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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-06-22 to 2016-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Tantalum pentachloride
- Physical state: solid at room temperature
- Colour: white
- Analytical purity: 99.9%
- Lot/batch No.: 144217
- Expiration date of the lot/batch: 2016-05-11
- Stability in water at room temperature: hydrolysis
- Storage condition of test material: at room temperature, protected from humidity
- Other: Due to its physical properties (fast hydrolysis and pH-shift) it was not possible to use the test item as delivered in a repeated dose toxicity study. In consultation with the sponsor it was decided that the test item was further processed by hydrolysis and neutralisation in order to prepare a formulation that was feasible for oral dose administration (for detailed information please see chapter "details on oral exposure").

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at start of the treatment period: males/females: 10-11 weeks
- Weight at study initiation: males: 246-294 g, females: 168-208 g
- Fasting period before study: no data
- Housing: in groups of two animals/sex/cage in IVC cages (type III H, polysulphone cages). During mating period males and females were housed together 1:1. After the confirmation of mating, females were kept individually during gestation/lactation period
- Diet (e.g. ad libitum): Altromin 1324 , ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulation (high dose) was prepared by slowly suspending the respective test item in Aqua ad iniectabilia/10M NaOH (approx. 4:1) for hydrolysis. The neutralising agent was used to adjust the pH-value to approximately 8. Both vehicle and neutralising agent were added to give the appropriate final concentration of the test item. The preparation procedure was performed on a magnetic stirrer while cooling on ice water.
Subsequent dose levels were achieved by further diluting the prepared test item formulation with the respective volume of vehicle.
The test item formulations were prepared at least once every ten days. Prepared formulations were stored at room temperature and protected from light.

PREPARATION OF THE SHAM CONTROL FORMULATION:
The sham control formulation was prepared by adding NaCl to Aqua ad iniectabilia, which based on a theoretically calculated amount of NaCl that may be assumed to be formed at higher dose groups (BSL study 145145 and 145146) during the dose formulation preparation. The calculation of NaCl in the high dose was made only once before the treatment initiation and the same calculated quantity of NaCl was used in every new batch of sham control formulation.

VEHICLE
- Vehicle used: Aqua ad iniectabilia
- Amount of vehicle (if gavage): 7 mL/kg bw
- Lot/batch no. (if required): 405559 and 405701

CHARACTERISATION of the NEUTRALISING AGENT
The pH-value of the test item was adjusted by sodium hydroxide solution (NaOH). The specifications provided by the supplier are listed as follows:
- Name: sodium hydroxide solution
- Concentration: 10M in H2O
- Batch No: 4F017374 and 0000557447

SHAM CONTROL FORMULATION:
- Name: NaCl
- Batch: 15A120009
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): females were kept individually during gestation/lactation period and males were returned to its original cage.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating) and in the last week of the study (gestation/lactation) from all groups (12 samples). However, the sham control samples were not analyzed for concentration of test item as it was not considered necessary. Samples were analysed using ICP-MS.
Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 (first week of pre-mating period) and 5 (gestation) (12 samples).
Samples for stability analysis were taken before the start of the study, 0 hours after the preparation and another sample 10 days after the preparation (at room temperature), from high and low dose formulations (4 samples).
Duration of treatment / exposure:
The animals were treated with the test item formulation, vehicle or sham control formulation on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
once daily, 7 days a week
Details on study schedule:
according to guideline
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
10 (6 per sex for the sham control)
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Details on study design:
- Dose selection rationale: based on the results of a previous dose range finding study
- Rationale for animal assignment: Random
Positive control:
not necessary

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals (except animal no. 36 of MD group, which was inadvertently missed) outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of administration and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.

FOOD CONSUMPTION: Yes
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes, see chapter (neuro)behavioural examination

HAEMATOLOGY: Yes
Haematological parameters from 5 randomly selected males and females (only lactating females were evaluated) from each group were examined at the end of the treatment as part of the sacrifice of the animals. In case of sham control animals haematology was measured on all males and lactating females. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. Parameters checked in table 1 were examined

CLINICAL CHEMISTRY: Yes
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) from each group were examined at the end of the treatment as part of the sacrifice of the animals. In case of sham control animals clinical biochemistry was measured on all males and lactating females. Blood from the abdominal aorta of the animals was collected in serum separator tubes. Parameters checked in table 3 were examined.

URINALYSIS: Yes
A urinalysis was performed with samples collected from 5 randomly selected males and females (only lactating females were evaluated) from each group as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded. In case of sham control animals urinalysis was made on all males and lactating females. Parameters checked in table 4 were examined.

(NEURO)BEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the lactation period in 5 randomly selected females (only lactating females were evaluated)
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy

OTHER:
BLOOD COAGULATION:
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) from each group were examined at the end of the treatment as part of the sacrifice of the animals. In case of sham control animals blood coagulation was measured on all males and lactating females. Blood from the abdominal aorta of the animals was collected in citrate coated tubes. Parameters checked in table 2 were examined.
Oestrous cyclicity (parental animals):
If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented.
Sperm parameters (parental animals):
Parameters examined male parental generation: testis weight, epididymis weight, for the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides
Litter observations:
LITTER OBSERVATIONS:
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 tattoo marked on the paws. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while female animals were sacrificed on the respective post-natal day 4. The surviving pups were sacrificed by decapitation on PND 4.
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole). The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was also recorded for one female sacrificed on day 26 post-coitum due to non-delivery.

