Registration Dossier

Administrative data

Description of key information

Repeat dose oral: Currently, seven different animal studies are available that have evaluated the repeated dose toxicity of (calcium) sulfate. None of the studies describes any severe toxicological effects after oral administration of (calcium) sulfate.

Repeat dose inhalation: Currently, four different animal studies are available that have evaluated the repeated dose inhalation toxicity of (calcium) sulfate. Besides the animal studies, several health surveillance data, mainly from gypsum miners are also available.

Repeat dose dermal: No studies on dermal toxicity of calcium sulphate are available

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and guideline
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: 254.2 - 297.8 g (males) and 182.7 - 208.2 g (females)
Route of administration:
oral: gavage
Duration of treatment / exposure:
35 days for male animals and 41-45 days for female animals
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control

Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
60
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
- Clinical observations performed and frequency: Clinical symptoms were observed once a day but were observed once a week in detail; a death rate was observed twice a day; and body weight was observed once a week and just before the necropsy, but in case of pregnant females, it was measured on the day 0, 7, 14, 20 of gestation period, date of delivery, and 4 days after the delivery; consumption rate of fodder was observed once a week except mating period.

Tests for sensory organ and reflex action: 5 animals were randomly selected from each test group. Both preyer reflex test and corneal reflex test were performed before necropsy and during lactation for males and females, respectively.

Behaviour test: 5 animals were randomly selected from each test group to do grip strength test in terms of behaviour test. This test was performed before necropsy and during lactation for males and females, respectively.

- Haematological and biochemical test of blood: randomly selected 5 male and female animals from each test group were fasted a day before necropsy for both tests. Animals were anesthetized using ether and cut the abdomen open to collect blood. In case of the haematological test, blood coagulation preventative chemicals for the test of blood coagulation and the calculation of blood-corpuscles were 3.2 % sodium citrate and EDTA- 2K, respectively. On the other hand, blood coagulation preventative chemical was not used for the biochemical test, but gathered blood was left itself in the room temperature then the sera were separated using a centrifuge. For haematological test, 6 following items were measured; Haematocrit, hemoglobin concentration, erythrocyte count, total and different leucocyte count, platelet count, prothrombin time, and active partial thromboplastin time. For biochemical test of blood, eleven following items were measured; sodium, potassium, glucose, total cholesterol, blood urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, aspatate aminotransferase, and total bilirubin.


Organs examined at necropsy: Organ weight: testes, epididymider (all males) liver, kidney, adrenals, thymus, spleen, brain and heart (5 male and female animals from each test group).

Fixation: 22 kinds of tissues were fixed to do histopathologic tests such as testes, epididymides, ovaries, accessory sex organs for all animals, brain (including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including peyer’s patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea, lungs, uterus, urinary bladder, lymph nodes (cervical mesenteric), peripheral nerve (sciatic or tibial), and bone marrow.




























Statistics:
Statistical decision tree, but in case of recovery group, either two-side Students t-test or two-side Apsin Welch t-test was used. In case of categorical data, two-sided Fishers exact test was used.
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 examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality: There was one death at day 8 for male, and each death on the day 7 and 14 for female in the treatment group of 300 mg/kg/day. These were occurred during the administration process, so it did not have relationship with test substance.

Body weight: In male groups, temporarily, a case of diminishment in the amount of body weight change was observed at week 2 within the
control group in which some clinical signs were observed such as damage to esophagus. Body weight loss for female animals was observed several times during lactation period in every treatment group including the control group. However, these were occurred temporarily because of the lactation.

- Clinical signs: In male control group, a case of salivation and bloodylike secretion was observed on the day 11 and 12. In the 1,000 mg/kg/day treatment group, a case of depilation, dcab and pus was observed on the left cheek between the day 25 and the closing day. However, the frequency of occurrence was low and no dose-response correlation. Thus these symptoms were not influenced by test substance. In female control group, a case of genitalia bloody-like secretion was observed at day 29. In the 100 mg/kg/day treatment group, each case of hypoactivity and depilation was observed on the day 8 and 9, and between day 44 and the closing day, respectively. However, these symptoms were disappeared in short, thus these did not have relationship with test substance.

Amount of fodder consumption: No crucial difference between the treatment group and the control group was observed for both male and female animals during test period. For recovery group, no significant change was observed within themselves.

Test of reflex action: Five male and female animals were randomly selected from each test group, in which no specific reaction was observed.

Grip strength test: For male animals, 6 animals were left out from the treatment group. For female animals, 5 animals were left out from the control group, and the treatment group. All things being considered, there was no dose-response correlation and was no illness at the related organs such as the cerebellum and muscle.

Organ weight: Both absolute weight of the liver and the left kidney were increased at the recovery group with administration of 1,000 mg/kg/day as compared with that of the control group within the recovery group. There was no histopathological illness at the organs, so increased organ weight did not have relationship with test substance.

