Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is no indication for fertility risks caused by barium chloride based on the results of the extended one generation study in rats in which a NOAEL of 30 mg/kg/day was derived.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-08-07 to 2020-07-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted on 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS
The study was performed in rats according to OECD guideline 443 in compliance with GLP. The test substance was administered by the oral route. The basic configuration of EOGRTS was performed as based on the toxicological profile of the substance there are no concern-driven scientific triggers for the performance of the F2 generation (extension of Cohort 1B), developmental neurotoxicity (DNT; cohorts 2A and 2B) and/or developmental immunotoxicity (DIT; cohort 3) cohorts.

- Premating exposure duration for parental (P0) animals: A premating exposure period of 10 weeks is used to cover the full spermatogenesis and folliculogenesis before the mating.

- Basis for dose level selection: The highest dose level was selected with the aim to induce some systemic toxicity, but not death or severe suffering of the animals. Dose levels were selected based on the data from the previous studies in rats. In embryo-fetal development toxicity study in rats (Theuns- van Vliet, 2014) performed at 10, 30 and 100 mg/kg bw/day, maternal toxicity was observed in the high dose group as evidenced by the spontaneous deaths of two animals on gestation day 21 and the conditional decline of another animal on gestation day 21. Based on the longer duration of the treatment period in the EOGRTS study (18 weeks versus 20 day of the 414 study), the dose levels were set at 3, 10 and 30 mg/kg bw/day for this study.

- Inclusion/exclusion of extension of Cohort 1B: According to column 2 (specific rules for adaptation from column 1) point 8.7.3 of the amended REACH Annex X, extension of cohort 1B to include the F2 generation shall be proposed by the registrant based on the following conditions being met (a and any of b(i), b(ii) or b(iii)). See also: Chapter R.7a: Endpoint sepcific guidance Version 5.0 - December 2016:
A. The substance has uses leading to significant exposure of consumers or professionals, taking into account, inter alia, consumer exposure from articles
No – The substance has no uses leading to significant exposure of consumers or professionals. The substance has only a limited professional use, which is not expected to affect many users.
B (i). The substance displays genotoxic effects in somatic cell mutagenicity tests in vivo which could lead to classifying it as Mutagen Category 2, or
No – The substance is not classified as Mutagen Category 1A or 1B or 2.
B (ii). There are indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure, or
No – The substance is not classified as a PBT or vPvB. The toxicokinetic behaviour of the substances gives no hints for very slow clearance, the NOAEC/LOAEC of subchronic studies are not more than 3 times lower than that the NOAEC/LOAEC from a subacute study. Therefore there are no indications that the internal dose for the substance will reach a steady state in the test animals only after an extended exposure.
B (iii) There are indications of one or more relevant modes of action related to endocrine disruption from available in vivo studies or non-animal approaches
No - There are no indications based on the available study results that endocrine disruption is a relevant mode of action for the substance.

Therefore, based on the above considerations, the registrant does not believe that there is a basis for extending cohort 1B to include the F2 generation.

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: The registrant does not believe there is a need to include cohorts 2A and 2B in the test design. This is based on:
• previous studies with the substance do not indicate neurotoxic effects such as changes in brain weight or in specific neural areas not secondary to body weight, changes in brain volume or specific neural areas or (histo)pathological findings in brain, spinal cord and/or nerves
• Behavioural observations during the subchronic toxicity study did not reveal any concerns for a neurotoxic potential of the test material.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3: The registrant does not believe there is a need to include cohort 3 in the test design. This is based on:
• the substance has not caused biologically significant changes in haematology/clinical chemistry and/or organ weight associated with immunotoxicity such as reduced leucocyte count in combination with reduced spleen weight in repeated dose studies
• the substance has not caused significant effects to immunology organs such as thymus atrophy in repeated dose studies

- Route of administration: The oral route has been selected as it is the most appropriate route of administration for substances to focus on the detection of hazardous properties on reproduction.
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: the animals were approximately 5 - 6 weeks old at receipt
- Weight at study initiation: 126 - 150 g at receipt
- Housing:
♦ From arrival to pairing and after mating (males), 5 animals per sex per cage in polysulfone solid bottomed cages measuring 59.5x38x20 cm.
♦ During mating, 1 male/1 female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mes lid and floor.
♦ After mating, during gestation, birth and lactation, the females were individually housed in solid bottomed cages (measuring 42.5x26.6x18.5 cm)
- Diet (e.g. ad libitum): A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019, Settimo Milanese, Italy) was offered ad libitum throughout the study, except during the fasting procedure necessary for clinical pathology investigations
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles, except in the case of urinalysis investigations.
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12h / 12h
No relevant deviations from these ranges were recorded during the study.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the test substance preparations, the required amount of test substance was dissolved in the vehicle (demineralized water). The preparations were made at up to weekly intervals (concentrations of 0.3, 1.0 and 3.0 mg/mL). All concentrations were expressed in terms of the test item as supplied.
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone a the same dose volume. The dose volume was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Details on mating procedure:
- M/F ratio per cage: 1 male/1 female
- Length of cohabitation: 14 days. If mating had not occurred after 14 days of cohabitation, the animals were separated without further opportunity for mating.
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy (GD0)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CONCENTRATION VERIFICATION:
Samples of the formulation preparations were analysed to check the concentration once during pre-test, in week 1 of P generation, last week of P generation/Week 1 of F1, last week of F1, for a total of 4 occasions. For each occasion, 2 replicates of approx. 10 mL were collected from the dosing formulations.The samples were analysed for barium chloride dihydrate content at the site Quinta Analytica s.r.o. using an ICP/MS method validated over the concentration range 0.1 - 6.0 mg/mL (ValRep-296-2019). The in-process validation included an 8 non-zero points standard calibration curve covering the range 0.1 – 6.0 mg/mL of BaCl2.2H2O, and six quality control samples (QCs) at three concentration levels. The difference between obtained concentration in samples and the nominal concentration is less than 15%. The difference between duplicate samples was <10% of their mean value. The concentration in the control samples was < LLOQ (0.1 mg/mL).

HOMOGENEITY: not determined since the test substance is completely dissolved in the vehicle.

