Bentonite, acid-leached

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented and scientifically accepted study

Data source

Reference

Reference Type:

publication

Title:

Chronic toxicological evaluation of dietary Novasil clay in sprague-dawley rats

Author:

Afriyie-Gyawu E, Mackie J, Dash B, Wiles M, Taylor T, Huebner H, Tang L, Guan H, Wang J & Phillips T

Year:

2005

Bibliographic source:

Food Additives and Contaminants. 22(3): 259-269

Materials and methods

Principles of method if other than guideline:

Prior to human intervention trials utilizing dietary NovaSil (NS) for the management of aflatoxins (AF) induced disease, potential adverse effects of NS needed to be determined. The objective of the study was to use a rodent model to evaluate the relative safety of long-term exposure to NS clay in the diet.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Novasil Clay

IUPAC Name:

Novasil Clay

Details on test material:

- Name of test material (as cited in study report): Novasil Clay (NS) -a naturally occuring calcium montmorillonite clay
- Source: Engelhard Chemical Corporation (Iselin, NJ)
- Analytical purity: All-trans retinol (>95% purity) and α-tocopherol

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (Houston TX)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: Male (102 - 163 g), females (79 - 135 g)
- Diet: Maintained on powdered feed (Teklad rodent diet 8604) ad libitum
- Water: ad libitum



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25
- Photoperiod (hrs dark / hrs light): 12 hr light/12 hr dark

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.25% (125-250 mg/kg/day), 0.5% (250-500 mg/kg/day), 1.0% (500-750 mg/kg/day) and 2.0% (equivalent to 1000-1500 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

10 animals per sex per dose with the exception of the 0.5% NS group which contained 9 males and 11 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dietary clay concentrations were based on the highest level previously allowed (2.5% w/w) by the Association of American Feed Control Officials (AAFCO) for use in pelleting processes and as an anti-caking agent in non-medicated livestock feeds.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


BODY WEIGHT: Yes
- Time schedule for examinations: Measured initially then once per week throughout the course of the study


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined : Yes


FOOD EFFICIENCY:
- Body weight gain in g: Yes



OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected after 28 weeks
- Anaesthetic used for blood collection: Yes (isoflurane)
- Parameters checked in table 5 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected after 28 weeks
- Parameters checked included the following: Total protein (TP), albumin (ALB), calcium (Ca), phosphorous (P), glucose (GLUC), blood urea nitrogen (BUN), creatinine (CRT), total bilirubin (T-BIL), alkaline phosphatase (ALP), creatine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), globulin (GLOB), A/G ratio, gamma glutamyl-transferase (GGT), amylase (AMYL) and cholseteroal (CHOL) (see table 5). In addition, serum micronutrients iron (Fe) and zinc were also quantified


URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see tables 3 and 4)

Other examinations:

Serum and hepatic vitamins A and E were analyzed

Statistics:

All experimental data were subjected to ANOVA. Data that did not fit the normal distribution were log transformed before analysis. Means were compared using Tukeys multiple comparison procedere and considered significant at p ≤ 0.05

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Throughout the study all rats with the exception of two males remained healthy and active with no noticable behavioural changes. No mortalities occurred.


BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant differences between treated and untreated rats

FOOD CONSUMPTION AND COMPOUND INTAKE
There were no statistically significant differences between treated and untreated rats with regards to total feed consumption (TFC), cumulative feed consumption (CFC) - calculated as the sum of feed consumption from initiation to each additional week

FOOD EFFICIENCY
There were no statistically significant differences between treated and untreated rats with regards to feed conversion efficiency (CFCE) -computed as the FCE from initiation to each additional four week period throughout the study



HAEMATOLOGY
Analysis of whole blood samples showed no significant differences in hematological parameters between rats fed diets containing NS compared to untreated controls in either males or females with the exception of males containing 0.25% NS which showed a significantly higher mean MCH. There was no evidence of hemoparasites. Erythrocytosis or anemia was not observed in the treated compared to untreated rats.


CLINICAL CHEMISTRY
Serum biochemistry indicated that Ca was slightly increased in the 0.5% and 2.0% NS-treated females compared to absolute controls. All other parameters were unchanged between NS-treated and untreated groups within both sexes. Serum and hepatic VA and VE levels were unchanged with dietary inclusion of NS at all treatment levels except a slight increase in VA in the 1.0% NS-treated females compared to untreated females. Serum Fe and Zn levels were also unaffected by the dietary inclusion of up to 2.0% NS except in 1.0% NS-treated males which showed increased Fe levels in comparison to the control. There was no trend toward dose-dependency observed in either case.


