Barium bis(dihydrogenorthophosphate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted Sept. 2009

Deviations:

yes

Remarks:

food consumption not evaluated, following organs were not examined: aorta, cervix, eye, harderian gland, lacrimal gland, peripheral nerve, skeletal muscle, vagina

GLP compliance:

not specified

Specific details on test material used for the study:

molecular weight: 244.28 g/mol

Species:

rat

Strain:

Fischer 344

Remarks:

F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Facility, Frederick, MD, USA
- Age at study initiation: 6 weeks
- Weight at study initiation: males/females: not specified
- Housing: 5 per cage (pylocarbonate cages)
- Diet: ad libitum (NIH-07 open stock mash diet; Zeigler Brothers, Inc., Gardners, PA, USA)
- Water: ad libitum
- Acclimation period: 12 - 14 days

DETAILS OF FOOD AND WATER QUALITY: diet contains less than 20 ppb barium

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23
- Humidity (%): 45 - 54
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared by mixing barium chloride dihydrate and water in a volumetric flask and stirring mechanically for 1 minute. Dose formulations were prepared weekly.
Stability studies of the 500 ppm dosed water solutions were performed using ultraviolet spectroscopy by the analytical chemistry laboratory. Stability of the dose formulations was confirmed for at least 3 weeks when stored in the dark at 25°C and for at least 3 days when stored exposed to air and light. No special handling was required during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dose formulation of barium chloride dihydrate were conducted at the study laboratory and the analytical chemistry laboratory using complexometric titration. During the 2-year studies, the dose formulations were analysed at least once every 8 weeks. The dose formulations were within 10% of the target concentrations. Results of analyses performed by the analytical chemistry laboratory were in good agreement with the results obtained by the study laboratory.

Duration of treatment / exposure:

104 weeks males
105 weeks females

Frequency of treatment:

daily

Post exposure period:

none

Dose / conc.:

500 ppm (nominal)

Remarks:

corresponding to 15 mg barium/kg bw/day for males and females

Dose / conc.:

1 250 ppm (nominal)

Remarks:

corresponding to 30 or 45 mg barium/kg bw/day for males or females, respectively

Dose / conc.:

2 500 ppm (nominal)

Remarks:

corresponding to 60 or 75 mg barium/kg bw/day for males or females, respectively

No. of animals per sex per dose:

60
10 animals per sex and dose were used for interim evaluation after 15 months of chemical administration

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Because of mortality, lower final mean body weights, decreased water consumption, and the presence of kidney lesions in male and female rats receiving 4000 ppm in a 13 week study (subchronic study), the high dose selected for the 2-year study was 2500 ppm. No further information on study design was stated.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily. The clinical observations were recorded initially, weekly for 13 weeks, then monthly and at interim evaluations. The interim evaluation was conducted with 10 males and 10 females per group after 15 months of chemical administration.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded initially, weekly for 13 weeks, then monthly and at interim evaluations. The interim evaluation was conducted with 10 males and 10 females per group after 15 months of chemical administration.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: once weekly by cage