STABILITY:
During the validation of the analytical method, the stability of the test substance formulations (0.3 and 3.0 mg/mL) was determined after storage of 11 days at room temperature (20±5°C) and at 2-8°C. Based on the results it was concluded that barium chloride dihydrate is stable for at least 11 days at both storage conditions.
Duration of treatment / exposure:
P-generation: Males were treated once a day for at least 10 weeks prior to pairing, during mating up to the day before sacrifice which was scheduled at the termination of weaning of the majority of the F1 animals, for a total of 119-125 days; Females were treated once a day for at least 10 weeks prior to pairing, during mating, gestation and post partum periods until Day 21 post partum, the day before sacrifice, for a total of 115-124 days.
Cohort 1A: Males and females were treated starting from post-natal Day 21 (PND 21) for a minimum of 10 weeks up to the day before necropsy (approximately 13 weeks of age).
Cohort 1B: Males and females were treated starting from the day of the selection post-natal Day 21 (PND 21) up to the day before necropsy (approximately 14 weeks of age).
Frequency of treatment:
once daily
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- F0 generation parental animals: 25/sex/group
- Cohort 1A: 20/sex/group
- Cohort 1B: 20/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the data from the previous studies in rats. In embryo-fetal development toxicity study in rats (Theuns-van Vliet, 2014) performed at 10, 30 and 100 mg/kg bw/day, maternal toxicity was observed in the high dose group as evidenced by the spontaneous deaths of two animals on gestation day 21 and the conditional decline of another animal on gestation day 21. Based on the longer duration of the treatment period in the EOGRTS study (18 weeks versus 20 day of the 414 study), the dose levels were set at 3, 10 and 30 mg/kg bw/day for this study.
- Rationale for animal assignment (if not random): At weaning, i.e. on PND 21, 40 male and 40 female pups/group, where possible from different litters, were randomly selected with the exception of obvious runts (i.e. animals with a body weight more than two standard deviations below the mean pup weight of the respective litter). 20 male and 20 female pupswere randomly allocated to each group of Cohort 1A and Cohort 1B, respectively.
- Fasting period before blood sampling for clinical biochemistry: yes, during the last week of treatment, blood samples for haematology and clinical chemistry were collected from 10 randomly selected animals/group/sex in the P-generation and Cohort 1A under condition of food and water deprivation.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily animals were checked for mortality; on working days animals were checked early in the morning and in the afternoon. At weekends and public holidays a check was carried out at approx. mid-day. During the treatment period all animals were checked daily for any abnormal clinical signs before the administration as well as approx. 1-1.5 hours after the administration
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and / or signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment (P generation only) and at least once a week from the start of treatment (Day 21 of age for Cohorts 1A and 1B) until termination, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena for at least 1 minute. Recorded signs included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. All observations were recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the day of allocation (Day 21 of age for Cohorts 1A and 1B) and then approximately at weekly intervals from the first day of treatment (Day 21 of age for
Cohorts 1A and 1B) to termination or until termination or until positive identification of mating (P generation). Females of P generation after mating were also weighed on Days 0, 7, 14 and 20 post coitum and on Days 1, 4, 7, 14 and 21 post partum. Body weight was also recorded in Cohorts 1A and 1B on the day when they attain puberty (completion of preputial separation or vaginal patency). All animals were weighed on the day of sacrifice.

FOOD CONSUMPTION:
Food consumption was recorded on the same days as body weights, at weekly intervals starting from Day 1 of dosing and up to pairing for P generation and starting from nominal Day 28 for F1 generation. In addition, for males of P generation the food consumption was recorded at weekly intervals from the end of the mating period to termination. Food consumption of females of P generation was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7, 14 and 21 post partum starting from Day 1 post partum.

WATER CONSUMPTION
- Time schedule for examinations: not measured

CLINICAL PATHOLOGY INVESTIGATIONS: Yes
- Time schedule for collection of blood: During the last week of treatment, samples of blood for haematology and clinical chemistry were collected from the retro-orbital sinus, from 10 males randomly selected from each group, For coagulation tests, blood was collected at necropsy from the abdominal vena cava under isoflurane anaesthesia, with no food and water deprivation.
As a part of the sacrificial procedure, and under condition of food deprivation, blood samples for haematology, coagulation tests and clinical chemistry were withdrawn from the abdominal vena cava of 10 females (for P generation females with viable litters, if possible), all randomly selected from each group. In addition, samples of blood for hormone determination (0.5 mL) were collected in the same condition from all females and males of each group for P generation.
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: yes, food deprivation for blood samples for haematology, coagulation test (females) and clinical chemistry.
- Haematology: Parameters checked in Table 1 were examined.
- Coagulation: Prothrombin time and activated partial thromboplastin time were investigated.
- Clinical chemistry: Parameters checked in Table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of treatment, individual overnight urine samples were collected from 10 males and 10 females (for P generation females with viable litters, if possible), all randomly selected from each group (the same animals selected for clinical pathology investigation under the same condition).
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.
- The following measurements were performed on urine: Volume (manually recorded), appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood. The sediment, obtained from centrifugation at approx. 3000 rmp for 10 minutes, was examined for epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components.

OTHER: During the last week of treatment, a measure of blood clotting time was performed once from 10 parental males and 10 parental females (females with viable litters, if possible), all randomly selected from each group.
Oestrous cyclicity (parental animals):
The assessment of oestrous cycles, with vaginal smears, was performed once daily from allocation to dosing.
Oestrous cycles were also monitored for at least 2 weeks before pairing (starting from Day 57 of treatment) up to positive identification of mating. The vaginal smear data was examined to determine the following:
1. anomalies of the oestrous cycle;
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also taken on the day of necropsy.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generation and in cohort 1A males (all groups):
During the necropsy procedure, examination of sperm parameters was performed in all males. At termination, the weight of one epididymal cauda was recorded for all P and F1 (Cohort 1A) males. One epididymis was used for enumeration of cauda epididymis sperm reserves. Sperm from the cauda epididymis was examined for evaluation motility and morphology. Sperm motility was evaluated immediately after sacrifice. The percentage of motile sperm was determined. At least 200 spermatozoa per sample were observed and classified as either normal (both head and midpiece/tail appear normal) or abnormal.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- General litter observations: Each litter was examined as soon as possible after parturition was considered complete (Day 0 post partum or PND 0) to establish the number and sex of pups (live and dead), stillbirths, live births, and the presence of gross anomalies (i.e. externally visible abnormalities, abnormal skin colour or texture; lack of milk in stomach; presence of dried secretions) and a qualitative assessment of body temperature (presence of cold pups), state of activity and reaction to handling. Observations were performed once daily for all litters.
- Body weight: All live F1 pups were individually weighed on Days 1, 4, 7, 14 and 21 post partum
- Anogenital distance: The anogenital distance (AGD) of each pup was measured on PND 1. The measure of AGD was normalized to the cube root of pups body weight measured on Day 1 post partum.
- Nipple count: The presence of nipples/areolae was checked on PND 13.
- Testes descent: Testes descent was checked on Day 21 post partum.

