  developmental toxicity study in rats is included in this dossier.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Quality of whole database:
Based on the general REACH information requirements, a testing proposal for an OECD 414 prenatal developmental toxicity study in rats is included in this dossier.

Justification for classification or non-classification

Administration of the registered substance up to the limit dose of 1000 mg/kg body weight per day did not result in statistical significant changes of reproductive and/or repeated dose toxicological parameters. Based hereupon classification and labelling of the registered substance concerning reproductive toxicity is not warranted. However, based on the general REACH information requirements, an additional testing proposal for an OECD 414 prenatal developmental toxicity study in rats is included in this dossier.