Fatty acid chlorides, C18 unsatd., reaction products with sodium N-methyltaurinate

Endpoint summary

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:

screening for reproductive / developmental toxicity

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

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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, 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 an OECD prenatal developmental toxicity study, the No Observed Adverse Effect Level (NOAEL) of test item for maternal and embryofoetal toxicity in Wistar female rats via oral route was found to be the highest dose level employed i.e., 1000 mg/kg body weight/day
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.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

The animal room was air-conditioned with adequate (above 10) air changes per hour The experimental room was continuously monitored for tem¬pera¬ture and relative humidity. The ranges for room tem¬pera¬ture and relative humidity were 20.5°C to 22.3°C and 51 to 65%, respectively. The animals were provided with a light cycle of 12 hours light and 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dosing per gavage commenced on day 5 of gestation and continued until day 19 of gestation. Dose volume was 10 mL per kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity analysis. Dose formulation anaylsis.

Details on mating procedure:

Animals were paired as one male and one female in the evening hours and following morning each female was examined for vaginal smear or the presence of a copulation plug in the vagina. The presence of sperm in the vaginal smear was taken as positive evidence of mating (Day 0 of gestation).The maximum mating period observed was of eight days. During the mating phase, animals were housed on one male: one female basis. After successful mating, the females were returned to their original cage and housed individually during gestation.

Duration of treatment / exposure:

Day 5 of gestation until day 19 of gestation

Frequency of treatment:

Once daily

Duration of test:

The dosing commenced on day 5 of gestation and continued until day 19 of gestation.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 female animals per dose group

Control animals:

yes, concurrent vehicle

Maternal examinations:

Clinical signs were recorded at same time(s) each day taking into consideration the peak period of anticipated effects after dosing. The condition of the animals was recorded including mortality, moribundity, pertinent behavioral changes, and all signs of overt toxicity. Body weights were examined during the acclimatization period once weekly, on first day of pairing and weekly thereafter as well as on gestation days 0, 3, 5, 8, 11, 14 17 and 20.

Ovaries and uterine content:

At termination, the uteri were removed and the pregnancy status of the animals was ascertained. Gravid uteri including the cervix were weighed. The uteri of females were examined for the presence and number of implantation sites and the number of corpora lutea in the ovaries were determined for pregnant animals. The uterine content was examined for number of resorption, live and/or dead foetuses. The degree of resorption was described in order to estimate the relative time of death of the conceptus. Uteri that appear non-gravid were further examined by 5% ammonium sulphide staining to reveal any early resorption or post implantation loss.

Fetal examinations:

Each foetus was subjected to external examination, which included all visible structures, surfaces and orifices (including the oral cavity). One-half of the fetuses (alternating foetuses within the litter) independent of sex were processed for skeletal alterations (odd number) and remaining half (even number) of each litter were examined for visceral (soft tissue) alterations. On completion of external examination the foetuses selected for skeletal evaluation were eviscerated and placed in 70% isopropyl alcohol.

Statistics:

Statistical analysis was performed using statplus program. All the data was checked for Normality with Shapiro-Wilk W test and for Homogeneity with Bartlett Chi-Square test. Each group of animals was subjected to Analysis of Variance (ANOVA). Values were given as mean ± standard deviation (SD).

Indices:

Pre-implantation loss, post-implantation loss, sex ratio, variation incidence

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In high dose (1000 mg/kg) group animals, burrowing followed by dullness and dyspnoea was observed shortly after dosing which subsided to normal after 3 hours post dosing from first day to last day of dosing. Only dullness was observed in some of the animals in intermediate dose (300 mg/kg) group following each gavage treatment which subsides to normal after 3 hours post dosing from first day to last day of dosing.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in any of the treated animal through out the scheduled treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference in the body weight and body weight gain (%) was observed in pregnant female animals of low (100 mg/kg), intermediate (300 mg/kg) and high dose (1000 mg/kg) groups. A statistically significant decrease in body weight was observed in high dose (1000 mg/kg) group when compared with low dose (100 mg/kg) group only on 20th day of gestation. However, this change was within the normal range with respect to age and sex of the strain of animals used. Hence, the changes in body weight and body weight gain were considered as biological variation without any toxicological relevance.
Detailed data see the tables in section "Any other information on results inc. tables"