The following parameters were examined in pups that were selected for the cohorts (1A and 1B):
- General clinical observations: Viability (morbidity and mortality) checks were performed twice daily (at weekends on public holidays, once a day). Cage side observation were conducted at least once daily during the dosing period (at approximately 1 to 1.5 hours post dose).
- Detailed clinical observations: Once weekly from the start of treatment (Day 21 of age) until termination, all animals were subjected to detailed clinical observations.
- Body weight: Body weights of all male and female animals were recorded at weekly intervals from the first day of treatment (Day 21). Additionally body weight was recorded on the day when they attain puberty (completion of preputial separation or vaginal patency).
- Clinical pathology investigations: During the last week of treatment, samples of blood for haematology and clinical chemistry were collected from the retro-orbital sinus under isoflurane anaesthesia, from 10 Cohort 1A males randomly selected from each group, under condition of food and water deprivation. In addition, samples of blood for hormone determination (0.5 mL) were collected in the same condition from all males of each group. Parameters checked in Table 1 and 2 were examined. For Cohort 1A females blood was collected as part of the sacrificial procedure, see section 'Postmortem examination offspring'.
- Food consumption: Food consumption was recorded on the same days as body weights, at weekly intervals starting from nominal Day 28 for F1 generation.
- Vaginal opening: The onset of vaginal opening was monitored starting from Day 28 of age until 100% occurrence. On the day of occurrence the body weight was recorded.
- Vaginal smears: A vaginal smear was taken daily in female pups, after the onset of vaginal patency (the occurrence of vaginal opening), until the first cornified smear is recorded (first oestrus), in order to determine the time interval between these two events. Oestrous cycles for all F1 females was also monitored, with vaginal smears performed once daily, for a period of two weeks, commencing around Post Natal Day (PND) 57. Vaginal smears were also taken on the day of necropsy.
- Balano preputial separation: The cleavage of the balano preputial skinfolds separation of males was checked once daily from Day 35 of age until 100% occurrence. On the day of occurrence the body weight was recorded.
- Urinalysis (Cohort 1A): Individual overnight urine was collected from F1 Cohort 1A study animals (10 animals/sex/group) during the last week of treatment and checked for volume, appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, urobilinogen and microscopic examination of sediment.
- Thyroid hormone assay (Cohort 1A): For Cohort 1A females, samples of blood for hormone determination (0.5 mL) were collected under condition of food deprivation from the retro-orbital sinus under isoflurane anaesthesia.
During the necropsy procedure of surplus F1 pups at PND 4 (after culling), blood samples (ca. 0.2 mL) were collected from 2 of the non selected pups (1 male and 1 female if possible) under light ether anaesthesia by intracardiac heart puncture for possible determination of T3, T4 and TSH concentrations. Blood (ca 0.5 mL) was also collected from F1 pups at PND22 (not selected for cohorts) under isoflurane anaesthesia from the abdominal vena cava from at least two pups (1 sample/sex) per litter. Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid Stimulating Hormone (TSH) by a multiplex assay, using Luminex Magpix system and the MILLIPLEX MAP Rat Thyroid Magnetic Bead Panel kit (Merk Millipore, cat. no. RTHYMAG-30K).
- Immunox analysis: From all males and all females of each treatment group of Cohort 1A, mesenteric lymph nodes were weighed. From 10 males and 10 females of each treatment group, one half of the spleen was subjected to spleen lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells). The remaining half of the spleen was preserved for histopathological evaluation

GROSS EXAMINATION OF DEAD PUPS:
Yes, pups found dead on PND 0 or at a later time or sacrificed for humane reasons were subjected to necropsy (external and internal examination) for the identification of possible defects and cause of death, with the exception of those excessively cannibalised or autolysed.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Males of the P generation were killed after the weaning of the majority of F1 females.
- Maternal animals: Females with live pups were killed on Day 22 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. The females showing no evidence of copulation were killed 25 days after the last day of the mating session. The females which did not give birth 25 days after positive identification of mating were killed shortly after.
All animals were weighed on the day of sacrifice.

GROSS NECROPSY
- The clinical history of adult animals 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 and the required tissue samples preserved in appropriate fixative and processed for histopathological examination (see Table 3).
The ovaries and uteri of each dam on Day 22 post partum were examined to determine:
♦ Number of corpora lutea on each ovary;
♦ Number of implantation sites.
Uteri of females without litters were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation. If implantations are noted after the immersion, the number of corpora lutea will be counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
- From all animals completing the scheduled test period, the organs indicated in Table 3 were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal. The tissues required for histopathological examination are listed insections in Table 3. 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 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
Microscopic examination was performed on the collected organs of all animals of the control and high dose groups and in the low and medium dose group animals that died or were killed in extremis during the treatment period. Since effects were observed in the spleens of the high dose males, histopathological examination of the spleen was extended to the males of the other dose groups.
Postmortem examinations (offspring):
SACRIFICE
- The F1 pups that were not selected for the cohorts (non-selected pups) at PND 4 or at weaning (PND 22) were sacrificed.
- The Cohort 1A and 1B pups were sacrificed on scheduled necropsy (approx. 13 and 14 weeks of age, respectively). All animals were weighed on the day of sacrifice. As a part of the sacrificial procedure, and under condition of food deprivation, blood samples for haematology and clinical chemistry were withdrawn under isoflurane anaesthesia from the abdominal vena cava of 10 Cohort 1A females, all randomly selected from each group. Parameters checked in Table 1 and 2 were examined. For Cohort 1A females, samples of blood for hormone determination (0.5 mL) were collected in the same conditions from the retro-orbital sinus under isoflurane anaesthesia. During the necropsy procedure blood samples from the vena cava were taken from 10 randomly selected Cohort 1A males and females per dose group for coagulation

GROSS NECROPSY
The clinical history of Cohort 1A and 1B animals 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 and the required tissue samples preserved in appropriate fixative and processed for histopathological examination (see Table 3 and 4). The pups of parental generation (P generation) not selected for cohorts, were sacrificed after weaning (on PND 22). All pups were subjected to gross necropsy (external and internal examination). For up to 10 pups per sex per group, from different litters (where possible), organs were weighed and retained in the 10% buffered formalin (See Table 5). In addition, thyroid and mammary tissues from the same male and female pups were preserved for possible further histological examination

HISTOPATHOLOGY / ORGAN WEIGTHS
From all Cohort 1A and 1B animals completing the scheduled test period, the organs indicated in Table 3 and 4 respectively, were dissected free of fat and weighed. The ratios of organ weight to body weight was calculated for each animal.
The tissues required for histopathological examination are listed insections in Table 3. 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 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
Microscopic examination was performed on the collected organs of all animals of the control and high dose groups and in the low and medium dose group animals that died or were killed in extremis during the treatment period.