Necropsy opinions: For male animals, in the control group within the recovery group, a case of left and right caput epididymis cyst was observed and the 1,000 mg/kg/day recovery group had symptom of right caput epididymis cyst. However, its frequency of occurrence was low and it was even observed at the control group within the recovery group, so it did not have relationship with test substance. For female animals, in the 300 mg/kg/day treatment group, each animal was dead on the day 7 and 14 and; each case of lung dark-red discolouration was observed, but white particle in a lobe of the lung was observed just from one of carcasses. A case of spleen white nodule was observed for an animal in the 300 mg/kg/day treatment group. There was a case of right adrenal gland white spots at the 1,000 mg/kg/day treatment group. In the control group within the recovery group, each case of right adrenal gland hemorrhagia and atrophy and liver adhesion with diaphragm was observed.

Analysis of haematological test of blood: In the 1,000 mg/kg/day male recovery group, segments were increased (p < 0.05) in contrast with that of the control group within the recovery group. Prothrombin time (PT) was decreased in the 1,000 mg/kg/day female recovery group in comparison with that of the control group within the recovery group. However, these symptoms were not observed in the definitive test groups, so these symptoms were not influenced by test substance. In addition, level of WBC (white blood cell) was increased (p < 0.001) in the 100 mg/kg/day female treatment group in contrast with that of the control group. However, its increased value was in the normal range and no dose-response correlation, so it did not have relationship with test substance.

- Analysis of biochemical test of blood: In the 100 mg/kg/day treatment group for male animals, BUN (Blood urea nitrogen) was decreased as compared with that of the control group. The male treatment groups with both administration of 300 mg/kg/day and the 1,000 mg/kg/day decreased in TP (Total protein), ALB (Albumin), AST (Aspatate aminotransferase), ALT (Alanine aminotransferase), BUN (Blood urea nitrogen), CREA (Creatinine), Na (Sodium), TCHO (Total cholesterol), and Cl (Chloride) as compared with those of the control group. In case of the 1,000 mg/kg/day male recovery group, the value of AST was decreased significantly in contrast with that of the control group within the recovery group.
No significant difference was found at every test item between female control and treatment group. The female recovery group with administration of 1,000 mg/kg/day decreased in AST in contrast with that of the control group within the recovery group, but TCHO and GLU (Glucose) were increased. In fact, decreased values of AST and ALT could be no toxicological effects. In addition, the changed values of AST, TCHO, and GLU in this
test were in the normal range and no histopathological opinion in terms of related organs, so these changed values were not influenced by test substance. However, decreased values of TP, ALB, BUN, and CREA were possibly influenced by excretion process or metabolism of test substance in relation to the kidney, and these symptoms were possibly recovered in 2 weeks from reversible effects.

- Histopathology: For male animals, in the control group, each case of heart focal inflammatory cell infiltration, submadibular lymph node blood absorption, liver mononuclear cell foci, and adrenal gland cortical vacuolation was observed. In the 300 mg/kg/day treatment group, two cases of pancreas vacuolation, and a case of liver mononuclear cell foci were observed. In the treatment group with administration of 1,000 mg/kg/day, there were three cases of liver mononuclear cell foci; and a case of heart focal inflammatory cell infiltration was found.

For female animals, in the control group, two cases of liver mononuclear cell foci, a case of kidney cortical scaring, and a case of pancreas vacuolation were observed. In the treatment group with administration of 100 mg/kg/day, one case of esophagus submucosal gland proliferation was observed. In the 300mg/kg/day treatment group, a case of trachea submucosal gland proliferation was observed. In the 1,000 mg/kg/day treatment group, each case of pancreas vacuolation and liver mononuclear cell foci was observed. However, these symptoms for both sexes were just subtle level and were occurred spontaneously, so there was no significant difference between the treatment group and the control group.















































Dose descriptor:
NOAEL
Effect level:
79 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
calcium sulfate anhydrous
Sex:
male
Basis for effect level:
other: see remarks
Dose descriptor:
LOAEL
Effect level:
237 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
calcium sulfate anhydrous
Sex:
male
Basis for effect level:
other: The LOAEL for calcium sulfate dihydrate was determined to be 300 mg/kg/day for males. The value has been corrected for calcium sulfate anhydrous
Dose descriptor:
NOAEL
Effect level:
790 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
calcium sulfate anhydrous
Sex:
female
Basis for effect level:
other: No effects were observed in females.
Remarks on result:
other: The NOAEL for calcium sulfate dihydrate was 1000 mg/kg/day and has been corrected for calcium sulfate anhydrous
Critical effects observed:
no
Conclusions:
The study was performed with calcium sulfate dihydrate and the results calculated for calcium sulfate anhydrous.

The NOAEL for calcium sulfate anhydrous was 79 mg/kg/day for males based on a significant decrease in TP (Total Protein), ALB (Albumin), BUN (Blood Urea Nitrogen), and CREA (Creatinine) at the 300 mg/kg b.w./day and 1,000 mg/kg b.w./day groups. No effects were observed in females.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: The study is provided as supporting information for calcium sulfate.
Qualifier:
according to
Guideline:
other: No data
Deviations:
not applicable
Principles of method if other than guideline:
Rats were fed on poultry growers mash containing 21% protein, 7.2% fat, 4.4% fibre and 5.8% ash supplemented with 5 g calcium sulfate. Effect on haematological parameters was monitored for 2 weeks and weights of rats recorded.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Department of biochemistry, University of Ilorin, Ilorin, Kwara State of Nigeria.
- Diet (ad libitum): feed worth 60 % body weightof the rat was given to each dietary group daily.
- Water (ad libitum): 500 mL per dietary group daily.