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the jugular vein of all rats at the 15 months interim evaluations.
- Anaesthetic used for blood collection: not specified
- Animals fasted: not specified
- How many animals: all rats
- Parameters examined: hematocrit, hemoglobin, erythrocytes, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, platelets, reticulocytes, nucleated erythrocytes, and leukocyte count and differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 15 months
- Animals fasted: not specified
- How many animals: 10 male and 10 female rats per group were randomly selected for interim evaluations after 15 months of chemical administration.
- Parameters examined: urea nitrogen, creatinine, calcium, phosphorus, alanine aminotransferase, creatine kinase, lactate dehydrogenase, sorbitol dehydrogenase, and γ-glutamyltransferase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Plasma and bone analyses at the 15 months interim evaluations: plasma from blood collected for clinical pathology was analyzed to determine plasma barium concentrations in rats. In addition, the left femur from eight male and eight female rats in the control and 2500 ppm groups were analyzed for barium, calcium, and phosphorus concentrations, and bone density.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adrenal gland, brain, heart, right kidney, liver, lung, ovary, spleen, right testis, uterus and thymus of rats were weighed at the 15 months interim evaluations.
HISTOPATHOLOGY: Yes
A complete histopathologic examination was performed on all animals. In addition to gross lesions, tissue masses, and associated lymph nodes, the tissues examined included: adrenal gland, brain, bone and marrow, clitoral gland, large intestine (cecum, colon, rectum), epididymis, esophagus, heart, kidney, liver, lung, mammary gland, mandibular lymph node, mesenteric lymph node, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach (forestomach and glandular), testis, thyroid gland, trachea, thymus, urinary bladder, and uterus.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier and is presented in the form of graphs. Statistical analyses for possible dose-related effects on survival used Cox’s method for testing groups for equality and Tarone life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. Analysis of continuous variables: Two approaches were employed to assess the significance of pair wise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which have approximately normal distributions, were analyzed using the parametric multiple comparison procedures of Dunnett and Williams. Clinical chemistry, hematology, neurobehavioral, and cardiovascular data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Dunn and Shirley. Jonckheere’s test was used to assess the significance of the dose response trends and to determine whether a trend sensitive test (Williams’ or Shirley’s test) was more appropriate for pair wise comparisons than a test that does not assume a monotonic dose-response trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance using the Mann-Whitney U test.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no chemical-related clinical findings noted in male or female rats.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Estimates of survival probabilities for male and female rats are shown in Table 5 in the attachment and in the Kaplan-Meier curves in Figure 1 in the attachment. Survival of exposed female groups was similar to that of the controls. The marginally increased survival of exposed male groups was probably due to a decreased incidence of leukemia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of male rats receiving 2500 ppm were slightly lower than controls from week 18 to the end of the study (Figure 2 and Table 6 in the attachment). Female rats that received 2500 ppm had mean body weights 5% to 11% lower than the controls beginning at week 49 (Figure 2 and Table 7 in the attachment). The final mean body weights of males receiving 500 and 1250 ppm and females receiving 500 ppm were similar to those of the controls. The final mean body weights of males that received 2500 ppm was 5% lower than that of the controls. The final mean body weights of females receiving 1250 and 2500 ppm were 6% and 11% lower than the controls, respectively.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Barium chloride dihydrate in drinking water caused a dose-related decrease in water consumption. Average water consumption (g/day) over the 2-year period for males was: 0 ppm, 21.2; 500 ppm, 20.2; 1250 ppm, 18.7; 2500 ppm,16.5; and over the 2-year period for females was: 16.2, 15.6, 14.9, and 12.1. The greatest effect on water consumption was observed in rats receiving 2500 ppm. Beginning as early as week 5, water consumption by these groups was substantially depressed (males: 11% to 30%; females: 19% to 33%; Tables K1 and K2 in the attachment).

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematologic parameters measured at the 15 months interim evaluation were considered to be within the range of normal values (Table H3).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry parameters measured at the 15 months interim evaluation were considered to be within the range of normal values (Table H3).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