OTHER:
- During the necropsy procedure, shortly after the death of each animal (except for those found dead), bone marrow samples were obtained from the femur of all Cohort 1A animals. Smears prepared from these samples were air dried, fixed in methanol, stained using a May-Grunwald-Giemsa procedure and stored. The evaluation of bone marrow smears was performed in 10 male and 10 female animals per group, randomly selected.
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. Statistical analysis of histopathological
findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n>5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups was assessed by the nonparametric version of theWilliams test. Further tests were used as considered appropriate. The criterion for statistical significance was p<0.05.
Reproductive indices:
The following reproductive indices were calculated:
♦ Male copulatory index: (No. of males with evidence of copulation/No. of animals paired) ×100
♦ Male fertility index: (No. of males which induced pregnancy/No. of males paired) ×100
♦ Female copulatory index: (No. of females with evidence of copulation/No. of animals paired) ×100
♦ Female fertility index: (No. of pregnant females/No. of females paired) ×100
♦ Pre- coital interval = Mean number of days between pairing and evidence of mating
Offspring viability indices:
♦ Pre-natal loss = No. of visible implantations - live litter size at birth / No. of visible implantations x 100
♦ Post-natal loss at birth = Total litter size at birth - live litter size / Total litter size x 100
♦ Pup loss (till PND 4 before culling) = Live litter size at birth - live litter size at PND 4 (before culling) / Live litter size at birth x 100
♦ Pup loss (till PND 21 after culling) = Live litter size at PND 4 (after culling) - live litter size at Day 7, 14, 21/live litter size at PND 4 (after culling) x 100.
♦ Sex ratios were calculated at birth, Day 4 post partum and Day 21 post partum are presented as the percentage of males per litter.
♦ Lactation index for F0 females after culling = No. of live pups at Day 21 post partum / Live litter size at Day 4 (after culling) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed during the study. Protruding or damaged eyes were observed in some male animals from different groups. This was ascribed to the bleeding technique. No clincal signs were observed in the females at any phase of the study.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Four cases of unscheduled deaths occurred during the study. One mid-dose male (10mg/kg/day) was found dead at Day 45 of the mating phase. One control female was found dead on Day 10 of the post partum phase. The occurrence of an abscess was the cause of the poor health condition which led the animal to death. One low dose female (3mg/kg/day) was sacrificed for welfare reasons at Day 2 of the post partum phase. The cause of the poor heath condition of this female was considered related to the post partum uterine haemorrage. One high dose female (30mg/kg/day) was sacrificed for welfare reasons at Day 22 of the gestation phase. This animal had acute inflammation in the uterus with purulent exudate (pyometra) that was the cause of the poor health condition.
All the unscheduled deaths which occurred during the study were considered incidental and unrelated to the treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Means of body weight and body weight gain of treated males and females were comparable to the control group throughout the study. Terminal body weight of treated animals of both sexes of the three groups (Groups 2, 3 and
4) was comparable to the concurrent control group (Group 1).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption in treated males and females was comparable to the control group during the whole study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean corpuscular haemoglobin concentration was statistically significantly lower than controls in males dosed at 30 mg/kg/day (2%). Due to the minimal severity, this finding was considered to be not adverse. The other statistically significant differences between control and animals receiving 10 mg/kg/day (mean corpuscular haemoglobin in males, basophils in both sexes) were not dose-related, therefore they were considered to be unrelated to treatment. No changes were recorded in the coagulation and blood clotting time.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant changes were recorded. The statistically significant differences between control and animals dosed at 10 mg/kg/day (alkaline phosphatase and sodium in males, gamma-glutamyl transferase in females) were not dose-related, therefore they were considered to be incidental.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No changes were recorded.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes considered to be associated with the oral administration of Barium chloride dihydrate were present in the spleen of males dosed with 30 mg/kg/day (high dose). There was an increased incidence and severity of extramedullary haematopoiesis when compared to concurrent controls (8 animals at the high dose vs 1 control), graded as moderate. The extramedullary haematopoiesis, also called compensatory reactivation or reactive extramedullary haematopoiesis, was considered a non adverse natural homeostatic response sometimes occurring independently or without an obvious underlying cause, and lacking serious clinical or diagnostic implications. A correlation to the change in the mean corpuscular haemoglobin concentration in males dosed at 30 mg/kg/day seen in high dose treated males was most likely present. The incidence and the severity of extramedullary haematopoiesis in the males treated with 3 and 10 mg/kg/day (low and mid-doses) and in the high dose treated females were comparable to the concurrent controls.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrous cycle and pre-coital interval of treated females were comparable to control females.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Qualitative testis staging in the high dose males did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle (Stages I- XIV). As regular layering in the germinal epithelium was noted, there was no treatment-related effect on the spermatogenic cycle.
Reproductive performance:
no effects observed
Description (incidence and severity):
- Differential follicle and corpora lutea counts per ovary did not reveal any relevant differences in all high dose females, when compared with their respective controls.
- The copulatory indices were 100% for all treated and control groups.
- The fertility indices were 96% for controls, 88% in low and mid-dose (3 and 10 mg/kg/day) and 92% in the high dose groups (30 mg/kg/day).
Results are provided in Table 6 and 7.
- Thyroid hormones: No changes were recorded in T3, T4 or TSH of parental animals.
Results are provided in Table 8.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest dose level
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed in Cohort 1A and 1B animals during the study.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: One mid-dose male dosed at 10 mg/kg/day was found dead at Day 27 of the dosing phase. No clinical signs were observed in this animal.
Cohort 1B: One mid-dose female at 10 mg/kg/day) was found dead at Day 57 and one control male died on Day 69 of the dosing phase. The cause of the death of these animals was not considered to be treatment-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Means of body weight and body weight gain of treated Cohort 1A and 1B males and female were comparable to the control group throughout the study. Terminal body weight of treated Cohort 1A and 1B animals of both sexes of the three groups (Groups 2, 3 and 4) was comparable to the concurrent control group (Group 1).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No relevant changes were observed in food consumption during the study for Cohort 1A and 1B animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Reticulocytes were statistically significantly higher than control in females dosed at 30 mg/kg/day (25%). Due to the absence of changes of the other red blood cell series (e.g. haematocrit, haemoglobin, erythrocytes), this finding was considered to be not adverse. In addition, mean neutrophils data were higher than controls in females dosed at 30 mg/kg/day (62% above controls). This finding was mostly due to the high values of two animals only, therefore the observed neutrophilia was considered to be unrelated to treatment.
The other statistically significant differences between control and animals receiving 10 mg/kg/day (platelets in males and mean corpuscular volume in females) were not dose related, therefore they were considered to be incidental
Prothrombin time was statistically significantly reduced in males of all treated groups (3% to 6%). A slight reduction of this parameter usually has no pathological significance, therefore this finding was considered to be not adverse
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
One male dosed at 30 mg/kg/day showed increases of alanine and aspartate aminotransferase (3.3 and 2.2 fold, respectively). Due to the minimal incidence, this finding was considered to be incidental.
In addition, gamma-glutamyl transferase was decreased in males dosed at 30 mg/kg/day (57% below controls). The decrease of this parameter has no pathological significance, therefore this finding was considered to be not adverse.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No changes were recorded.
Sexual maturation:
no effects observed
Description (incidence and severity):
Balanopreputial separation: There were no effects on the mean age of balanopreputial separation in Cohort 1A and 1B males.
Vaginal opening: There were no effects on the mean age of vaginal opening in Cohort 1A and 1B females.
Time to first estrous: There were no effects on the mean time from vaginal opening to first oestrous in Cohort 1A and 1B females.
Estrous cycles: There were no effects on means estrous cycle parameters in Cohort 1A and 1B females.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant increase in the anogenital distance (AGD) performed on Day 1 post partum (normalised to the cube root of the body weight), was observed in male pups receiving 10 and 30 mg/kg/day (approximately +6% and +7%, respectively). No significant differences were observed in female pups. Since the increases were minimal and mean values of all groups were within the range of theERBC historical control data (0.55 - 3.14 mm/g1/2 for males), these variations were not considered to be adverse. Results are provided in Table 9.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were retained in male pups on Day 22 post partum, since they were not observed.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- F1 pups on Day 22: No treatment-related intergroup differences were observed in absolute and relative organ weights of F1 pups sacrificed on Day 22 post partum.
- Cohort 1A and 1B: No treatment-related organ weight changes were seen. All other organ weight variations between control and treated animals were considered incidental, having no histopathological correlate and unrelated to the dose, and to be within the physiological range of Sprague Dawley SD rats of this age.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- Cohort 1A and 1B: No gross pathology treatment-related changes were noted in Cohort 1A and 1B animals. All observed changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
- Necropsy findings in decedent pups, in pups sacrificed on Days 4 and 22 post partum: Autolysed or cannibalized thoracic and/or abdominal organs were observed in the majority of decedent pups both in control and treated groups, without any possible macroscopic examination of internal organs. No significant abnormalities were recorded at the external and internal examination in pups sacrificed on Days 4 and 22 post partum. Tip of tail missing or damaged tail and right kidney pelvic dilatation were noted in individual pups from different groups.
Histopathological findings:
no effects observed
Description (incidence and severity):
Following histopathological evaluation, no treatment-related changes were noted. All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Other effects:
no effects observed
Description (incidence and severity):
- Thyroid hormone determination (Cohort 1A): No changes were recorded in T3, T4 or TSH.
- Spermatogenic cycle (Cohort 1A and 1B): Qualitative testis staging in the high dose males did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle (Stages I- XIV). As regular layering in the germinal epithelium was noted, there was no treatment-related effect on the spermatogenic cycle.
- Morphological evaluation of the ovaries (Cohort 1A and 1B: No treatment-related findings were detected following morphological evaluation of the ovaries.
- Enumaration of ovarian follicles (Cohort 1A and 1B): Differential follicle and corpora lutea counts per ovary did not reveal any relevant differences in all high dose females, when compared with their respective controls.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Clinical observations performed weekly for neurotoxicity assessment (observation of animals at removal from the cage and in an open arena) were comparable between treated and control animals for Cohort 1A and 1B.
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
No statistically significant differences were observed in the frequencies of all cell populations in any treatment group of both sexes. Expression of surface markers was similar for all groups for both genders.
In conclusion, no sign of alteration in the immune cell distribution was observed in splenocytes of animals treated with the test item at any dose level, when compared to controls and to historical control data.
- Sex-ratio: Sex ratio was comparable between treated and control groups.
- Enumeration of ovarian follicles: Differential follicle and corpora lutea counts per ovary did not reveal any relevant differences in all high dose females, when compared with their respective controls.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed in reproductive and developmental parameters and general toxicity at any of the dose levels
Reproductive effects observed:
no