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption of pregnant female animals at low (100 mg/kg), intermediate (300 mg/kg) and high dose (1000 mg/kg) groups was not significantly different throughout the treatment period. A statistically significant decrease in feed consumption was observed in high dose (1000 mg/kg) group when compared with control (0 mg/kg), low (100 mg/kg) and intermediate dose (300 mg/kg) groups only on day 11th of gestation, and in control (0 mg/kg) group animals only on day 14th of gestation. These changes were within the normal range with respect to age and sex of the strain of animals used. Hence, these changes in feed consumption were considered as biological variation and thus not of toxicological relevance.
Detailed data see the tables in section "Any other information on results inc. tables"

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

Description (incidence and severity):

Daily observations revealed no indications of general and functional pertinent behavioral changes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormality was observed in any of the female animals from control, low, intermediate and high dose groups. Bursal cyst was observed in right ovary of one female animal of low dose group as an incidental finding.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

No abortion noted througout the study period.
Data see tables in section "Any other information on results inc. tables"

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No pre- and/or post-implantation losses observed.
Data see tables in section "Any other information on results inc. tables"

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No litter losses by resorption observed.
Data see tables in section "Any other information on results inc. tables"

Early or late resorptions:

no effects observed

Description (incidence and severity):

No late resorptions observed.
Data see tables in section "Any other information on results inc. tables"

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses observed.
Data see tables in section "Any other information on results inc. tables"

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No changes on pregnancy duration observed.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): No effects on pregnancy duration observed.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All the female animals of control, low, intermediate and high dose groups showed positive evidence of mating. At necropsy, three females each of control, high and low dose groups and four females of intermediate dose group were found non pregnant. As all dose groups are comparably involved and no dose response occurred, this finding is not of toxicological relevance.
Detailed data see tables in section "Any other information on results inc. tables"

Other effects:

no effects observed

Description (incidence and severity):

No significant difference was observed in the sex ratio of foetuses in low, intermediate and high dose groups or other reproductive indices.
Detailed data see tables in section "Any other information on results inc. tables"

Details on maternal toxic effects:

No maternal effects of toxicological relevance observed throughout the study period.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: overall findings

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No significant difference was observed in litter and foetus weight of low, intermediate and high dose groups.
Detailed data see tables in section "Any other information on results inc. tables"
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No changes in fetal/pub body weights recorded.
Detailed data see tables in section "Any other information on results inc. tables"

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No significant differences were observed in early and late resorptions and in the number of live foetuses in all treatment groups.
Detailed data see tables in section "Any other information on results inc. tables"

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant difference was observed in the sex ratio of foetuses in low, intermediate and high dose groups.
Detailed data see tables in section "Any other information on results inc. tables"

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No significant difference was observed in litter size and the foetus weights in low, intermediate and high dose groups.
Detailed data see tables in section "Any other information on results inc. tables"

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External examination of foetuses revealed one small sized foetus in control, six in low dose, eight in intermediate dose and three in high dose group. However, statistical significant differences in respective body weights were not observed. As the external variations observed were randomly distributed across the groups and in the absence of any dose response relationship, these findings were considered as incidental and of no toxicological relevance.
Detailed data see tables in section "Any other information on results inc. tables"

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal examination of foetuses revealed variations common in type and distribution for the strain of rats used. They were isolated and when expressed on a foetus/litter basis, no statistical significant differences between treatment groups and control animals were observed.
Skeletal examination at the dose levels of 100, 300 and 1000 mg/kg body weight thus did not indicate any test item-related effects.
Detailed data see tables in section "Any other information on results inc. tables"

Visceral malformations:

no effects observed

Description (incidence and severity):

Variations observed during visceral examination of foetuses revealed variations common in type and incidence for the strain of rats used and were randomly distributed across all groups. A dose response relationship was not observed. Therefore these findings were considered as incidental findings and of no toxicological relevance.
Detailed data see tables in section "Any other information on results inc. tables"

Other effects:

no effects observed

Description (incidence and severity):

No effects observed

Details on embryotoxic / teratogenic effects:

No effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

SUMMARY OF MORTALITY AND CLINICAL SIGNS

	Group
	1	2	3	4
Dose (mg/kg bw)	0	100	300	1000
Mortality	Nil	Nil	Nil	Nil
Clinical signs	Nil	Nil	Dullness	Excessive burrowing, Dullness, Dyspnoea

 