At the end, there was no increased incidences of non-neoplastic lesions that could be related to test substance.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no increased incidences of neoplasms in rats receiving barium chloride dihydrate.
Multiple organs: Mononuclear cell leukemia was not observed in males at 15 months (table A1 in the attachment). However, at 2 years, there was a significant negative trend in the incidence of mononuclear cell leukemia in males and the incidences in exposed male groups were significantly decreased (Table 9 and A3 in the attachment). The decreased incidence of this lethal neoplasm may account for the marginal increase in survival of exposed males. The incidences of mononuclear cell leukemia in exposed females were similar to that in the controls (0 ppm, 15/50; 500 ppm, 13/50; 1250 ppm, 9/50; 2500 ppm, 9/50; table B3 of the attachment).
Adrenal gland: A significant negative trend in the incidence of adrenal medulla pheochromocytoma (benign or malignant) was observed in male rats (13/49, 11/50, 12/49, 6/50; table A3 in the attachment). The incidence of this neoplasm in the 2500 ppm males was significantly decreased. The incidences of adrenal medulla hyperplasia in exposed male rats were similar to that in the controls (7/49, 11/50, 5/49, 9/50; Table A5 in the attachment). Incidences of adrenal medulla pheochromocytoma and hyperplasia in exposed females were similar to those in controls (Tables B3 and B4).
Mammary gland: A significant negative trend in the incidence of mammary gland neoplasms (fibro-adenoma, adenoma, or carcinoma) was observed in female rats (17/50, 21/50, 13/50, 11/50; table B3 in the attachment).

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Plasma barium and bone analysis: Plasma barium levels were significantly increased in males receiving 2500 ppm and all exposed groups of females (Table 8 in the attachment).
The density of femoral bone in rats that received 2500 ppm was similar to that of the controls (Table 8 in the attachment). Barium levels in all portions of femoral bone were approximately 400 times greater in males and females receiving 2500 ppm than in controls (Table 8).
Calcium levels in the upper portion of the femoral bone of male and female rats receiving 2500 ppm were slightly but significantly lower than those of the controls. Phosphorus levels in the femoral bone in exposed males and females were similar to those in the controls.

Key result

Dose descriptor:

NOAEL

Effect level:

2 500 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 60 and 75 mg barium/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at the highest dose level

Key result

Critical effects observed:

no

Based on the water consumption data the average daily dose of barium chloride dihydrate received by rats was 15, 30, or 60 mg barium/kg bw for males, and 15, 45, or 75 mg barium/kg bw for females.

Conclusions:

There was no evidence of increased neoplasm incidences that could be attributed to barium chloride dihydrate administration in both sexes of rats. Thus, the highest applied concentration of 2500 ppm represents a NOAEL (corresponding to barium doses of 60 and 75 mg/kg bw/d to male and female rats, respectively).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the analogue justification attached to chapter 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

rat

Effect level:

2 000 ppm

Based on:

test mat.

Remarks:

equivalent to 110 and 115 mg barium/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

mortality

Dose descriptor:

NOAEL

Remarks:

mouse

Effect level:

2 000 ppm

Based on:

test mat.

Remarks:

equivalent to 205 and 200 mg barium/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

mortality

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Drinking water containing 125, 500, 1000, 2000, or 4000 ppm barium chloride dihydrate was estimated to deliver daily doses of 10, 30, 65, 110, or 200 mg barium/kg bw to male rats and 10, 35, 65, 115, or 180 mg barium/kg bw to female rats.

Drinking water containing 125, 500, 1000, 2000, or 4000 ppm barium chloride dihydrate was estimated to deliver daily doses of 15, 55, 100, 205, or 450 mg barium/kg bw to male mice and 15, 60, 110, 200, or 495 mg barium/kg bw to female mice.

Conclusions:

The no observed adverse effect level (NOAEL) for source substance barium chloride dihydrate in drinking water for rats and mice was estimated to be approximately 2000 ppm based on increased mortality and renal toxicity. The dose of 2000 ppm corresponds to NOAEL values of 110 and 115 mg barium/kg bw/day to male and female rats, respectively and of 200 and 205 mg barium/kg bw/day for male and female mice, respectively. As explained in the analogue justification the target and the source substances are considered to be similar in their toxicological behvior. Therefore, it is considered that the target and the source substances are unlikely to lead to differences in repeated toxicity.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted Sept. 2009

Deviations:

yes

Remarks:

food consumption not evaluated, following organs were not examined: aorta, cervix, eye, harderian gland, lacrimal gland, peripheral nerve, skeletal muscle, vagina