Table 6. Mating and fertility data of the P generation

Dose level (mg/kg/day)

0

3

10

30

Oestrous cycles of F0 females before pairing (± SD)

3 (± 1.08)

3 (± 0.95)

3 (± 1.04)

3 (± 0.94)

Number of animals paired (M + F)

25 + 25

25 + 25

25 + 25

25 + 25

Number of females mated

25

25

25

25

Copulatory index, males and females (%)

100

100

100

100

Mean number of days to mate (days ± SD)

2 (± 1.57)

3 (± 2.38)

2 (± 1.46)

3 (± 2.39)

Number of pregnant females

24

22

22

23

Fertility index, male and female (%)

96

88

88

92

 

Table 7. Reproductive data of the P generation

Dose level (mg/kg/day)

0

3

10

30

Number of females with liveborn

23

22

22

22

Gestation length (days ± SD)

22.3 (± 0.64)

22.4 (± 0.49)

22.4 (± 0.49)

22.3 (± 0.48)

Implantations (mean ± SD)

15.2 (± 4.37)

16.4 (± 1.89)

16.9 (± 2.40)

16.1 (± 2.48)

Total litter size at birth (mean ± SD)

13.6 (± 4.96)

14.8 (± 3.36)

14.3 (± 3.59)

13.9 (± 2.91)

Live litter size at birth (mean ± SD)

13.3 (± 4.85)

14.5 (± 3.33)

14.1 (± 3.50)

13.8 (± 3.00)

Pre-natal loss (% ± SD)

11.7 (± 19.1)

12.2 (± 16.6)

16.5 (± 17.5)

14.4 (± 12.3)

Post-natal loss at birth (% ± SD)

1.93 (± 4.68)

2.32 (± 4.45)

1.16 (± 2.57)

0.81 (± 2.63)

Post-natal loss on Day 4 p.p. (% ± SD)

5.97 (± 10.3)

8.40 (± 12.3)

5.22 (± 7.26)

4.56 (± 5.52)

Post-natal loss on Day 7 p.p. (% ± SD)

2.70 (± 9.29)

2.02 (± 5.34)

1.91 (± 5.12)

2.27 (± 8.69)

Post-natal loss on Day 14 p.p. (% ± SD)

4.17 (± 11.4)

2.02 (± 5.34)

3.33 (± 7.96)

2.73 (± 8.83)

Post-natal loss on Day 21 p.p. (% ± SD)

4.67 (± 11.4)

2.02 (± 5.34)

3.33 (± 7.96)

3.18 (± 8.94)

Sex ratio of F1 pups at birth(% ± SD)

55.9 (± 19.5)

52.1 (± 14.9)

50.5 (± 15.1)

55.2 (± 17.5)

Sex ratio of F1 pups on Day 4 p.p. 

(% ± SD)

54.9 (± 15.6)

50.7 (± 15.8)

52.1 (± 15.9)

56.2 (± 19.5)

Sex ratio of F1 pups on Day 21 p.p. 