BODY WEIGHTS (G) – SUMMARY

FEMALE

Group	Body weights (G)
	Gestation Day
		0	3	5	8	11	14	17	20
Group 1	Mean	227.25	232.75	238.29	246.21	256.15	266.79	281.79	306.38
	SD	10.01	9.73	9.59	9.57	11.22	12.63	18.22	25.60
	Min	210.2	214.4	218.2	224.8	232.2	245.2	251.4	263.1
	Max	244.2	250.2	253.8	260.4	270.2	290.2	323.4	352.1
	N	21	21	21	21	21	21	21	21
Group 2	Mean	228.79	235.81	242.44	250.46	259.14	270.67	286.48	310.64
	SD	10.46	10.67	11.56	11.34	12.33	15.51	15.11	17.30
	Min	212.4	218.3	224.1	232.1	238.2	243.4	260.2	274.3
	Max	244.6	251.4	267.1	273.8	282.2	294.1	310.5	338.2
	N	21	21	21	21	21	21	21	21
Group 3	Mean	225.28	231.71	238.18	246.19	256.07	266.52	279.71	298.19
	SD	11.39	10.20	10.37	11.19	11.86	13.16	14.74	19.09
	Min	209.1	218.1	224.0	230.4	238.2	247.1	259.1	269.2
	Max	245.3	249.2	256.8	270.4	281.3	292.1	310.1	332.1
	N	20	20	20	20	20	20	20	20
Group 4	Mean	224.95	231.49	238.42	246.10	255.90	266.32	278.81	293.96^#
	SD	10.61	10.47	10.32	9.68	10.54	11.75	13.28	14.84
	Min	210.6	220.1	226.3	233.2	240.1	249.2	259.2	268.1
	Max	246.8	252.8	255.2	260.2	271.1	286.2	304.1	321.4
	N	21	21	21	21	21	21	21	21

^#Significant at p ≤ 0.05 level with group 2

BODY WEIGHT GAIN (%) - SUMMARY

FEMALE

Group	Body weights Gain (%)
	Gestation Day
		3	5	8	11	14	17	20
Group 1	Mean	2.43	4.88	8.39	12.77	17.44	24.02	34.91
	SD	1.00	1.61	2.44	3.48	3.90	6.33	10.90
	N	21	21	21	21	21	21	21
Group 2	Mean	3.08	5.99	9.52	13.30	18.32	25.28	35.84
	SD	1.43	2.74	3.27	3.15	4.59	5.24	6.22
	N	21	21	21	21	21	21	21
Group 3	Mean	2.89	5.77	9.33	13.72	18.35	24.19	32.41
	SD	1.26	1.65	2.48	2.98	3.00	3.38	6.34
	N	20	20	20	20	20	20	20
Group 4	Mean	2.92	6.02	9.47	13.84	18.50	24.03	30.82
	SD	1.21	1.92	2.82	3.75	4.95	5.01	6.90
	N	21	21	21	21	21	21	21

 

FEED CONSUMPTION - SUMMARY

FEMALE

Group	FEED CONSUMPTION (G/ANIMAL/DAY)
	Gestation Day
		0 - 3	3 - 5	5 - 8	8 - 11	11 - 14	14 - 17	17 - 20
Group 1	Mean	17.40	19.26	20.26	21.99	23.99	24.16	23.85
	SD	1.48	3.45	1.87	1.72	2.15	2.79	2.88
Group 2	Mean	18.06	19.15	19.62	21.91	23.09	23.43	22.91
	SD	2.03	3.96	2.76	2.48	1.48	1.82	2.88
Group 3	Mean	17.59	19.97	19.47	22.04	23.23	23.55	23.38
	SD	2.53	5.38	3.28	2.58	2.40	2.26	3.17
Group 4	Mean	17.04	17.93	18.71	19.10*^#@	21.41*	22.31	22.10
	SD	2.10	4.12	3.00	3.57	3.17	2.42	2.54

*Significant at p ≤ 0.05 level with group 1

^#Significant at p ≤ 0.05 level with group 2

^@Significant at p ≤ 0.05 level with group 3

PREGNANCY STATUS

GROUP	G1	G2	G3	G4
DOSE (mg/kg bw)	0	100	300	1000
FEMALE ANIMALS MATED	24	24	24	24
PREGNANT FEMALE ANIMALS	21	21	20	21

 

MACROSCOPIC FINDINGS- FEMALE

GROUP	G1	G2	G3	G4
DOSE (mg/kg bw)	0	100	300	1000
ANIMALS EXAMINED	24	24	24	24
ANIMALS WITHOUT ABNORMALITY	24	24	24	24
ANIMALS AFFECTED	0	0	0	0

MATERNAL DATA - SUMMARY

 