Route of administration:
oral: drinking water
Vehicle:
not specified
Details on oral exposure:

DIET PREPARATION
- 5 g calcium sulfate was mixed with water and added to poultry growers mash, containing 21 % protein, 7 % fat, 4.4 % fibre and 5.8 % ash.


Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Dose / conc.:
5 other: g (nominal water)
No. of animals per sex per dose:
5/sex/group
Control animals:
yes, concurrent no treatment
Details on study design:
No data
Positive control:
No data
Observations and examinations performed and frequency:
Haematological parameters: at the end of the 2nd week animals were sacrificed. Blood was withdrawn and anticoagulant K+EDTA was added to the blood to prevent clotting (2.0 mg/mL of blood). Leukocytes (counted vistually), differential counts, haemoglobin (cyanmethaemoglobin method) and packed cell volume (microhaematocrit technique) were determined. Each experiment was conducted in triplicate.

Body weights were noted.
Sacrifice and pathology:
No data
Other examinations:
No data
Statistics:
Data are presented as mean values with their respective standard errors and relationship between the diets and the growth of the rats has been determined.
Clinical signs:
not examined
Mortality:
not examined
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):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Contrary to the increase in weight observed in all dietary treatments, weight loss was observed in all rats given heavy metal laced diets, with the exception of calcium sulfate. Increase in weight was observed in the control and in all dietary groups (B - E) given feed supplemented with calcium sulfate.

No deaths were reported for rats fed calcium sulfate.

The effects of heavy metals on the haematological parameters :
male rats had the highest WBC counts (27400 ± 6.23/mm3) in the control dietary group A, whilst the least value of 2900/mm3 was recorded in the female rats of dietary group E. The value of RBC was higher in the heave-metal-laced diets than in metal-free diets. The value of packed cell volume was higher in dietary groups B, D and E while the values obtained for the control and dietary group C were lower. Haemoglobin was higher in the rats fed heavy metal-laced diets than those fed on metal-free diets. Higher RBC and PCV were observed in the rats of heavy metal-laced feed experiments.

Monocyte counts for heavy metal-free feeds were greater thanfor heavy-metal laced ones. The lymphocyte/neutrophil ratio in heavy metal-laced experiments, in dietary group E was 3.55:1 for females and 1.43:1 for males.
Remarks on result:
other: see remarks
Remarks:
Contrary to the increase in weight observed in all dietary treatments, weight loss was observed in all rats given heavy metal laced diets, with the exception of calcium sulfate. Increase in weight was observed in the control and in all dietary groups (B - E) given feed supplemented with calcium sulfate.
Critical effects observed:
not specified

Table1: Growth response of the albino rats, Rattus norvegicus fed on additional protein supplements for 7 weeks and heavy metals for 2 weeks 

Heavy metal

(A) (g)

(B) (g)

(C) (g)

(D) (g)

(E) (g)

Control

Growers mash

Growers mash

+ Blood meal

Growers mash

+ Fish meal

Growers mash

+ Soybean meal

Growers mash

+ Prawn meal

Male

Female

Male

Female

Male

Female

Male

Female

Male

Female

CaSO4

1.34

± 0.98

1.20

± 0.02

1.50

 ± 0.21

1.63

 ± 0.18

1.79

± 0.45

1.91

 ± 0.29

2.00

 ± 0.20

2.04

 ± 0.11

2.545

 ± 0.12

2.35

 ± 0.31

Each value is a mean of three values

Table 2: Growth response of the albino rats, Rattus norvegicus fed on additional protein supplements within 7 weeks.

Weeks

(A) (g)

(B) (g)

(C) (g)

(D) (g)

(E) (g)

Control

Growers mash

Growers mash

+ Blood meal

Growers mash

+ Fish meal

Growers mash

+ Soybean meal

Growers mash

+ Prawn meal

1

11.53

±0.61

9.17

±1.17

17.59

±0.41

18.50

±1.50

14.77

±0.24

18.00

±1.00

19.45

±0.67

11.27

±1.13

14.92

±1.64

12.75

±0.72

2

15.83

±1.68

11.59

±0.49

18.76

±0.03

19.28

±0.83

17.08

±0.63

19.53

±1.47

20.45

±0.67

14.84

±1.56

20.09

±0.92

17.10

±0.10

3

16.54

±0.98

13.20

±1.02

21.05

±0.78

20.73

±0.18

18.59

±0.91

2.13

±1.94

22.45

±1.20

17.41

±1.91

23.25

±0.36

17.95

±0.95

4

17.02

±0.68

14.52

±0.11

22.48

±1.02*

22.25

±0.97*

20.96

±2.76*

25.17

±1.63*

24.60

±3.07*

19.89

±2.21*

26.03

±0.95

19.83

±0.59

5

17.80

±0.79

15.57

±0.35

24.50

±0.61

24.21

±0.99

22.76

±2.65*

26.67

±2.54*

26.85

±2.16

22.11

±2.66

27.50

±1.79*

22.78

±1.84*

6

19.52

±0.42

15.92

±0.19

25.81

±1.17

25.92

±1.29

26.20

±1.55*

27.93

±2.53*

30.17

±.368

23.23

±2.78

29.86

±2.80*

26.08

±0.82*

7

21.57

±1.58*

17.40

±0.59*

27.64

±1.64

28.29

±1.47

29.39

±1.61

30.73

±3.12

32.19

±2.25*

27.25

±4.08*

32.60

±3.40

28.71

±0.48

Each value is a mean of three values ± standard error of the mean. The values in the same row by * are significantly correlated at 0.01 level.