(% ± SD)

52.3 (± 13.2)

52.8 (± 12.5)

50.7 (± 12.4)

54.3 (± 15.2)

Sex ratio: percentage of male pups

Table 8. Thyroid hormone concentrations in P generation animals (mean± SD)

Dose level (mg/kg/day)

0

3

10

30

Males P generation

T3 (nmol/L)

0.843 (± 0.323)

0.821 (± 0.291)

0.675 (± 0.157)

0.715 (± 0.215)

T4 (nmol/L)

45.7 (± 7.2)

42.6 (± 6.9)

43.9 (± 7.3)

42.2 (± 4.7)

TSH (ng/mL)

8.17 (± 1.68)

10.5 (± 4.75)

7.37 (± 2.47)

9.89 (± 3.70)

Females P generation

T3 (nmol/L)

0.912 (± 0.236)

0.745 (± 0.210)

0.821 (± 0.245)

0.735 (± 0.210)

T4 (nmol/L)

31.8 (± 4.7)

29.8 (± 6.9)

34.1 (± 6.5)

34.0 (± 8.5)

TSH (ng/mL)

7.82 (± 3.89)

7.93 (± 2.89)

8.48 (± 2.67)

6.70 (± 1.50)

Males Cohort 1A

T3 (nmol/L)

0.853 (± 0.123)

0.856 (± 0.183)

0.834 (± 0.147)

0.897 (± 0.201)

T4 (nmol/L)

48.5 (± 10.4)

53.1 (± 5.5)

47.8 (± 9.9)

49.8 (± 7.9)

TSH (ng/mL)

7.05 (± 3.12)

8.27 (± 4.52)

7.30 (± 2.54)

8.37 (± 2.01)

Females Cohort 1A

T3 (nmol/L)

1.070 (± 0.181)

0.993 (± 0.206)

1.055 (± 0.074)

0.994 (± 0.155)

T4 (nmol/L)

29.1 (± 6.2)

35.0 (± 7.2)

30.1 (± 6.6)

31.3 (± 3.5)

TSH (ng/mL)

5.11 (± 1.77)

5.72 (± 1.63)

6.07 (± 1.76)

5.74 (± 1.35)

 

Table 9. Anogenital distance on Day 1 p.p. of F1 pups

Dose level (mg/kg/day)

0

3

10

30

Males

Anogenital distance (normalized, mm/g1/3± SD)

2.12 (± 0.41)

2.14 (± 0.45)

2.24* (± 0.31)

2.26* (± 0.39)

Females

Anogenital distance (normalized, mm/g1/3± SD)

1.33 (± 0.25)

1.26 (± 0.30)

1.35 (± 0.27)

1.29 (± 0.26)

* = mean value of group is significantly different from control

Conclusions:
Based on the results of the present study, the dosage of 30 mg/kg/day was considered the NOAEL for general and reproductive toxicity and pups development in parental generation and for general toxicity in Cohorts 1A and 1B.
Executive summary:

In a GLP compliant Extended One Generation Study performed according to OECD 443, Barium dichloride dihydrate was adminstered daily by oral gavage to Sprague-Dawley rats. The animals (25 per sex/dose) were exposed to 3, 10 and 30 mg/kg bw/day barium dichloride dihydrate dissolved in demineralized water. The control group received demineralized water. F0 Males were treated for 10 weeks prior to pairing, through the mating period and thereafter until the day before necropsy, for a total of 119 -125 days. F0 females were treated for 10 weeks to pairing, during pairing and throughout the gestation and lactation period until Day 21 post partum, for a total of 115 -124 days. One male male and one female pup were selected and assigned to Cohort 1A and Cohort 1B to follow the toxicity effect in the F1 generation adminstered from weaning (Day 21 of age) up to 13 and 14 weeks, respectively. During the lactation period, the F1 animals were not administered with test item directly. Cohort 1A was allocated for primary assessment of effects upon reproductive systems and of general toxicity and Cohort 1B for histopathology follow-up assessment of the reproductive system.

Results parental generation (F0)

Four cases of unscheduled deaths occurred during the study. All the unscheduled deaths which occurred during the study were considered incidental and unrelated to the treatment. At the daily and weekly clinical observation, no signs considered adverse and no effect in the neurotoxicity assessment were observed. Body weight, body weight gain and food consumption were unaffected by treatment. No changes that could be considered adverse were seen in haematology, coagulation and clinical chemistry parameters of treated animals compared to controls. No changes were recorded in thyroid hormones determination.

No treatment-related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. Sperm analysis performed in all treated males was comparable to controls. Enumeration of ovarian follicles performed in control and high dose females did not reveal any relevant differences. Copulatory and fertility indices did not show any treatment-related differences among treated and control groups. Implantation, pre-natal loss, litter data and sex ratio did not show any changes of toxicological relevance. The number of females with live pups on Days 21/22 post partum was: 20 in the control, 21 in the low dose (3 mg/kg/day), 21 in the mid-dose (10 mg/kg/day) and 22 in the high dose (30 mg/kg/day) groups.

No differences considered treatment-related were observed in the anogenital distance between control and treated groups both for male and female pups. No nipples were observed in male pups. Clinical signs and findings at necropsy, as well as organ weights did not reveal any treatment-related or adverse effect in pups.

A minimal statistically significant increase in the absolute and relative spleen mean weights was induced by treatment with Barium chloride dihydrate in the high dose males of the parental generation, when compared to controls. Changes in the spleen correlated microscopically with a higher severity of extramedullary haematopoiesis. Gross pathology evaluation revealed no treatment-related changes.

Changes considered to be associated with the oral administration of Barium chloride dihydrate were present in the spleen of males dosed with 30 mg/kg/day (high dose) and consisted of increased incidence and severity of moderate extramedullary haematopoiesis. This change was considered a non-adverse natural homeostatic response sometimes occurring independently or without an obvious underlying cause.

Results Cohorts 1A and 1B

No treatment-related mortality occurred in Cohort 1A and Cohort 1B animals. At the daily and weekly clinical observation, no signs of toxicological relevance and no effect in the neurotoxicity assessment were observed in treated males and females of both Cohorts. Body weight, body weight gain and food consumption of both sexes of both Cohorts were unaffected by treatment. No changes were recorded in the haematology, coagulation, clinical chemistry and urinalysis

parameters between treated and control animals of Cohort 1A.

Oestrous cycle, vaginal opening and balano-preputial skin folds separation did not reveal differences considered adverse in Cohort 1A and Cohort 1B animals. Enumeration of ovarian follicles performed in control and high dose females did not reveal any relevant differences in Cohort 1A and Cohort 1B. The staging of spermatogenic cycle performed did not show any treatment-related effects in Cohort 1A and Cohort 1B.

No relevant changes were seen in bone marrow evaluation and in thyroid hormones determination (in Cohort 1A). No sign of alteration in the immune cell distribution was observed in splenocytes of treated animals of Cohort 1A. No treatment-related changes were noted at spermanalysis (in Cohort 1A), organ weights, gross pathology and the histopathological examination (in Cohort 1A).

Conclusion

In conclusion, signs of effects related to treatment with Barium chloride dihydrate, when administered to rats by oral route at dose levels of 3, 10 and 30 mg/kg/day, were observed in animals dosed at 30 mg/kg/day. These effects (minimal statistically significant increase in the absolute and relative spleen mean weights associated to increased incidence and severity of moderate extramedullary haematopoiesis), observed in high dose parental males, were considered a non-adverse natural homeostatic response sometimes occurring independently or without an obvious underlying cause. In the absence of degenerative changes, this was considered an adaptive and not adverse change.