GLP compliance:

not specified

Specific details on test material used for the study:

molecular weight: 244.28 g/mol

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Facility, Frederick, MD, USA
- Age at study initiation: 7 weeks
- Weight at study initiation: not specified
- Housing: 1 per cage (pylocarbonate cages)
- Diet: ad libitum (NIH-07 open stock mash diet; Zeigler Brothers, Inc., Gardners, PA, USA)
- Water: ad libitum
- Acclimation period: 15 - 16 days

DETAILS OF FOOD AND WATER QUALITY: diet contains less than 20 ppb barium

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23
- Humidity (%): 46 - 59
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared by mixing barium chloride dihydrate and water in a volumetric flask and stirring mechanically for 1 minute. Dose formulations were prepared weekly.
Stability studies of the 500 ppm dosed water solutions were performed using ultraviolet spectroscopy by the analytical chemistry laboratory. Stability of the dose formulations was confirmed for at least 3 weeks when stored in the dark at 25°C and for at least 3 days when stored exposed to air and light. No special handling was required during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dose formulation of barium chloride dihydrate were conducted at the study laboratory and the analytical chemistry laboratory using complexometric titration. During the 2-year studies, the dose formulations were analysed at least once every 8 weeks. The dose formulations were within 10% of the target concentrations. Results of analyses performed by the analytical chemistry laboratory were in good agreement with the results obtained by the study laboratory.

Duration of treatment / exposure:

103 weeks males
104 weeks females

Frequency of treatment:

daily

Post exposure period:

none

Dose / conc.:

500 ppm (nominal)

Remarks:

corresponding to 30 or 40 mg barium/kg bw/day for males and females, respectively

Dose / conc.:

1 250 ppm (nominal)

Remarks:

corresponding to 75 or 90 mg barium/kg bw/day for males and females, respectively

Dose / conc.:

2 500 ppm (nominal)

Remarks:

corresponding to 160 or 200 mg barium/kg bw/day for males and females, respectively

No. of animals per sex per dose:

60
10 animals per sex and dose were used for interim evaluation after 15 months of chemical administration

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Because of mortality, lower final mean body weights, decreased water consumption, and the presence of kidney lesions in male and female rats receiving 4000 ppm in a 13 week study (subchronic study), the high dose selected for the 2-year study was 2500 ppm. No further information on study design was stated.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily. The clinical observations were recorded initially, weekly for 13 weeks, then monthly and at interim evaluations. The interim evaluation was conducted with 10 males and 10 females per group after 15 months of chemical administration.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded initially, weekly for 13 weeks, then monthly and at interim evaluations. The interim evaluation was conducted with 10 males and 10 females per group after 15 months of chemical administration.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: once weekly by cage

