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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-11-24 to 2013-12-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 2001-01-22
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
, 2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2013-07-09
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Barium chloride dihydrate
- Molecular formula: BaCl2 2H2O
- Physical state: white crystalline powder
- Storage condition of test material: ambient temperature (15-25°C)

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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

Administration / exposure

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
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

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
- 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 weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 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 and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
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
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
- 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.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
- 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.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

open allclose all
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 dihydrate)
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Remarks:
(barium chloride)
Effect level:
25.6 mg/kg bw/day
Based on:
other: barium dichloride
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Remarks:
(barium chloride)
Effect level:
>= 85.3 mg/kg bw/day
Based on:
other: barium dichloride
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: mortality

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
FOETUS WEIGHT AND SEX
- mean foetus weight was comparable in all groups

Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
- mean percentages male littermates was comparable in all groups
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Description (incidence and severity):
- foetal external, visceral, and skeletal examinations did not reveal any treatment-related effects.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

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).