Conclusions:
Contrary to the increase in weight observed in all dietary treatments, weight loss was observed in all rats given heavy metal laced diets, with the exception of calcium sulfate. Increase in weight was observed in the control and in all dietary groups (B - E) given feed supplemented with calcium sulfate.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Data from literature in which the test parameters documented do not totally comply with relevant guidelines. Study used as supporting information for repeat dose toxicity.
Principles of method if other than guideline:
Rats were fed a basal diet or diets containing extra MnSO4, MgSO4 or Na 2SO4 in three experiments.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Weanling male rats were housed in individual cages and given tap water and their respective diets ad libitum.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The basal diet was designated as the cornstarch diet in all three experiments and contained 67 % cornstarch, 24 % vitamin-free casein, 5 % Crisco, 4 % salt mixture and vitamins. The salt mixture contained 3.8 % magnesium sulfate (anhydrous) and 0.02 % manganous sulfate monohydrate, but no sodium sulfate.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
None specified
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily. Treatment was added to diet which was supplied ad libitum.
Dose / conc.:
138 other: mmol/kg of feed
Remarks:
Highest dose level: See section on study design.


No. of animals per sex per dose:
In each of the three experiments there were 6 rats in each of the dietary groups
Control animals:
yes, plain diet
Details on study design:
For diets high in sulfates of magnesium, magnesium and sodium, part of the cornstarch diet was replaced respectively by extra manganous sulfate, extra magnesium sulfate, or sodium sulfate. In experiment a, the amount of respective sulfate salt added over and above that in the salt mixture for MnSO4, MgSO4 and Na2SO4 groups was 0.88 mmole/kg. This level was fed throughout the experiment. In experiment b, the respective sulfates were added initially at the level (over and above that in the basal diet) of 8.64 mmole/kg of feed. This level was progressively increased at the same times as for the MnSO4, MgSO4 or Na2SO4 groups in experiment b by doubling it on the 9th day of the feeding period to 17.28 mmole/kg, doubling this on the 17th day to 34.56, doubling the latter on the 25th day to 69.12 and then maintaining the level for the remaining 4 - 6 days. The final percentage by weight in the diet of the major sulfate constituent was 1.2 % MnSO4•H2O, 1 % MgSO4 and 1.0 % Na2SO4 for the respective high sulfate diets in experiment b. In experiment c, the extra sulfates were added at the level of 138 mmole/kg of feed throughout the experiment.
Positive control:
None specified
Observations and examinations performed and frequency:
In experiments a and c, feed intakes and feed:gain ratios were obtained for each week and for the total 4-week period. In all experiments, records were kept of any diarrhoea that occurred. The rats were weighed at the beginning of the 4-week period, at the end of each week and at the end just before killing them.
Sacrifice and pathology:
The gastrointestinal organs were freed from mesentery and excised. The small intestine and the colon plus rectum were hung full length, each with a 5 g weight attached to the lower end. When these organs had straightened out completely the lengths were measured. The organs were cleaned examined, dried and weighed.
In experiment c, a small snip of stomach was removed for histological examination before drying the organ, so that the dry weights of the stomach do not reflect the true weight as in experiments a and b.
After 3.5 weeks of the feeding period, blood was obtained from the tail for red and white blood cell counts and for determination of haemoglobin by oxyhaemoglobin method. At the time of killing the rats, blood was collected from the severed great vessels in the neck and the serum from this blood was analyzed for alkaline phosphatase, inorganic phosphate and protein.
Many tissues were preserved for later analysis and examination but it has not yet been possible to arrange for completion of this part of the study.
Other examinations:
In experiment c, additional observations were made as suggested by certain developments during the feeding period. These included visual observations of the teeth and of the volume of urine. Also the amount of water drunk during the first 48 h of the third week was recorded.
Statistics:
In experiments b and c, the numerical results were analyzed statistically by analysis of variance, the hypothesis in every case being that the groups were not equal. In those cases where the F value indicated that there was a difference among the groups at the 5 % level or less, a multiple range test was applied to determine which among the groups differed from any other. The results of the test are indicated in the tables for the 5 % and 1 % levels of probability and are referred to as significant if P< 0.05.
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):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
No data were presented for experiment a since at the low levels of sulfate salt additions in the experiment, all quantities examined were similar in all groups. Also, there were no significant differences among the groups in body weight gains within experiment b, so these gain details were also omitted.
Gains in body weight, intakes of feed and water and feed:gain ratios:
In experiment c, the rate in the MnSO4 group as compared to the rats in the other groups gained less weight, ate less feed, required more feed per gram of gain and drank much more water. Although MgSO4-fed rats suffered chronic diarrhoea they did not drink more water than the rats fed the cornstarch or Na2SO4 diets. The MgSO4-fed rats ate significantly less food than the Na2SO4-fed rats; they tended to eat less feed than the cornstarch diet rats also, but this latter tendency was significant only in the 1st week of the experiment.