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the jugular vein of all mice at the 15 months interim evaluations.
- Anaesthetic used for blood collection: not specified
- Animals fasted: not specified
- How many animals: all mice
- Parameters examined: hematocrit, hemoglobin, erythrocytes, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, platelets, reticulocytes, nucleated erythrocytes, and leukocyte count and differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 15 months
- Animals fasted: not specified
- How many animals: 10 male and 10 female mice per group were randomly selected for interim evaluations after 15 months of chemical administration.
- Parameters examined: urea nitrogen, creatinine, calcium, phosphorus, alanine aminotransferase, creatine kinase, lactate dehydrogenase, sorbitol dehydrogenase, and γ-glutamyltransferase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Plasma and bone analyses at the 15 months interim evaluations: plasma from blood collected for clinical pathology was analyzed to determine plasma barium concentrations in mice.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adrenal gland, brain, heart, right kidney, liver, lung, ovary, spleen, right testis, uterus and thymus of mice were weighed at the 15 months interim evaluations.
HISTOPATHOLOGY: Yes
A complete histopathologic examination was performed on all animals. In addition to gross lesions, tissue masses, and associated lymph nodes, the tissues examined included: adrenal gland, brain, bone and marrow, clitoral gland, large intestine (cecum, colon, rectum), epididymis, esophagus, heart, kidney, liver, lung, mammary gland, mandibular lymph node, mesenteric lymph node, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach (forestomach and glandular), testis, thyroid gland, trachea, thymus, urinary bladder, and uterus.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier and is presented in the form of graphs. Statistical analyses for possible dose-related effects on survival used Cox’s method for testing groups for equality and Tarone life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. Analysis of continuous variables: Two approaches were employed to assess the significance of pair wise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which have approximately normal distributions, were analyzed using the parametric multiple comparison procedures of Dunnett and Williams. Clinical chemistry, hematology, neurobehavioral, and cardiovascular data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Dunn and Shirley. Jonckheere’s test was used to assess the significance of the dose response trends and to determine whether a trend sensitive test (Williams’ or Shirley’s test) was more appropriate for pair wise comparisons than a test that does not assume a monotonic dose-response trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance using the Mann-Whitney U test.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no chemical-related clinical findings noted in male or female mice.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival of male and female mice receiving 2500 ppm was significantly lower than that of the controls. The reduction in survival of female mice that received 2500 ppm was first observed at week 15 of the study and by the end of the 2 years only 26% of the animals were alive. Survival in male mice receiving 2500 ppm was noticeably decreased by week 65. The reduced survival of exposed mice was attributed to chemical-related renal lesions. Estimates of survival probabilites for male and female mice are shown in Table 12 and in the Kaplan-Meier curves in Figure 3 in the attachment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final mean body weights of males and females exposed to 2500 ppm were 9% and 12% lower than those of controls (Figure 4 and Tables 13 and 14 in the attachment). Animals killed moribund or dying before the end of the study had moderate to marked weight loss.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Water consumption by exposed mice was similar to that by the controls (Table K3 and K4 in the attachment).

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no significant differences in hematology parameters between control and exposed mice (Table H4).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no significant differences in clinical chemistry between control and exposed mice.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

At the 15 months interim evaluation the absolute and relative spleen weights of female mice exposed to 2500 ppm were significantly lower than those of controls (Table F6).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidney: The incidence of nephropathy was significantly increased in male and female mice receiving 2500 ppm (Table 16, C4 and D4 in the attachment). The nephropathy was morphologically distinct from the spontaneous degenerative lesions that are commonly observed in aging B6C3F mice. The nephropathy was characterized by extensive regeneration of conical and medullary renal tubule epithelium, tubule dilatation, hyaline cast formation, multifocal interstitial fibrosis, and, in some kidneys, glomerulosclerosis. These lesions were accompanied by the presence of irregularly shaped aggregates of brown crystals located both within renal tubule lumens and in the interstitium. Some of the crystals appeared granular, while others had both straight and rounded edges and ranged in size from 8 to 50 pm. They were weakly anisotropic. Their location was difficult to determine because of their size and the possibility that dislocation had occurred with sectioning. However, most appeared to be located in tubule lumens of both the cortex and medulla, and in the lumen of the renal pelvis. The quantity of the crystals ranged from few to numerous in a particular kidney. While the chemical composition of the crystals is unknown, they may consist of precipitated barium or barium salts. The kidney lesions were considered to be the cause of the morbidity or death for most of the animals which did not survive to the end of the study.
Hematopoietic system: In 2500 ppm male and/or female mice there were increased incidences of lymphoid depletion of the spleen (males: 0 ppm, 0/50; 500 ppm, 8/50; 1250 ppm, 4/48, 2500 ppm, 9/50; females: 2/50, 2/53, 0/50, 11/52; Tables C4 and D4), thymus (males: 0/39, 0/42, 2/44, 5/35; females: 1/43, 1/46,0/47, 12/38), and mesenteric lymph node (males: 0/49, 1/47, 0/46, 4 /39; female: 0/49, 0/49, 0/49, 10/39). Because the majority of the thymic and splenic lymphoid lesions occured in the 2500 ppm animals that were found dead or killed moribund relatively early in the study, it is likely that these lesions are the result of debilitation associated with nephropathy.
Liver: There was a significant negative trend in the incidence of hepatocellular adenoma in male mice and the incidence in the 2500 ppm group was significantly lower than in the controls (24/51, 20/50, 15/48, 8/50; Table C3 in the attachment). The incidence of hepatocellular carcinoma in exposed males was similar to that in the controls.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No increased incidences of neoplasms were observed in exposed mice. The incidences of many neoplasms were lower in female mice exposed to 2500 ppm than in the controls because of the marked reduction in survival of this group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Plasma analyses: There were dose-related significant increases in plasma barium levels in exposed groups of male and female mice (table 15).