ORGAN WEIGHTS: Yes
The wet weight of the organs (Table 5) of 5 sacrificed adult males and 5 females (only lactating females were evaluated) randomly selected from each group was recorded as soon as possible. Paired organs were weighed together. In case of sham control animals the wet weight of the organs of all males and lactating females were recorded. In addition reproductive organs of all animals were weighed and preserved.

HISTOPATHOLOGY: Yes
A full histopathology was carried out on the preserved organs and tissues according to Table 6 of 5 randomly selected male and female animals (only lactating females were evaluated) of the control and high dose groups which were sacrificed at the end of the treatment period.
Because of possible treatment-related changes noted in the high dose group, stomach from animals of the low- and mid-dose groups (groups 3 and 4, respectively) pre-selected for the examination of full list organs/tissues, as well as stomach from all sham control animals was examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.

GROSS NECROPSY
- all animals were subjected to gross necropsy.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test and additionally Tukey test for absolute liver weight of females as the Dunnett test indicated statistical significance for SC and MD groups compared to control. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).
Reproductive indices:
Copulation, fertility, delivery and viability
Offspring viability indices:
Viability index,

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not specified
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Description (incidence and severity):
Furthermore, by the detailed testicular examination, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis as well.
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred in the control, sham control or any of the dose groups during the treatment period of this study.
Slight to severe salivation was noted in some males and females of the HD group and one male of the SC group on single occasion of treatment. Furthermore, moving the bedding was observed transiently in all males and all females of the HD and the SC group and in one male of the MD group. The clinical signs salivation and moving the bedding were immediately after the dose administration and therefore were considered to be a sign of discomfort caused due to test item treatment with no toxicological relevance.
Isolated incidence of abnormal breathing irrespective of the dose group on single occasion of treatment were considered to be incidental.
Alopecia or crust on various body parts, vocalisation or partial regurgitation of formulation were noted in isolated males and/or females of the dose groups and/or control groups. These clinical signs were transient in appearance and showed up irrespective of the groups. Therefore, they were considered to be incidental. None of the females showed signs of abortion or premature delivery.
During the weekly detailed clinical observation, no relevant differences between the groups were found.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In both males and females, there was no test item treatment related effect on body weight in the dose groups during the study period. There were no statistically significant differences between the dose groups, sham control group and the control group. There was no test item treatment related effect on food consumption in male. In females there was statistically significantly lower food consumption (up to 11% lower compared to controls) observed in the female LD, the MD and the HD group during the first week of treatment showing dose response relationship. However, with the progress of the study no such changes in food consumption was observed. Hence, the finding was not considered to be an adverse effect.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no test item treatment related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group. However, a slightly reduced copulation index (number of copulated females / number of pairs) of 90 % in the MD group compared to 100 % in all other groups. In the absence of dose response relationship the finding was not considered to be of toxicological relevance. The viability index was marginally lower in the HD group (99.29%) as compared to the control group (100%). This was due to the death of one single pup (no. 3) of female no. 85. As this finding was limited to a single pup it was considered incidental.

PRECOITAL INTERVAL AND DURATION OF GESTATION
There were no effects on the duration of precoital interval and the duration of gestation in the dose groups and sham control group, when compared to the control group

PRE-and POST-NATAL DATA
There were no test item treatment related effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0 and PND 4) and percentage of pre- and post-implantation loss in the dose groups and sham control group, when compared to the control group

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males, there were no statistically significant differences in the absolute and relative organ weights of the dose groups and sham control group when compared to the corresponding control group. In females, there was a statistically significantly higher absolute and relative liver weight in the MD group (higher by 13 to 16 % vs control) and a higher absolute liver weight in the sham control (higher by 15% vs control) compared to the control group animals. In the absence of a dose response relationship and an absence of macroscopic and microscopic findings, this was not considered to have toxicological relevance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no gross lesions that could be attributed to treatment with the test item

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histomorphologic changes considered to be due to systemic toxic effects of the test item were not observed in any organs and tissues examined in this study. However, local irritative effects which were considered to be related to the properties of the test item formulation were observed in the stomach. They consisted of mucous neck cell hypertrophy with/without increased submucosal inflammatory cell infiltrate in the glandular stomach of animals from Group 5 (High-dose group). The same changes were also observed in Group 2 (Sham control group), and there were no clear differences in incidence and severity between the sham control group and the high-dose group. Even only NaOH or NaCl, depending upon the concentration used, can act as an irritant to cause mucosal necrosis in the stomach, especially in the glandular stomach. Moreover, it is known that mucous neck cell hypertrophy/proliferation occurs as a response to mild irritation to mucosa. It is likely that increased inflammatory cell infiltrate recorded in some animals is also a response to irritation to mucosa. Thus, the gastric lesions were deemed not to be directly related to the test item.