No treatment-related changes were observed in reproductive and developmental parameters in Parental generation and for general toxicity in Cohorts 1A and 1B at any of the dose levels investigated.

Based on the results of the present study, the dosage of 30 mg/kg/day was considered the NOAEL for general and reproductive toxicity and pups development in Parental generation and for general toxicity in Cohorts 1A and 1B.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is fully acceptable
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP compliant Extended One Generation Study performed according to OECD 443, Barium dichloride dihydrate was adminstered daily by oral gavage to Sprague-Dawley rats at dose levels of 0, 3, 10 and 30 mg/kg bw/day. No treatment-related changes were observed in reproductive and developmental parameters in Parental generation and for general toxicity in Cohorts 1A and 1B at any of the dose levels investigated. For this reason the dosage of 30 mg/kg/day was considered the NOAEL for general and reproductive toxicity and pups development in Parental generation and for general toxicity in Cohorts 1A and 1B.

Effects on developmental toxicity

Description of key information
Developmental toxicity: a NOAEL of 85.3 mg BaCl2/kg was derived in an oral developmental toxicity study according to OECD 414.
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-11-24 to 2013-12-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2001-01-22
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
, 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2013-07-09
Limit test:
no
Specific details on test material used for the study:
- Analytical purity: ≥ 98.5%
- Batch No.: 9284
- Expiration date of the batch: 01 September 2015
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS- RccHan: WIST strain
- Source: Harlan, Horst, the Netherlands
- Age at study initiation: approx. 12 weeks of age
- Weight at study initiation: control group: 197.9 - 230.9 g; low dose group: 198.1 - 230.2 g; mid dose group: 190.9 - 232.5 g; high dose group: 191.6 - 239.4 g
- Housing: animals were housed in Macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the quarantine and acclimatization periods, the animals were housed in groups of 4 per sex. Mated females were housed individually in Macrolon cages.
- Diet (ad libitum): cereal-based (closed formula) rodent diet (Rat & Mouse No.3 Breeding Diet; RM3) (supplier: SDS Special Diets Services, Witham, England)
- Water (ad libitum): domestic mains tap-water
- Quarantine period: 9 days (upon arrival the rats were quarantined and checked for overt signs of ill health and abnormalities. During the quarantine period, serological examinations of the microbiological status of the rats were conducted in a random sample.)
- Acclimation period: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Relative humidity: exceeded 65% for short times only during cleaning activities
- Air changes: about 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: demineralized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item in the vehicle were prepared weekly, and stored in a refrigerator (2-10°C) in the dark in aliquots sufficient for one day. The vehicle for dosing the controls was similarly stored.
The solutions of the test item in the vehicle were prepared by stirring on a magnetic stirrer. Subsequently, 8 aliquots (7 days plus 1 extra) were taken per dose level according to the daily volume required for each dosing. These aliquots were taken under continuous stirring. On each subsequent day, one aliquot for each group was removed from the refrigerator and allowed to equilibrate to ambient temperature prior to dosing.

A dosing volume of 10 mL/kg was applied for all animals, which was adjusted based on the latest body weight. After gestation day 14 dose volumes were not adjusted.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From all three batches of the test items prepared in the study, samples were taken immediately after preparation and stored in a refrigerator until analysis. The following analyses were conducted by Inductively coupled plasma atomic emission spectrometry (ICP-AES) analysis. The test item was quantified using barium as a marker component:
- Homogeneity: the homogeneity of the test substance in the experimental test items was demonstrated in the first batch prepared, by analysing three samples (taken at different locations in the gavage liquid container) of each level.
- Concentration: the concentration of test substance at each level was determined in all three batches of test items prepared in the study.
- Stability: samples of the low-dose, mid-dose and high-dose level were analysed in the first batch prepared in the study at t=0 and after storage in the refrigerator (2 – 10 °C) for twelve days.

Results:
- Homogeneity: the relative standard deviations between the mean content at three different locations was < 5% in the low, mid and high dose level. Therefore barium chloride dihydrate was considered to be homogeneously distributed in each test.
- Stability: upon storage at refrigerator temperature from 22 November 2013 till 4 December 2013, the relative difference in test substance concentration between t=0 and t=4 days was -3.6, +1.5 and +4.2% in the low, mid and high dose level, respectively. And all the dose levels met the criteria for stability (relative difference ≤10%). Therefore it was concluded that there was no loss of test substance from any tests items during storage for twelve days in the refrigerator.
- Content: the content of barium chloride dihydrate determined in the test items are compared with the intended content. The relative difference between the mean determined content and the intended content was between 1.5 and 2.5% at all nominal levels of 1, 3 and 10 mg/ml which was within the acceptance criteria (relative difference ≤10%). Therefore, the actual content was considered to meet the intended level in each test item.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 2 females : one male
- Length of cohabitation: until a sperm positive smear was detected
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy
Duration of treatment / exposure:
gestation day 0 up to and including gestation day 20
Frequency of treatment:
daily
Duration of test:
25 days
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
24 mated female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels have been selected in consultation with the study monitor on the basis of a dose range finding study with the test item in pregnant rats.
During the dose range finding study groups of 5 mated females, were administered different dose levels of the test substance by gavage from gestation day 0 up to gestation day 21. A dose volume of 10 mL/kg body weight was applied and demineralized water was used as vehicle and control item. Dose levels of 0, 50, 175 and 250 mg/kg were administered.
Based on the preterm death of 3/5 females in the high dose group and 2/5 females in the mid dose group after a single dose, dosing was discontinued in both groups.
All surviving animals in the mid and high dose group were re-allocated to a new mid dose group and received 100 mg/kg body weight barium chloride from gestation day 2 onwards.
On gestation day 21 the animals were sacrificed and caesarean section was performed.
In-life parameters included clinical signs, morbidity, mortality, body weight and food consumption. At sacrifice uterus weight, number of corpora lutea, number of implantation sites, early and late resorptions, number of live and dead foetuses and foetus weight were recorded. In addition, foetuses were examined for external abnormalities/malformations and dams were observed for gross anatomical changes.