Dose descriptor:

NOAEL

Effect level:

2 500 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 160 and 200 mg barium/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at highest dose

Critical effects observed:

no

Concentrations of 500, 1250, and 2500 ppm barium chloride dihydrate delivered estimated daily doses of 30, 75, or 160 mg barium/kg bw to males and 40, 90, or 200 mg barium/ kg bw to females.

Conclusions:

There was no evidence of repeated dose toxicity or increased neoplasm incidences that could be attributed to barium chloride dihydrate adminstration in both sexes of mice. Thus, the concentration of 2500 ppm represents a NOAEL (corresponding to barium doses of 160 and 200 mg/kg bw/day to male and female mice, respectively).

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1994

Reference 2

Reference Type:

publication

Title:

Subchronic Toxicity of Barium Chloride Dihydrate Administered to Rats and Mice in the Drinking Water

Author:

D.D. Dietz, M.R, Elwell, W.E. Davis, Jr., E.F. Meirhenry

Year:

1992

Bibliographic source:

Fundamental and Applied Toxicology 19, 527-537

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted Sept. 1998

Deviations:

yes

Remarks:

parameters of clinical biochemistry were limited, no ophthalmologic examination, spleen, ovaries and uterus not weighed

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Barium chloride dihydrate

IUPAC Name:

Barium chloride dihydrate

Test material form:

solid

Details on test material:

- State of aggregation: solid, white crystalline
- purity: 99 - 100%
- Lot/batch No.: 123120 and 423103 (obtained from J.T. Baker Chemical Company, Phillipsburg, NJ, USA)

Specific details on test material used for the study:

molecular weight: 244.28 g/mol

Test animals

Species:

rat

Strain:

Fischer 344

Remarks:

F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Inc., Gilroy, CA, USA
- Age at study initiation: 32 days
- Weight at study initiation: males: 116 - 143 g, females: 101 - 113 g
- Housing: 5 per cage (polycarbonate cages)
- Diet: ad libitum (NIH-07 open-formula pellets diet; Zeigler Brothers, Inc., Gardners, PA, USA)
- Water: ad libitum
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: diet contains less than 20 ppb barium

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 40 - 62
- Air changes (per hr): 13.5
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared by mixing barium chloride dihydrate and water in a volumetric flask and stirring mechanically for 1 minute. Dose formulations were prepared weekly.
Stability studies of the 500 ppm dosed water solutions were performed using ultraviolet spectroscopy by the analytical chemistry laboratory. Stability of the dose formulations was confirmed for at least 3 weeks when stored in the dark at 25°C and for at least 3 days when stored exposed to air and light. No special handling was required during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dose formulation of barium chloride dihydrate were conducted at the study laboratory and the analytical chemistry laboratory using complexometric titration. The dose formulations were analysed at the initiation and midpoint of the 13 week-studies. The dose formulations were within 10% of the target concentrations. Results of analyses performed by the analytical chemistry laboratory were in good agreement with the results obtained by the study laboratory. Drinking water levels of 125, 500, 1000, 2000, or 4000 ppm barium chloride dihydrate were estimated to deliver daily doses of 10, 30, 65, 110, or 200 mg barium/kg body weight to males and 10, 35, 65, 115, or 180 mg barium/kg body weight to females.

