Barium 3,5,5-trimethylhexanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985-10-09 to 1987-10-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1994

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The toxicology study was conducted by administering barium chloride dihydrate (99% pure) in drinking water to B6C3F1 mice for 2 years. Groups of 60 males and 60 females received barium chloride dihydrate in the drinking water at concentrations of 0, 500, 1250, or 2500 ppm for 103 (males) or 104 weeks (females), corresponding to average daily doses of 30, 75, or 160 mg barium/kg body weight for males and 40, 90, or 200 mg barium/kg body weight for females.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Barium chloride dihydrate
- EC number: 233-788-1
- Physical state: white, crystalline solid
- Analytical purity: > 99 %
- Lot No.: 423103
- Stability: The chemical should be stable over a wide range of temperatures.
- Purity and water content: The purity and water content of the bulk chemical were reanalyzed every 4 months during the 2-year studies at the study laboratory by complexometric titration and weight loss on drying. The results indicated that the purity and moisture content of the barium chloride dihydrate did not change during the 2-year studies.
No further information on test material was stated.

Test animals

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)
- Age at study initiation: 7 weeks
- Housing: one per cage; Cages: Polycarbonate (Lab Products, Inc., Rochelle Park, NJ), changed twice weekly; Bedding: BetaChips, hardwood laboratory bedding (Northeastern Products, Corp., Warrensburg, NY), changed twice weekly; Cage filters: Nonwoven polyester (Snow Filtration, Cincinnati, OH), changed once every 2 weeks; Racks: Stainless steel (Lab Products, Inc., Rochelle Park, NJ), changed once every 2 weeks; Cages were rotated every 2 weeks.
- Diet (ad libitum): NIH-07 open stock mash diet (Zeigler Brothers, Inc., Gardners, PA) The diet used contained less than 20 ppm barium.
- Water (ad libitum)
- Acclimation period: Rats were quarantined for 15 (males) or 16 (females) days before the beginning of the studies. Five mice of each sex were randomly selected and evaluated for evidence of disease. Serology samples were collected for viral screening. The health of the animals was monitored during the studies according to the NTP Sentinel Animal Program.

ENVIRONMENTAL CONDITIONS
- Temperature: 21°C - 23°C
- Relative humidity: 46%- 59%
- Air changes: 10/hour
- Photoperiod (hrs dark / hrs light): Fluorescent light: 12 hours/day
No further information on test animals was stated.

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 during the 2-year study
Stability studies of the 500 ppm dosed water solutions were performed using ultraviolet spectroscopy. 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.
No further information on oral exposure was stated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dose formulations of barium chloride dihydrate were conducted at the study laboratory and the analytical chemistry laboratory (Midwest Research Institute (Kansas City, MO) 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:

The distilled water containing the barium chloride dihydrate was available ad libitum (7 days per week).

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 1250, or 2500 ppm (m/f)
Basis:
nominal in water

No. of animals per sex per dose:

60 males / 60 females

Control animals:

yes, concurrent vehicle

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 4,000 ppm in a 13 week study(subchronic study), the high dose selected for the 2-year study was 2,500 ppm.
- exact concentrations calculated for the cation: 30, 75, and 160 mg Ba/kg bw to males and 40, 90, and 200 mg Ba/kg bw to females
No further information on study design was stated.

Positive control:

No data

Examinations

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 inerim evaluations. The interim evaluation was conducted with 6 to 10 males and females per group after 15 months of chemical administration.

DETAILED CLINICAL OBSERVATIONS: No data

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 6 to 10 males and 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 data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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 data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the jugular vein of all mice at the 15 month interim evaluations.
- How many animals: Six to ten male and female mice per group were randomly selected for interim evaluations after 15 months of chemical administration.
- Parameters examined: hemoglobin, hematocrit, erythrocytes, mean erythrocyte volume, mean erythrocyte hemoglobin, mean erythrocyte hemoglobin concentration, platelets, reticulocytes, nucleated erythrocytes, and leukocyte count and differential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the jugular vein of all mice at the 15 month interim evaluations.
- How many animals: Six to ten male and 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, and γ -glutamyltransferase

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: Plasma and Bone Analyses
At the 15-month interim evaluations, plasma from blood collected for clinical pathology was analyzed to determine plasma barium concentrations in mice.
No further information on observations and examinations performed and frequency were stated.