ORGAN WEIGHTS
The relaitve organ weights (ROW) for organs measured as the ratio of wet organ weight (WOW) to final body weights (FBW) was not significantly different in rats consuming treated versus untreated diets.

GROSS PATHOLOGY
At necropsy there were no obvious gross abnormalities in the major organs of interest (liver, kidneys, lungs, heart, brain, spleen, tibia, uteri and ovaries) in NS-treated rats.


HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological evaluation of H & E stained sections of the major organs showed no substantial differences between NS treatment groups and untreated controls. Any lesions present were interpreted as background lesions and were of similar incidence and severity in NS-treated and control rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 - 1 500 mg/kg bw/day (nominal)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Total feed consumption (TFC), total body weight gain (TBWG) and feed conversion efficiency (FCE) of S-D rats following dietary ingestion of 0 -2.0% NS for 28 weeks

Treatment group (% NS)	TFC^a(g)	TBWG^a(g)	FCE^a
Males
Abs. Control	4305.5 ± 55.3	339.2 ± 8.8	12.74 ± 0.24
0.25%	4286.4 ± 66.6	341.9 ± 5.5	12.55 ± 0.21
0.5%	4581.9 ± 149.5	350.7 ± 11.4	13.11 ± 0.35
1.0%	4688.5 ± 199.9	349.9 ± 12.7	13.41 ± 0.34
2.0%	4622.2 ± 177.2	348.0 ± 7.2	13.28 ± 0.40
Females
Abs.Control	3102.4 ± 82.7	156.4 ± 7.9	20.07 ± 0.64
0.25%	3204.1 ± 84.6	164.5 ± 7.0	19.70 ± 0.70
0.5%	3185.5 ± 68.4	160.4 ± 4.4	19.95 ± 0.53
1.0%	3195.8 ± 68.1	173.9 ± 7.1	18.59 ± 0.67
2.0%	3212.0 ± 50.5	172.4 ± 6.9	18.89 ± 0.79

^aData represent mean values ( ±SEM)

Table 2: Relative organ weights of S-D rats following the dietary addition of 0 -2.0% NS for 28 weeks

Organ	Treatment group
	Abs Control	0.25%	0.5%	1.0%	2.0%
ROWs of males
Brain	3.75 ± 0.07	3.85 ± 0.06	3.72 ± 0.08	3.78 ± 0.11	3.78 ± 0.10
Heart	2.95 ± 0.05	2.88 ± 0.06	2.98 ± 0.07	2.79 ± 0.06	2.90 ± 0.05
Kidneys	5.95 ± 0.16	5.83 ± 0.13	5.91 ± 0.12	5.77 ± 0.12	5.75 ± 0.16
Liver	3.31 ± 0.10	3.28 ± 0.09	3.27 ± 0.10	3.22 ± 0.07	3.25 ± 0.09
Lungs	3.36 ± 0.08	3.60 ± 0.09	3.68 ± 0.12	3.44 ± 0.09	3.28 ± 0.14
Spleen	1.67 ± 0.06	1.78 ± 0.07	1.74 ± 0.04	1.76 ± 0.05	1.75 ± 0.06
Tibia	1.67 ± 0.02	1.65 ± 0.02	1.63 ± 0.02	1.70 ± 0.04	1.71 ± 0.03
ROWs of females
Brain	6.17 ± 0.13	6.07 ± 0.13	6.19 ± 0.10	6.02 ± 0.14	5.92 ± 0.09
Heart	3.20 ± 0.05	3.37 ± 0.06	3.26 ± 0.07	3.28 ± 0.11	3.24 ± 0.07
Kidneys	6.00 ± 0.11	5.91 ± 0.10	5.84 ± 0.12	5.63 ± 0.10	5.84 ± 0.07
Liver	2.94 ± 0.05	2.83 ± 0.04	2.85 ± 0.04	2.90 ± 0.05	2.83 ± 0.06
Lungs	4.72 ± 0.15	4.89 ± 0.14	4.64 ± 0.12	4.58 ± 0.09	4.65 ± 0.11
Spleen	2.06 ± 0.08	1.98 ± 0.09	2.02 ± 0.06	1.99 ±0.04	1.91 ± 0.09
Tibia	2.05 ± 0.12	1.93 ± 0.06	1.98 ± 0.07	2.03 ± 0.05	2.09 ± 0.05
Uterus/ovaries	2.47 ± 0.18	2.35 ± 0.10	2.76 ± 0.19	2.70 ± 0.24	2.64 ± 0.17