HAEMATOLOGY and COAGULATION
In males and females, no test item treatment related effects were observed for haematological parameters. However, there was a statistically significantly increase of large unstained cells (LUC) in male sham control group compared to control animals. But considering that no statistically significant changes in LUC of dose groups compared to control animals and also no dose response relationship was observed, no effect in LUC was considered. There was also statistically significantly lower RBC and monocyte counts in female MD groups. In the absence of dose response relationship, the findings were not considered to be of toxicological relevance. All mean and most of the individual values were within the historical control data range. There was also higher LUC in female MD and HD group, but in the absence of statistical significance, the finding was not considered adverse.
Blood coagulation was not affected in males due to test item treatment. In females there was statistically significantly longer prothrombin time (PT) in the HD group compared to the corresponding controls. This finding was within the historical control range and therefore was not considered to be adverse.

CLINICAL CHEMISTRY
There were no test item treatment related effects on clinical biochemistry parameters. All parameters were within the historical control data range.

URINALYSIS
The urinalysis performed in male and female animals revealed no test item treatment related effect.

(NEURO)BEHAVIOUR
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse toxicity observed in the study

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

LITTER DATA:
There were no test item treatment related effects on litter data including total number of pups born, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups and sex ratio on PND 0 and PND 4. There were no statistically significant changes noted for these litter data

LITTER WEIGHT DATA
There were no effects on pup mean weight, total litter weight, male and female litter weight on PND 0 and PND 4. There were no statistically significant change in dose groups compared to corresponding controls.

PUP SURVIVAL DATA
There were no effects on the survival of the pups from PND 1 through PND 4 in the dose groups and sham control group, when compared to the control group. A marginally higher mean mortality of pups between PND 1 and PND 4 was observed in the HD group (0.71%) compared to the control group (0.00%). This outcome did not achieve statistical significance and was attributed to the death of one single pup of one single dam on PND 1. Thus, it was considered incidental and not related to the treatment with the test item.

PUP EXTERNAL FINDINGS
No test item related gross external abnormalities of toxicological relevance were observed in the pups of any of the groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse toxicity observed in the study

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test no adverse effects were found after oral administration of Tantalum pentachloride in male and female Wistar rats and in the male and female pups. Based on the results, the NOAEL is considered to be 1000 mg/kg bw/day.
Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) Tantalum pentachloride (99.9%) was administered orally (after hydrolysis and neutralization) to 10 male and female Wistar rats/dose in water by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day. The animals were treated with the test item formulation on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

No adverse effects of Tantalum pentachloride after hydrolysis and neutralisation were found up to the dose level of 1000 mg/kg body weight/day.

There were no clinical signs of toxicological relevance in the dose groups and sham control group when compared to the control group. However, salivation and/or moving the bedding were observed transiently in all males and females of the HD and/ or SC group. These clinical signs were noted immediately after the dose administration, therefore, were considered to be signs of discomfort caused due to treatment.

There were no effects on the survival of the pups from PND 1 through PND 4 in the dose groups and sham control group, when compared to the control group. However, one single pup (pup no. 3 of dam 85) was found dead in the HD group, which was considered incidental.

There were no test item treatment related effects on clinical biochemistry parameters. There were no effects on urine parameters of males and females of dose groups compared to controls. Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance.

There were no test item treatment related effects on absolute and relative organ weights for males and females. Statistically significant differences were found in the weights of some organs (thyroid/parathyroid glands, prostate including seminal vesicles and coagulating glands, pituitary gland, liver and thymus of males and/ or females of dose and/ or control groups), which in the absence of a dose response relationship and also in the absence of macroscopic and microscopic findings were not considered to have toxicological relevance.

Under the conditions of this study, treatment-related histomorphologic changes were observed in the stomach of Group 5 (High-dose group). They consisted of mucous neck cell hypertrophy with/without increased submucosal inflammatory cell infiltrate in the glandular stomach. The same changes were also observed in Group 2 (Sham control group), and there were no clear differences in incidence and severity between the sham control group and the high-dose group. It was considered that these histologic changes were due to local stimuli to the glandular stomach mucosa, which could be associated with properties of the dose formulation in the intra-gastric environment, and the gastric lesions were deemed not to be directly related to the test item. The test item produced no histomorphologic evidence of toxicological properties in the male and female reproductive organs including testes, epididymides, prostate glands, coagulating glands, seminal vesicles, ovaries, uterus with cervix and vagina. Furthermore, by the detailed testicular examination, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis as well. The remainder of findings recorded were within the range of normal background lesions, which may be recorded in animals of this strain and age, or were incidental lesions that were not related to treatment with the test item.

There were no treatment-related effects found regarding to mortality, clinical signs, functional observations, histopathology, organ weights, reproduction, breeding data and pup development up to 1000 mg/kg bw/day. Based on the results, the NOAEL for maternal and reproductive/developmental toxicity is considered to be 1000 mg/kg bw/d. This study is classified as acceptable and satisfies the guideline requirement for an oral repeated dose toxicity study in rat.