Parameter	Group	G1	G2	G3	G4
	Dose (mg/kg bw)	0	100	300	1000
	No. of Dams	21	21	20	21
Gravid Uterine Weight	Mean	43.9637	53.2278	45.9287	49.9408
	SD	16.0935	10.1451	14.7209	18.3332
No. of CL	Mean	11.96	12.92	12.92	12.58
	SD	2.73	1.77	2.34	2.80
No. of Implantation	Mean	8.13	9.92	8.38	9.13
	SD	4.24	4.15	4.83	4.76
No. of Foetuses	Mean	8.38	10.14	9.00	9.71
	SD	3.35	2.31	3.36	3.98
Early Resorption	Mean	0.79	0.88	0.88	0.54
	SD	0.88	1.15	1.08	0.88
Late Resorption	Mean	0.00	0.17	0.00	0.08
	SD	0.00	0.64	0.00	0.28
Pre implantation loss	Mean	3.83	3.00	4.54	3.46
	SD	4.31	3.41	4.32	3.71
	%	30.90	24.41	35.89	29.12
Post implantation loss	Mean	0.79	1.04	0.92	0.63
	SD	0.88	1.20	1.10	1.01
	%	12.59	14.49	12.01	9.14
Dam with resorption/s	Number	13	12	12	8

LITTER DATA - SUMMARY

 

Parameter	Group	G1	G2	G3	G4
	Dose (mg/kg bw)	0	100	300	1000
	No. of Dams	24	24	24	24
No. of Litters		21	21	20	21
Total No. of Foetuses		176	213	180	204
Mean Litter Size		8.38	10.14	9.00	9.71
No. of Live Foetuses	Number	176	213	179	204
	Mean	8.38	10.14	8.95	9.71
	SD	3.35	2.31	3.32	3.98
No. of Dead Foetuses	Number	0	0	1	0
	Mean	0	0	0.05	0
	SD	0.00	0.00	0.22	0.00
No. of Live Male Foetuses	Number	93	96	96	101
	Mean	4.43	4.57	4.80	4.81
	SD	2.58	1.89	2.12	2.52
No. of Live Female Foetuses	Number	83	117	83	103
	Mean	3.95	5.57	4.15	4.90
	SD	1.69	2.13	2.08	2.41

GROUP MEAN FOETUS WEIGHTS

 

Group		Foetus weight/Litter (G)	Foetus Weight (G)
			Male	Female
Group 1	Mean	3.3814	3.3354	3.4330
	SD	0.6200	0.6853	0.5369
	N	176	93	83
Group 2	Mean	3.3860	3.4788	3.3099
	SD	0.5341	0.5384	0.5205
	N	213	96	117
Group 3	Mean	3.3906	3.4430	3.3300
	SD	0.6227	0.6619	0.5720
	N	179	96	83
Group 4	Mean	3.3245	3.3427	3.3066
	SD	0.7148	0.7441	0.6879
	N	204	101	103

 

GROUP MEAN SEX RATIO

 

Group		Sex ratio (%) Male
Group 1	Mean	49.08
	SD	17.36
	N	176
Group 2	Mean	45.26
	SD	16.60
	N	213
Group 3	Mean	54.83
	SD	17.99
	N	180
Group 4	Mean	50.21
	SD	17.08
	N	204

 

EXTERNAL FINDINGS

 

Group		G1	G2	G3	G4
Dose (mg/kg bw)		0	100	300	1000
No. of Litter Examined		21	21	20	21
No. of Foetuses Examined		176	213	179	204
Variation Incidence – Number (%)
No. of Foetus with Variations	Total Variations	1(0.57)	6(2.82)	8(4.47)	3(1.47)
	a)Foetus small in size	1(0.57)	6(2.82)	8(4.47)	3(1.47)

VISCERAL FINDINGS

 

Group		G1	G2	G3	G4
Dose (mg/kg bw)		0	100	300	1000
No. of Litter Examined		21	21	20	21
No. of Foetuses Examined		84	103	84	97
Variation Incidence – Number (%)
No. of Foetus with Variations	Total Variations	19(22.62)	18(17.48)	15(17.86)	22(22.68)
	Spleen: Small in size	8(9.52)	7(6.80)	5(5.95)	7(7.22)
	Spleen: Pale	5(5.95)	4(3.88)	4(4.76)	8(8.25)
	Ureters: Convoluted	6(7.14)	7(6.80)	6(7.14)	7(7.22)