Volume of urine:
In experiment c, rats in the MnSO4 group voided more urine than rats in the other groups, as shown by much larger pools of urine on the waxed papers placed beneath the rats. The increase in volume was estimated as at least two-fold, but exact measurements were not made.

Teeth:
During the 3rd week of experiment c, it was noted that the front surfaces of the incisor teeth of 5 out of 6 rats in the MnSO4 group were white or very pale yellow in colour. Only one rat in the MnSO4 group had incisors of the normal orange-yellow colour, whereas every rat in the other groups had normally coloured incisors. These differences persistsed until the end of the experiment.

Blood findings in experiment c:
Haemoglobin in the MnSO4 group was significantly lower than in the other three groups. Only one rat in the MnSO4 group had a haemoglobin content falling within the range of the other three groups; this was the same rat which had normally coloured incisor teeth. The numbers of red blood cells were signifcantly greater in the MnSO4 group than in the cornstarch or Na2SO4 groups. The differences among the groups in numbers of white blood cells were not significant statistically. The differences in serum protein was not significant; however three values in the MnSO4 group were slightly below the normal range of 6.2 - 7.1 g/100mL. The MgSO4 group exceeded all other groups in serum alkaline phosphatase activity. Serum inorganic phosphate was lower in the MnSO4 group than in the other groups.

Diarrhoea:
In experiment b there were no cases of diarrhoea in the cornstarch group, one slight case in the MnSO4 group and two slight cases in the Na2SO4 group lasting only 1 day each. In the MgSO4 group, only two of the six rats had no diarrhoea, three had only slight diarrhoea (one for 1 day, another on 2 non-successive days, and the third on 3 non-successive days) while the remaining rat had slight diarrhoea on 6 different days (including 4 in succession) during the middle periods of the experiment and moderate diarrhoea for the three successive days following the last increase in MgSO4 level. Also, on certain other days when diarrhoea did not occur, the faeces of this rat and one other rat in the MgSO4 group were soft and had a variety of unusual shapes: some pellets were extra long but of normal thickness, some were very thin width but of normal length and some showed the lines of "working over" plainly. Also, several of the pellets had several small sideways projections at one end and thus resembled cloves. The pellets were black in all groups.
In experiment c, there was no diarrhoea in the MnSO4 group and only 1 instance of slight diarrhoea lasting 1 day on the cornstarch group. Only one rat in the Na2SO4 group had any diarrhoea at all. This occurred on 4 different days (including 3 in succession) in about the middle of the feeding period, and was slight in each instance. Diarrhoea began in all the rats of the MgSO4 group after 1 - 3 days of commencing the feeding and continued until the end of the experiment. Once established, diarrhoea in this group was severe on most days during the 4th week. However there was no clear-cut indication of remission of the MgSO4-produced diarrhoea, such as occurs with the diarrhoea produced by various sugar-containing diets.

Appearance of organs:
In experiment b, the cecal contents in the MgSO4 group were quite runny (althought the liquid contained some solid material) as compared to the more pasty or waxy cecal contents in the other groups. In the MgSO4 group of experiment c, the contents of the cecum and colon were runny and the cecum was noticeably enlarged compared to the cecum in the other groups. The tissues around the rectum in the MgSO4 group seemed to be tougher and more difficult to separate from the rectum than is usual, perhaps indicating that some adaptation occurred external to the organ itself.

Weights and lengths of organs:
In experiment b, the cecum was significantly heavier in the MgSO4 group than in the cornstarch and Na2SO4 groups but the differences were not very large. In experiment c, the MnSO4 group was exceeded by the other 3 groups in final body weight in the absolute weight of the stomach. The absolute weight of the small intestine also tended to be least in the MnSO4 group significantly exceeded the other three groups in the weight of the stomach and small intestine in relation to body weight. The MgSO4 group exceeded the other groups in the absolute and relative weights of the cecum. MnSO4 exceeded Na2SO4 in the relative cecal weight. The absolute weights of the colon plus rectum did not differ significantly among the groups, but the relative weights of this organ were significantly greater in the MnSO4 group than in the other groups. Most of the differences in weights of organs in experiment c appear to be related to the smaller size and body weight attained by the rats in the MnSO4 group; there are difficulties in interpreting differences in weights of organs in such a situation. In experiment c, the colon plus rectum was longer in the MgSO4 group than in the cornstarch or MnSO4 groups. The ratio of weight to length of this organ, like the absolute weight, did not differ significantly among the groups.
Dose descriptor:
other: effect level
Effect level:
138 other: mmol/kg of feed
Sex:
male
Basis for effect level:
other: The major effects occurred at the highest level of sulfates.
Critical effects observed:
not specified

Gains in body weight, intakes of feed and water, and feed: gain rates in experiment c:

Dietary group

Gain in 4 wk

(gm)

Feed intake in 4 wk

(gm)

Over-all feed : gain ratio for 4 wk

(gm/gm)

Water intake 1st48 h of 3rdwk

(mL)

Cornstarch

186

(178 – 199)

386

(375 – 396)

2.08

(1.97 – 2.16)

48

(36 – 58)

MnSO4

109*

(101 – 124)

301*

(280 – 319)

2.76*

(2.52 – 2.89)

83*

(53 – 99)

MgSO4

180

(162 – 195)

356†

(324 – 391)

1.98

(1.84 – 2.12)

48

(37 – 55)

Na2SO4

192

(180 – 209)

408

(371 – 453)

2.13

(2.03 – 2.19)

51

(40 – 60)

Figures given above are averages; ranges in parentheses. Average b. wt at start was 55 gm in each of the groups.