Duration of treatment / exposure:

95 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 ppm

Remarks:

corresponding to 10 mg barium/kg bw/day for males and females

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

corresponding to 30 and 35 mg barium/kg bw/day for males and females, respectively

Doses / concentrations 3

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 65 mg barium/kg bw/day for males and females

Doses / concentrations 4

Dose / conc.:

2 000 ppm

Remarks:

corresponding to 110 and 115 mg barium/kg bw/day for males and females, respectively

Doses / concentrations 5

Dose / conc.:

4 000 ppm

Remarks:

corresponding to 200 and 180 barium mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: A dose range finding study was performed with male and female F433/N rats. Groups of five male and five female rats received 0, 125, 250, 500, 1000, or 2000 ppm of barium chloride dihydrate in distilled drinking water for 15 days. No chemical-related deaths occurred among male or female rats. One male rat that received 2000 ppm was accidentally killed on day 14. While the final mean body weights of male and female rats receiving barium chloride dihydrate were within 5% of the controls, the mean body weight gain of male rats receiving 2000 ppm was 18% lower than that of controls. Water consumption by male and female rats that received 2000 ppm was slightly lower than that by the controls (<= 16%) during week 2. Drinking water levels of 125, 250, 500, 1000, or 2000 ppm barium chloride dihydrate were estimated to deliver daily doses of 10, 15, 35, 60, or 110 mg barium/kg bw. There were no chemical-related clinical findings of toxicity or lesions noted at necropsy. Motor activity, grip strength, and thermal sensitivity were not affected in exposed rats. No significant differences in absolute or relative organ weights were observed in rats receiving barium chloride dihydrate. No biologically significant differences in the serum levels of potassium, phosphorus, and calcium or hematology parameters were observed in exposed rats.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once weekly
- Cage side observations were not further described.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: at study initiation, once weekly, and at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: once weekly by cage

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 13 weeks
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: not specified
- How many animals: all rats
- Parameters examined: hematocrit, hemoglobin, erythrocytes, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, platelets, nucleated erythrocytes, and leukocyte count and differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the 13-week
- Animals fasted: not specified
- How many animals: all rats
- Parameters examined: barium, sodium, potassium, calcium, and phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before exposure and after 45 and 90 days of exposure to dosed drinking water
- Dose groups that were examined: all dose groups
- Battery of functions tested: spontaneous motor activity, forelimb and hindlimb grip strenght, thermal sensitivity, startle response to acoustic and air-puff stimuli, and hindlimb foot splay

IMMUNOLOGY: No

OTHER: Cardiovascular studies were performed on each rat before exposure and after 45 and 91 days of exposure. The studies included electrocardiogram recordings and analysis and blood pressure measurements.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adrenal gland, brain, heart, right kidney, liver, lung, right testis, and thymus were weighed.
HISTOPATHOLOGY: Yes
A complete histopathologic examination was performed on all control animals and all rats receiving 4000 ppm. In addition to gross lesions, the tissues examined included: adrenal gland, brain, epididymis, esophagus, heart, kidney, large intestine (cecum, colon, rectum), liver, lung, mammary gland, mandibular lymph node, mesenteric lymph node, nose, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle, skin, small intestine, spleen, sternebrae (including marrow), stomach, testis, thyroid gland, trachea, thymus, urinary bladder, and uterus. In addition, the kidney, liver, spleen, and thymus of rats receiving 2000 ppm; and the adrenal gland, heart, and salivary gland of female rats receiving 2000 ppm were examined microscopically.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier and is presented in the form of graphs. Statistical analyses for possible dose-related effects on survival used Cox’s method for testing groups for equality and Tarone life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. Analysis of continuous variables: Two approaches were employed to assess the significance of pair wise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which have approximately normal distributions, were analyzed using the parametric multiple comparison procedures of Dunnett and Williams. Clinical chemistry, hematology, neurobehavioral, and cardiovascular data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Dunn and Shirley. Jonckheere’s test was used to assess the significance of the dose response trends and to determine whether a trend sensitive test (Williams’ or Shirley’s test) was more appropriate for pair wise comparisons than a test that does not assume a monotonic dose-response trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance using the Mann-Whitney U test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