Sacrifice and pathology:

Complete necropsy performed on all animals. The animals were anestetised by carbon dioxide asphyxiation. The adrenal gland, brain, heart, right kidney, liver, lung, ovary, spleen, right testis, uterus and thymus of mice were weighed at the 15-month interim evaluations. At necropsy, all organs and tissues were examined for gross lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin for microscopic examination. A complete histopathologic examination was performed on all mice. In addition to gross lesions, tissue masses, and associated lymph nodes, the tissues examined included: adrenal gland, brain, bone and marrow, large, intestine (cecum, colon, rectum), epididymis, esophagus, gallbladder,heart, kidney, 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 (duodenum, jejunum, ileum), spleen, stomach (forestomach and glandular), testis, thyroid gland, trachea, thymus, urinary bladder, and uterus.
no further information on sacrifice and pathology were stated.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing groups for equality and Tarone (1975) 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 (1955) and Williams (1971, 1972). Clinical chemistry, hematology, neurobehavioral, and cardiovascular data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Dunn (1964) and Shirley (1977). Jonckheere’s test (Jonckheere, 1954) 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 (Hollander and Wolfe, 1973).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Survival of male and female mice receiving 2,500 ppm was significantly lower than that of the controls. The reduction in survival of female mice that received 2,500 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 2,500 ppm was noticeably decreased by week 65. The reduced survival of exposed mice was attributed to chemical-related renal lesions.
There were no significant clinical findings of organ-specific toxicity.

BODY WEIGHT AND WEIGHT GAIN
Final mean body weights of males and females exposed to 2,500 ppm were 9 % and 12 % lower than those of controls. Animals killed moribund or dying before the end of the study had- moderate to marked weight loss.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Water consumption by exposed mice was similar to that by the controls. Concentrations of 500, 1,250, and 2,500 ppm barium chloride dihydrate delivered estimated daily doses of 30,75, or 160 mg bariumkg body weight to males and 40, 90, or 200 mg barium/ body weight to females.

HAEMATOLOGY
There were no significant differences in hematology parameters between control and exposed mice.

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

ORGAN WEIGHTS
At the 15-month interim evaluation the absolute and relative spleen weights of female mice exposed to 2,500 ppm were significantly lower than those of controls.

GROSS PATHOLOGY/HISTOPATHOLOGY: NEOPLASTIC/ HISTOPATHOLOGY: NON-NEOPLASTIC
No increased incidences of neoplasms were observed in exposed mice. The incidences of many neoplasms were lower in female mice exposed to 2,500 ppm than in the controls because of the marked reduction in survival of this group.
Kidney: The incidence of nephropathy was significantly increased in male and female mice receiving 2,500 ppm. 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, glomerulcsclerosis. 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 2,500 ppm male and/or female mice there were increased incidences of lymphoid depletion of the spleen (male: 0 ppm, 0/50; 500 ppm, 8/50; 1,250 ppm, 4/48,2,500p pm, 9/50; female: 2/50,2/53,0/50,11/52; thymus (male: 0/39, 0/42, 2/44, 5/35; female: 1/43, 1/46,0/47, 12/38), and mesenteric lymph node (male: 0/49, 1/407/, 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 2,500 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 2,500 ppm group was significatnly lower than in the controls (24/51, 20/50, 15/48, 8/50). The incidence of hepatocellular carcinoma in exposed males was similar to that in the controls.

OTHER FINDINGS Plasma and Bone Analyses
There were dose-related significant increases in plasma barium levels in exposed groups of male and female mice.

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

LOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

other: barium

Sex:

male

Basis for effect level:

other: Calculated as Ba2+

3

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

other: barium

Sex:

female

Basis for effect level:

other: Calculated as Ba2+

4

Dose descriptor:

NOAEL

Effect level:

1 250 ppm

Based on:

test mat.

Sex:

male/female

5

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

other: barium

Sex:

male

Basis for effect level:

other: Calculated as Ba2+

6

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day (nominal)

Based on:

other: barium

Sex:

female

Basis for effect level:

other: Calculated as Ba2+

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The exposure concentration of 2500 ppm barium chloride to rats represents the NOAEL value for nonneoplastic effects (corresponding to Ba doses of 60 and 75 mg/kg bw/d to male and female rats, respectively). However, male and female mice exposed to the concentration of 2500 ppm showed an increased incidence of nephropathy. Therefore, the corresponding doses of 160 and 200 mg/kg bw/d to male and female mice, respectively, are LOAEL values. Thus, in the 2-year study in mice the NOAEL values for nonneoplastic effects is 1250 ppm corresponding to doses of 75 mg Ba/kg bw/d to male and 90 mg Ba/kg bw/d to female mice, respectively.

Executive summary:

In mice, the two-year survival of both sexes that received 2500 ppm was significantly lower than that of the controls due to renal toxicity. Final body weights of 2500 ppm males and females were lowered. No chemical-related haematological and clinical findings were observed. No increased incidences of neoplasms were observed in exposed mice. The incidence of nephropathy was significantly increased in male and female mice receiving 2500 ppm. In 2500 ppm male and female group the relative and absolute spleen weights were lower than the controls and incidences of lymphoid depletion were increased. The plasma barium concentrations were significantly increased in all rats and mice except the 500 ppm male rats after exposure for 15 months. Barium levels in bone of rats from the 2500 ppm groups were about 400 times greater than those in the controls.