* significantly different from the other three grops (P < 0.01).

† significantly different from the Na2SO4 group (0.01 < P < 0.05).

Blood and serum in experiment c:

Dietary group

Tail blood after 3.5 weeks of feeding the diets

Serum from blood from vessels in the neck, obtained at killing

Hemoglobin

(g/100 mL)

Red blood cells (millions/cu mm)

White blood cells (thousands/cu mm)

Protein

(g/100 mL)

Alkaline phosphatase

(Bodansky U/100 mL)

Inorganic phosphate

(mg P/100mL)

Cornstarch

14.4

(13.2 – 16.3)

6.91

(6.0 – 7.6)

9.19

(1.75 – 15.45)

6.7

(6.4 – 6.9)

84

(73 – 91)

5.3

(4.4 – 6.20

MnSO4

11.6*

(10.0 – 14.0)

8.80

(7.4 – 11.0)

16.21

(11.95 – 21.20)

6.3

(5.7 – 7.0)

85

(81 – 89)

3.8§

(3.6 – 3.8)

MgSO4

14.6

(14.1 – 15.4)

7.74

(6.9 – 8.75)

12.23

(6.7 – 16.6)

6.8

(6.6 – 7.0)

99

(90 – 106)

6.0

(4.8 – 7.0)

Na2SO4

14.6

(14.0 – 15.2)

6.94

(5.85 – 8.05)

6.94

(2.20 – 23.4)

6.5

(6.3 – 6.9)

78

(66 – 83)

5.3

(4.8 – 5.8)

Values give nare averages; ranges are in parentheses. Six rats per group, except that the values and statistical analyses for phosphatase amd phosphate are for 4 of the 6 rats in each group. The determinations of phosphatase in the sera of the other 2 rats of each group were ruined. The pooled sera of these reamining 2 rats of each group yielded values of 6.1, 3.7, 6.3 and 5.8 mg P/100 mL for hte cornstarch, MnSO4, MgSO4 and Na2SO4 gropus respectively.

* Significantly different from the other three groups (P < 0.01)

† Significantly different from cornstarch and Na2SO4 (0.01 < P < 0.05)

‡ Significantly different from Na2SO4 (P<0.01) and from cornstarch and MnSO4 (0.01 < P <0.05).

§ Significantly different from MgSO4 (P < 0.01) and from cornstarch and Na2SO4 (0.01 < P < 0.05)

Body wieght, weights of cleaned dried organs and length of small intestine and colon plus rectum in experimentsb and c:

Dietary group

B wt

Weights of cleaned dried organs

Length of organs

Stomach

Small Int.

Cecum

Colon+ rectum

Small Int.

Colon+ rectum

g

mg

mg

mg

mg

cm

cm

Experiment b

Cornstarch

211

(193 – 235)

214

(196 – 235)

945

(877 – 996)

65

(52 – 74)

144

(130 – 164)

90

(96 – 105)

17.2

(16.5 – 18.5)

MnSO4

210

(187 – 234)

231

(211 – 243)

904

(760 – 993)

73

(64 – 84)

150

(133- -163)

100

(95 – 112)

17.6

(16.5 – 19.5)

MgSO4

216

(171 – 237)

222

(184 – 244)

922

(844 – 1007)

80*

(63 – 90)

145

(124 – 166)

98

(91 – 103)

17.8

(17.0– 18.5)

Na2SO4

211

(172 – 246)

128

(189 – 247)

875

(881 – 1174)

63

(51 – 75)

142

(125 – 162)

96

(87 – 101)

17.1

(15.5 – 18.5)

Experiment c

Cornstarch

248

(232 – 260)

222

(202 – 238)

1011

(881 – 1174)

82

(70 – 97)

162

(145 – 184)

104

(99 – 107)

17.4

(16.4 – 10.0)

MnSO4

171†

(156 – 190)

179‡

(139 – 210)

915

(828 – 1025)

68

(60 – 75)

157

(141 – 180)

101

(94 – 105)

17.4

(15.5 – 19.0)

MgSO4

243

(228 – 260)

217

(198 – 236)

1029

(961 – 1116)

161†

(140 – 183)

165

(147 – 201)

105

(98 – 111)

19.7§

(18.5 – 21.0)

Na2SO4

256

(236 – 281)

229

(213 – 244)

1047

(1000 – 1134)

80

(71 – 90)

156

(144 – 167)

107

(104 – 112)

18.0

(16.0 – 19.5)

Values given are averages; ranges are in parentheses. Various groups are compared only within each experiment. In experiment c, weight of stomach is low in ggropus due to removal of a small sample for histological examination. In experiment c, weights of small intestine were found to be different by analysis of variance (0.01 < P < 0.05) but no differences between single means were found using the multiple range test.