Results:
Oral administration of 0, 50, 100, 175 and 250 mg/kg barium chloride to mated females resulted in:
- the preterm death of 3/5 animals in the 250 mg/kg group and 2/5 animals in the 175 mg/kg group after a single oral dose.
- the spontaneous death of one animal in the 100 mg/kg group on gestation day 21. This animal was found dead before cesarean section and had 11 dead foetuses. This animal had received one dose of 250 mg/kg on gestation day 0 and daily doses of 100 mg/kg from gestation day 2 to 21.
- limited clinical observations in the 250 and 175 mg/kg group, including hunched posture an piloerection.
- no effect on body weight or body weight gain, food consumption, mean number of corpora lutea, implantation sites, early and late resorptions and the mean number of live foetuses.
- although based on a limited number of litters (four in the 50 mg/kg group and three in the 100 mg/kg group) an effect on foetus weight could not be ruled out.
- no foetuses showing external malformations
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days (GD) 0, 3, 6, 10, 14, 17 and 21

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (gestation days 0-3, 3-6, 6-10, 10-14, 14-17 and 17-21)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
The females were killed by decapitation after CO2/O2 anaesthesia on gestation day 21 and examined for gross abnormalities.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Weight of empty uterus: Yes
- Weight of ovaries: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Gross evaluation of placentas: Yes
For seemingly non pregnant females (part of) the uterus was stained with Na2SO3 in order to visualize possible implantation sites (Salewski E, 1964). Upon staining non pregnancy was confirmed for these females.
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Further examinations:
- number of live and dead foetuses
- sex of the foetuses
- live foetuses (individually) and corresponding placentas
- foetal weight
Statistics:
Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 or p<0.01.
Continuous data were subjected to the ‘Decision tree for continuous data’ and dichotomous data to the ‘Decision tree for dichotomous data’.
Indices:
For each group the following indices were calculated:
- female fertility index = (no. of pregnant females/no. of inseminated females) x 100
- pre-implantation loss = [(no. of corpora lutea – no. of implantation sites) / no. of corpora lutea] x 100
- post-implantation loss = [(no. of implantation sites – no. of live foetuses) / No. of implantation sites] x 100
- gestation index = (no. of females with live foetuses/no. of females pregnant) x 100
- sex ratio = (no. of live male foetuses/no. of live foetuses) x 100
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: treatment-related death

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITIES
- two animals in the high dose group were found dead on day 21 of gestation. Both animals were pregnant and all foetuses were dead. Although death was not preceded by clinical signs, growth retardation or gross anatomical observations at necropsy that could clarify the death of these animals, the death of these rats is ascribed to treatment.
- one animal in the high dose group felt cold and was weakened and showed piloerection on gestation day 21. Upon necropsy this animal showed hydrothorax, haemorrhages in the liver and haemorrhagic discharge in the vagina. Also the death of this high-dose rat is ascribed to treatment.
- the spontaneous death of two rats, and the conditional decline of one rat on day 21 of gestation were considered to be treatment-related and to represent severe maternal toxicity in the high dose group.
- all foetuses were dead in the above three rats. The foetal deaths observed in these animals are considered to be related to the severe maternal toxicity in the high-dose group.

BODY WEIGHT AND BODY WEIGHT CHANGE
- no effects were observed on body weight. A slightly, but statistically significantly reduced body weight gain was observed in the high dose group as compared to the control group during the first three days of dosing. This was considered to be related to treatment and recovered thereafter.
- no effects on body weight or body weight gain were observed in the low dose group and the mid dose group as compared to the control group.

FOOD CONSUMPTION
- no effects were observed on food consumption.

REPRODUCTIVE PERFORMANCE
- 23, 22, 23 and 22 pregnant females in the control group, low dose, mid dose and high dose group, respectively.
- reproduction indices were comparable for the control, low dose, mid dose and high dose group
- no effects were noted in mean number of corpora lutea, mean number of implantation sites, preimplantation loss, mean number of early resorptions, late resorptions and mean number of live foetuses.

MACROSCOPY
- no treatment-related effects were observed

FEMALE REPRODUCTIVE ORGANS
- mean ovary weight, mean full and empty uterus weight were comparable in all groups
- mean carcass weight and net body weight change were comparable in all groups
- mean placenta weight was comparable in all groups
Dose descriptor:
NOAEL
Remarks:
(barium chloride dihydrate)
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Remarks:
(barium chloride)
Effect level:
25.6 mg/kg bw/day
Based on:
other: bariumdichloride
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FOETUS WEIGHT AND SEX
- mean foetus weight was comparable in all groups
- mean percentages male littermates was comparable in all groups

FOETAL EXAMINATION
- foetal external, visceral, and skeletal examinations did not reveal any treatment-related effects.
Dose descriptor:
NOAEL
Remarks:
(barium chloride dihydrate)
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects observed at the highest dose level
Dose descriptor:
NOAEL
Remarks:
(barium chloride)
Effect level:
>= 85.3 mg/kg bw/day
Based on:
other: barium chloride
Basis for effect level:
other: No effects observed at the highest dose level
Remarks on result:
other: NOAEL calculated for barium chloride from the barium chloride dihydrate data.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Daily administration of barium chloride dihydrate at dose levels of 0, 10, 30 or 100 mg/kg body weight to pregnant rats from gestation day 1 up to and including gestation day 20, resulted in maternal toxicity as evidenced by the spontaneous deaths of two animals on gestation day 21 and the conditional decline of another animal on gestation day 21 in the high dose group. No developmental toxicity was observed.
The NOAEL for maternal toxicity was therefore 30 mg/kg body weight (recalculated for barium chloride: 25.6 mg/kg bw/day). In absence of developmental effects the NOAEL for prenatal developmental toxicity in the rat was ≥ 100 mg/kg body weight (recalculated for barium chloride: ≥ 85.3 mg/kg bw/day).
Endpoint:
developmental toxicity
Data waiving:
other justification
Justification for data waiving:
other:
Species:
rabbit
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
85.3 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is fully acceptable
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity of barium chloride dihydrate was evaluated in a recent prenatal developmental toxicity study by daily administration of the test item at dose levels of 0, 10, 30 or 100 mg BaCl2 * 2 H2O/kg body weight to pregnant rats from gestation day 1 up to and including gestation day 20. No effects on body weights, food consumption and clinical signs were observed. Maternal toxicity was evidenced by the spontaneous deaths of two animals on gestation day 21 only and the conditional decline of another animal on gestation day 21 in the high dose group (100 mg BaCl2 * 2 H2O/kg bw).

No developmental toxicity or treatment-related observations, whatsoever in external, visceral and skeletal foetal examinations were observed in any dose level. The NOAEL for maternal toxicity was therefore 30 mg/kg body weight barium chloride dihydrate (25.6 mg/kg bw barium chloride). In absence of developmental effects, the NOAEL for prenatal developmental toxicity in the rat was ≥ 100 mg/kg body weight barium chloride dihydrate (≥85.3 mg/kg bw barium chloride).

Furthermore, tentative NOAEL values for developmental toxicity of 4,000 ppm and 2,000 ppm for rats and mice, respectively, are also reported in the study by Dietz et al. (1992). However, these NOAELs are of limited value to evaluate the potential for barium to induce developmental effects because the study design did not include prenatal exposure of the female animals to barium dichloride dihydrate. Therefore, this study has to be considered as inadequate for the assessment of the potential to induce developmental toxicity and cannot be used in a regulatory context.

Justification for classification or non-classification

In accordance to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for effects on fertility and development.

Additional information