Three males and one female that received 4000 ppm died during the last week of the study. The cause of these deaths was not apparent histologically, but the deaths were considered to be chemical related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The final mean body weights and mean body weight gains of male and female rats receiving 4000 ppm were significantly lower than those of the controls (final mean body weights: 13% and 8% lower; mean body weight gains: 18% and 24% lower) (see table 4 in the attachment).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water consumption by male and female rats that received 4000 ppm was lower than that by controls; these groups consumed approximately 70% of that consumed by the controls.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum phosphorus levels in males receiving 2000 and 4000 ppm and in females receiving 500, 1000, 2000, and 4000 ppm were significantly higher than those in controls (table H2 in the attachment). Elevations in serum phosphorus levels may have been caused by renal tubule damage. However, due to the minimal to mild severity of this lesion, it is more likely that the elevated values were due to an artifact from hemolysis of collected blood samples.
Significantly decreased sodium levels in 4000 ppm male rats and calcium levels in 1000 ppm males did not occur in a dose-related manner and thus were not considered to be clearly related to barium chloride dihydrate exposure.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute and relative kidney weights of females that received 2000 and 4000 ppm and the relative kidney weight of males that received 4000 ppm were significantly greater than those of the controls. The findings in the 4000 ppm group were associated with chemical-induced renal lesions (table F2 in the attachment). The differences in the absolute and/or relative weights in other organs could be attributed to the decrease in mean body weights observed in 4000 ppm male and female rats.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Chemical-related kidney lesions occurred in three male and three female rats receiving 4000 ppm. None were observed in the controls or in any of the remaining exposure groups. Grossly, the kidneys were pale and had roughened surfaces.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

A slight but significant decrease in undifferentiated motor activity in rats that received 4000 ppm was observed at day 90 of the study (table G2 in the attachment). A marginal decrease in this parameter was observed at day 90 of the study in all other exposed groups of rats except in 1000 ppm females.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, the kidney lesions appeared as a minimal to mild, focal to multifocal dilatation of the proximal convoluted tubules in the outer medulla and the renal cortex. The tubule epithelial cells were usually low cuboidal cells with a decreased cytoplasmic volume, yet they contained a nucleus of typical size. Tubule dilatation observed in this study was different from the common spontaneous lesions observed in the kidney of rats. In this study, early lesions of nephropathy were observed in virtually all males and in small numbers of females in all treatment groups as well as the controls.
Additionally, minimal to mild atrophy of the spleen and/or thymus was observed in small numbers of male and female rats that received 4000 ppm.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Cardiovascular examination: No significant alterations in blood pressure or electrocardiogramm recordings were found up to and at the highest dose of 4000 ppm (table G2 in the attachment).

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 ppm

Based on:

test mat.

Remarks:

equivalent to 110 and 115 mg barium/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

mortality

2

Dose descriptor:

LOAEL

Effect level:

4 000 ppm

Based on:

test mat.

Remarks:

equivalent to 200 and 180 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

mortality

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

180 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) for barium chloride dihydrate in drinking water for rats was estimated to be approximately 2000 ppm based on the final mean body weights, mean body weight gains, decreased water consumption, mortality, and renal toxicity. The dose of 2000 ppm corresponds to NOAEL values of 110 and 115 mg barium/kg bw/day to male and female rats, respectively. Therefore, a LOAEL of 4000 ppm is derived which corresponds to values of 200 and 180 mg/kg bw/day for males and females, respectively.