Note: the ROWs were calculated by dividing the wet weight of each organ by the final body weight of the rat just before termination. ROW values for all organs in both sexes have been multiplied by a factor of 103 except the liver which is multiplied by 102. Data are reported as mean values ( ±SEM) 

Table 3: Histological findings for male S-D rats following the dietary ingestion of 0 -2.0% NS for 28 weeks

Treatment group	Incidence (group mean severity*)
	Control	0.25%	0.5%	1.0%	2.0%
Liver
Focal inflammation	4 (1.0)	3 (1.0)	3 (1.0)	5 (1.0)	3 (1.0)
Basophilic focus of hepatoceluular alteration	0	0	1 (1.0)	0	0
Kidneys
Focal interstitial inflammation/fibrosis	10 (1.2)	10 (1.1)	10 (1.1)	10 (1.2)	10 (1.1)
Focal cortical tubular epithelial hyperplasia	10 (2.0)	10 (2.2)	10 (2.1)	10 (2.0)	10 (1.8)
Focal tubular dilatation	10 (1.6)	10 (1.8)	10 (2.1)	10 (1.7)	9 (1.9)
Focal mineralization	4 (1.0)	3 (1.0)	2 (1.0)	3 (1.0)	4 (1.0)
Heart
Focal interstitial inflammation	1 (1.0)	2 (1.0)	0	1 (1.0)	0
Lungs
Focal vacuolated alveolar macrophage accumulation	5 (1.4)	4 (1.5)	4 (1.8)	5 (1.2)	3 (2.0)
Focal eosinophilic interstitial inflammation	1 (1.0)	0	2 (1.5)	0	1 (1.0)
Focal interstitial inflammation other	0	0	0	0	0
Focal arteriolar mineralization	1 (1.0)	1 (1.0)	2 (1.0)	2 (1.0)	3 (1.0)
Focal osteosis	1 (1.0)	2 (1.0)	1 (1.0)	0	1 (1.0)
Brain	0	0	0	0	0
Stomach
Focal gland dilation/epithelial attenuation	4 (1.3)	2 (1.5)	2 (1.0)	1 (1.0)	0
Intestine (duodenum, jejunum, ileum, colon)
Skin
Focal perivascular inflammation	0	0	0	1 (2.0)	0

Note: * Group mean severity was calculated by adding severity scores 1 -4 for rats with lesions and dividing the number of rats with lesions

Table 4: Histological findings for female S-D rats following the dietary ingestion of 0 -2.0% NS for 28 weeks

Treatment group	Incidence (group mean severity*)
	Control	0.25%	0.5%	1.0%	2.0%
Liver
Focal inflammation	5 (1.0)	6 (1.0)	6 (1.0)	2 (1.0)	7 (1.0)
Basophilic focus of hepatoceluular alteration	0	0	1 (1.0)	0	0
Kidneys
Focal interstitial inflammation/fibrosis	5 (1.0)	8 (1.1)	7 (1.0)	5 (1.0)	7 (1.0)
Focal cortical tubular epithelial hyperplasia	6 (1.0)	7 (1.4)	8 (1.3)	7 (1.1)	8 (1.3)
Focal tubular dilatation	6 (1.0)	8 (1.5)	8 (1.1)	9 (1.1)	9 (1.2)
Focal mineralization	1 (1.0)	1 (1.0)	0	2 (1.0)	2 (1.0)
Heart
Focal interstitial inflammation	1 (1.0)	1 (1.0)	0	2 (1.0)	0
Lungs
Focal vacuolated alveolar macrophage accumulation	2 (1.0)	5 (1.2)	5 (1.4)	4 (1.3)	5 (1.0)
Focal eosinophilic interstitial inflammation	0	1 (1.0)	0	0	0
Focal interstitial inflammation other	0	0	0	0	1 (1.0)
Focal arteriolar mineralization	0	0	0	0	0
Focal osteosis	1 (1.0)	0	1 (1.0)	0	1 (1.0)
Brain	0	0	0	0	0
Stomach
Focal gland dilation/epithelial attenuation	2 (1.0)	2 (1.0)	0	0	1 (1.0)
Intestine (duodenum, jejunum, ileum, colon)	0	0	0	0	0
Skin
Focal perivascular inflammation	2 (1.0)	0	0	1 (1.0)	0
Ovary
Paraovarian cyst	1 (2.0)	0	1 (1.0)	0	0
Uterus	0	0	0	0	0