* Significantly different from Na2SO4 and cornstarch (0.01 < P < 0.05)

† Significantly different from the other 3 groups (P < 0.01)

‡ Significantly different from cornstarch and Na2SO4 (P < 0.010 and from MgSO4 (0.01 < P < 0.05)

§ Significantly different from cornstarch and MnSO4 (0.01 < P < 0.05)

Conclusions:
The major effects occurred in the experiment with the highest level of extra sulfates (138 mmole/kg of feed). The MgSO4-fed rats ate less feed than the Na2SO4 -fed rats. Also, in the MgSO4 group, the activity of serum alkaline phsophatase was greater and the cleaned dried cecum was considerably heavier than in the other groups and the colon plus rectum was slightly longer compared to rats of the other groups in the basal and the MnSO4 groups. MnSO4 fed rats compared to rats of the other groups in this experiment ate less feed, gained less body weight, required more feed per gram of gain, drank more water, voided more urine, showed depigmentation of the labial surface of the incisor teeth, had less haemoglobin and serum inorganic phosphorous and had cleaned dried stomachs of lighter absolute weight. The erythrocyte count was higher in the MnSO4 group than in the basal or Na2SO4 groups.
Executive summary:

Rats were fed a basal diet or diets containing extra MnSO4, MgSO4 or Na2SO4, in three experiments. The major effects occurred in the experiment with the highest level of extra sulfates (138 mmole/kg of feed). The MgSO4-fed rats ate less feed than the Na2SO4 -fed rats. Also, in the MgSO4 group, the activity of serum alkaline phsophatase was greater and the cleaned dried cecum was considerably heavier than in the other groups and the colon plus rectum was slightly longer compared to rats of the other groups in the basal and the MnSO4 groups. MnSO4 fed rats compared to rats of the other groups in this experiment ate less feed, gained less body weight, required more feed per gram of gain, drank more water, voided more urine, showed depigmentation of the labial surface of the incisor teeth, had less haemoglobin and serum inorganic phosphorous and had cleaned dried stomachs of lighter absolute weight. The erythrocyte count was higher in the MnSO4 group than in the basal or Na2SO4 groups.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study is provided as supporting information only
Principles of method if other than guideline:
90 day study to investigate the effects of drinking water with different sulfate contents.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: drinking water
Duration of treatment / exposure:
90 days
Frequency of treatment:
Water was provided ad libitum
Dose / conc.:
10 mg/L drinking water
Remarks:
low sulfate group
Dose / conc.:
280 mg/L drinking water
Remarks:
medium sulfate group
Dose / conc.:
1 595 mg/L drinking water
Remarks:
high sulphate group
No. of animals per sex per dose:
25
Details on study design:
Rats received drinking water with different sulfate contents.
Control group A received tap water containing 9 to 10 mg/L sulfate
Group B (low sulfate) received Bagats natural mineral water contaning less than 10 mg/L sulfate
Group C (medium sulfate) got Vittel Grande Source containing 280 mg/L sulfate
Group D (high sulfate) received Vittel Hepar with 1595 mg/L sulfate
Observations and examinations performed and frequency:
Food consumption and individual body weight were recorded weekly. Water consumption was measured daily. 20 male and 20 female rats of each group were fasted for 16 hours prior to blood sampling. Whole blood was analysed for red blood cells, white blo
od cells, haemoglobin, and haematocrit. Plasma analysis included blood urea nitrogen, glucose, triglycerides, total cholesterol, phospholipids and alkaline phosphatase.
Sacrifice and pathology:
All rats were inspected macroscopically and the weights of liver, kidneys, adrenals, brain, and testis recorded. Tissue samples were taken from stomach, duodenum, ileum, caecum, colon, kidneys, liver, adrenals, gonads, heart, lung, thyroid, pancreas, thymus, spleen, bladder and aorta.
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):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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:
not examined
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOEL
Effect level:
1 595 mg/L drinking water
Sex:
male/female
Basis for effect level:
other: no gastrointestinal, hematological, blood chemistry, histological effects
Critical effects observed:
no

Table 1: Body weight and food conversion efficiency (Mean value ± S.E.M)

Group

n

Males

Initial body weight

Body weight 4 weeks

Body weight 12 weeks

Food conversion efficiency

A

20

75.9 ± 1.2

284.9 ± 3.6

458.8 ± 7.0

0.35

B

20

75.7 ± 1.2

281.4 ± 4.7

445.8 ± 6.6

0.34

C

20

75.9 ± 1.2

276.9 ± 3.5

438.3 ± 6.5*

0.33

D

20

75.3 ± 1.2

282.3 ± 5.1

451.9 ± 9.3

0.33

Females

A

20

69.1 ± 1.5

181.2 ± 2.3

261.5 ± 3.2

0.25

B

20

68.6 ± 1.6

192.1 ± 3.9

269.3 ± 5.8

0.25

C

20

69.1 ± 1.5

188.8 ± 3.9

262.2 ± 5.8

0.25

D

20

68.5 ± 1.7

188.6 ± 3.9

266.8 ± 5.9

0.26

* = P 0.05

Table 2: Plasma chemistry (Mean value ± S.E.M)