SKELETAL FINDINGS

Group	G1	G2	G3	G4
Dose (mg/kg bw)	0	100	300	1000
No. of Litter Examined	21	21	20	21
No. of Foetuses Examined	92	110	95	107
Variation Incidence – Number (%)
Hyoid
Not ossified	1(1.09)	0	0	0
Incompletely ossified	1(1.09)	0	0	4(3.74)
Bipartite	1(1.09)	0	0	0
Absent	1(1.09)	0	0	0
Thoracic Vertebra
Dumbbell shaped	21(22.83)	26(23.64)	18(18.95)	34(31.78)
Bipartite	3(3.26)	10(9.09)	12(12.63)	4(3.74)
Lumbar Vertebra
Not ossified	0	1(0.91)	0	0
Dumbbell shaped	0	1(0.91)	0	0
Sacral Vertebra
Not ossified	1(1.09)	0	0	0
Incompletely ossified	1(1.09)	0	0	0
Caudal Vertebra
Not ossified	6(6.52)	4(3.64)	0	5(4.67)
Sternal centers (1)
Not ossified	10(10.87)	11(10.00)	9(9.47)	12(11.21)
Incompletely ossified	10(10.87)	6(5.45)	12(12.63)	14(13.08)
Sternal centers (2)
Not ossified	5 (5.43)	3(2.73)	0	5(4.67)
Incompletely ossified	4(4.35)	0	3(3.16)	0
Misshapen/Irregular shaped	0	2(1.82)	1(1.05)	0
Manubrium
Not ossified	11 (11.96)	5(4.55)	1(1.05)	7(6.54)
Incompletely ossified	3(3.26)	0	4(4.21)	0

 

SKELETAL FINDINGS (CONTD.)

Group	G1		G2	G3	G4
Dose (mg/kg bw)	0		100	300	1000
No. of Litter Examined	21		21	20	21
No. of Foetuses Examined	92		110	95	107
Variation Incidence – Number (%)
Xyphiod
Not ossified	25 (27.17)		27(24.55)	41(43.16)	41(38.32)
Incompletely ossified	13(14.13)		17(15.45)	8(8.42)	4(3.74)
Ribs - Left
Waviness	1 (1.09)		0	0	9(8.41)
Ribs - Right
Waviness	1 (1.09)		0	0	10(9.35)
Supernumerary	0		0	0	1(0.93)
Metacarpals - Left
Not ossified		0	2(1.82)	0	1(0.93)
Incompletely ossified		1(1.09)	0	0	0
Metacarpals - Right
Not ossified		0	2(1.82)	0	1(0.93)
Incompletely ossified		1(1.09)	0	1(1.05)	0
Metatarsal - Left
Not ossified		1(1.09)	4(3.64)	0	1(0.93)
Incompletely ossified		3(3.26)	0	0	0
Metatarsal - Right
Not ossified		1(1.09)	4(3.64)	0	2(1.87)
Incompletely ossified		3(3.26)	0	0	0
Pubis - Left
Incompletely ossified		0	0	1(1.05)	0
Pubis - Right
Not ossified		0	0	1(1.05)	0

 

                                                                

Conclusions:

The No Observed Adverse Effect Level (NOAEL) of test item HOSTAPON TPHC for maternal and embryofoetal toxicity in Wistar female rats via oral route was found to be the highest dose level employed i.e., 1000 mg/kg body weight/day

Executive summary:

The test item,HOSTAPON TPHC(formulated in distilled water) was administered once daily by oral gavage to three treatment groups of twenty four pregnant female Wistar rats per group from day 5 to day 19 of gestation at dose levels of 100, 300 and 1000 mg/kg body weight/day. A control group of twenty four females were administered with vehicle (distilled water)alone.

Female animals treated at 1000mg/kg bw/day(high dose) showed burrowing followed by dullness and dyspnoea while female animals treated at 300 mg/kg bw/day (intermediate dose) showed only dullness shortly after each administration. These findings recovered to normal after 3 hours post dosing each day. These temporary effects can be regarded unspecific in nature due to minor local irritative effects caused by test item administration.

The evaluation of the reproductive organs of the dam, pre and post implantation loss, gravid uterine weight, litter size, viability of the foetus, litter weight and foetus weight, sex ratio of the foetus and the external, visceral and skeletal examination of the foetus revealed that HOSTAPON TPHC did not cause any maternal as well as embryo foetal toxicity up to the highest dose of 1000 mg/kg bw/day tested.

Effect on developmental toxicity: via oral 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 developmental toxicity.

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 OECD 414 prenatal developmental toxicity study in rats is included in this dossier.