Note: *Group mean severity was calculated by adding severity scores 1 -4 for rats with lesions and dividing by the number of rats with lesions

Table 5: Hematology of S-D rats following the dietary administration of 0 -2.0% NS for 28 weeks

Hematological parameter	Treatment group
	Abs control	0.25%	0.5%	1.0%	2.0%
Males
WBC/µl	5840 ± 713	6180 ± 637	5667 ± 464	8120 ± 369	5365 ± 704
Neutrophils (%)	14.1 ± 3.0	14.1 ± 3.3	14.1 ± 2.7	10.3 ± 1.6	13.9 ± 2.3
Lymphocytes (%)	83.2 ± 2.9	81.9 ± 3.5	83.6 ± 3.1	82.2 ± 1.8	83.7 ± 2.6
Monocytes (%)	1.8 ± 0.2	1.8 ± 0.5	1.4 ± 0.2	1.0 ± 0.0	1.3 ± 0.3
Easinophils (%)	2.3 ± 0.5	3.2 ± 0.7	2.3 ± 0.7	2.4 ± 0.4	2.5 ± 0.8
RBCs/µl (x 10^6)	7.5 ± 0.1	7.4 ± 0.1	7.5 ± 0.1	7.5 ± 0.1	7.5 ± 0.1
Hb (g/dl)	13.4 ± 39.5	13.6 ± 0.1	13.6 ± 0.2	13.6 ± 0.1	13.5 ± 0.2
PCV (%)	39.5 ± 52.6	39.9 ± 0.3	40.4 ± 0.5	40.0 ± 0.4	39.7 ± 0.5
MCV (fl)	52.6 ± 17.8	54.3 ± 0.6	53.7 ± 0.3	53.6 ± 0.6	53.0 ± 0.4
MCH (pg)	17.8 ± 0.2	18.6 ± 0.2*	18.1 ± 0.1	18.2 ± 0.2	18.0 ± 0.1
MCHC (g/dl)	33.9 ± 0.3	34.2 ± 0.2	33.7 ± 0.2	33.9 ± 0.3	34.0 ± 0.2
Females
WBC/µl	3930 ± 431	4250 ± 487	3936 ± 356	3950 ± 452	3411 ± 486
Neutrophils (%)	17.7 ± 2.6	13.5 ± 1.7	13.0 ± 2.7	13.9 ±1.1	14.0 ± 2.7
Lymphocytes (%)	79.7 ± 2.9	83.4 ± 2.1	85.0 ± 2.8	78.4 ± 6.6	82.8 ± 3.1
Monocytes (%)	1.2 ± 0.2	2.3 ± 0.5	1.3 ± 0.3	1.0 ± 0.0	1.8 ± 0.6
Easinophils (%)	2.2 ± 0.6	3.1 ± 0.7	1.9 ± 0.2	1.8 ± 0.3	2.2 ± 0.4
RBCs/µl (x 10^6)	6.5 ± 0.1	6.4 ± 0.1	6.5 ± 0.1	6.2 ± 0.1	6.5 ± 0.1
Hb (g/dl)	12.4 ± 0.2	12.4 ± 0.2	12.6 ± 0.1	12.3 ± 0.2	12.5 ± 0.2
PCV (%)	36.5 ± 0.6	36.1 ± 0.5	37.2 ± 0.5	35.7 ± 0.3	36.5 ± 0.7
MCV (fl)	56.4 ± 0.6	56.8 ± 0.5	57.1 ± 0.5	57.2 ± 0.1	56.3 ± 0.5
MCH (pg)	19.1 ± 0.2	19.5 ± 0.2	19.4 ± 0.2	19.7 ± 0.1	19.3 ± 0.1
MCHC (g/dl)	33.9 ± 0.2	34.3 ± 0.1	33.9 ± 0.1	34.4 ± 0.1	34.3 ± 0.1

Note: Data are repoted as mean values ( ±SEM). * Indicates statistical significance compared to controls (p ≤ 0.05); WBC, white bllod cells; RBC, red blood cells; Hb, hemoglobin; PCV, percent corpuscular volume; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; and MCHC, mean corpuscular hemoglobin concentration

Applicant's summary and conclusion

Conclusions:

Results suggested that dietary inclusion of NovaSil at levels as high as 2.0% (w/w) which is ~equivalent to 1000-1500 mg/kg/day does not result in overt toxicity.