Group

n

Males

Blood urea nitrogen mmol/l

Glucose mmol/l

Triglycerides

Cholesterol (total) mmol/l

Alkaline phosphatase mU/ml

Total protein g/l

A

20

6.29 ± 0.25

6.86 ± 0.15

1.09 ± 0.08

2.39 ± 0.13

59.0 ± 2.7

69 ± 0.6

B

20

6.24 ± 0.32

6.80 ± 0.11

0.96 ± 0.06

2.11 ± 0.10

56.8 ± 2.6

68 ± 0.8

C

20

5.63 ± 0.26

6.48 ± 0.13

1.08 ± 0.09

2.27 ± 0.11

54.0 ± 2.5

68 ± 0.5

D

20

5.79 ± 0.23

6.82 ± 0.23

0.98 ± 0.07

2.20 ± 0.08

58.1 ± 3.5

66 ± 0.6

Females

A

20

6.80 ± 0.32

6.85 ± 0.13

0.70 ± 0.07

2.36 ± 0.11

37.6 ± 2.5

72 ± 0.9

B

20

6.42 ± 0.18

6.86 ± 0.16

0.77 ± 0.09

2.33 ± 0.10

34.8 ± 2.5

70 ± 0.7

C

20

6.29 ± 0.32

6.53 ± 0.11

0.80 ± 0.07

2.38 ± 0.10

38.3 ± 2.6

72 ± 0.9

D

20

5.80 ± 0.20

7.21 ± 0.21

0.71 ± 0.09

2.37 ± 0.12

37.1 ± 2.0

69 ± 1.0

Table 3: Haematological results (Mean value ± S.E.M)

Group

n

Males

Red blood cells (10  6/mm3)

Haemoglobin (g%)

Haematocrit (%)

White blood cells (10  3/mm3)

Prothrombin time (s)

A

20

7.37 ± 0.08

15.9 ± 0.2

51 ± 0.5

10.9 ± 0.5

12.7 ± 0.1

B

20

7.26 ± 0.13

15.4 ± 0.1

49 ± 0.5

7.8 ± 0.6

12.3 ± 0.1

C

20

7.29 ± 0.10

15.8 ± 0.2

50 ± 0.6

7.1 ± 0.4

12.2 ± 0.1

D

20

7.29 ± 0.11

15.6 ± 0.2

50 ± 0.5

7.1 ± 0.4

12.2 ± 0.1

Females

A

20

6.76 ± 0.09

15.5 ± 0.1

49 ± 0.4

4.8 ± 0.3

12.6 ± 0.2

B

20

5.88 ± 0.38

15.0 ± 0.1

47 ± 0.5

4.1 ± 0.3

12.3 ± 0.1

C

20

6.57 ± 0.10

15.2 ± 0.1

48 ± 0.4

4.5 ± 0.2

11.9 ± 0.1

D

20

6.56 ± 0.10

14.8 ± 0.1

47 ± 0.3

3.6 ± 0.2

12.2 ± 0.2

Table 4: Absolute organ weights (Mean value ± S.E.M)

Group

n

Males

Liver (g)

Kidney (g)

Adrenals (mg)

Brain (g)

Testis

A

20

12.0 ± 0.3

2.8 ± 0.1

52 ± 2

1.6 ± 0.02

3.6 ± 0.0

B

20

11.5 ± 0.3

2.8 ± 0.1

55 ± 2

1.6 ± 0.03

3.4 ± 0.0

C

20

11.0 ± 0.3*

2.8 ± 0.1

56 ± 2

1.6 ± 0.02

3.5 ± 0.1

D

20

11.2 ± 0.3

2.9 ± 0.1

58 ± 2

1.5 ± 0.02

3.5 ± 0.1

Females

A

20

6.5 ± 0.1

1.7 ± 0.1

72 ± 2

1.4 ± 0.02

B

20

6.5 ± 0.1

1.7 ± 0.0

71 ± 3

1.5 ± 0.01

C

20

6.5 ± 0.1

1.7 ± 0.1

70 ± 3

1.4 ± 0.02

D

20

6.4 ± 0.1

1.7 ± 0.1

73 ± 2

1.5 ± 0.01

* = P 0.05

All treatments were well tolerated and promoted the expected growth. The determination of several haematological and blood chemistry parameters gave comparable results in both sexes of treated and control animals. Observation of the animals during the treatment period did not show any diseases or ill effects. Organ weights were in the physiological range and macroscopic inspection showed a completely normal pattern. Subsequent microscopic evaluation of the organs did not indicate any deleterious effects of the treatments. An observation of interest was a trend towards lower blood urea nitrogen (BUN) values with high sulfate intake. This observation however only became significant in later stages of survivmg rats. Taking into account the ten times higher liquid intake of rat compared to man and the continuous administration of the water it can be considered that drinking water containing sulfate in excess of 200 mg/L is safe.

Conclusions:
The study provides evidence that low, medium and high sulphate content in drinking water do not cause any gastro-intestinal disturbance and the hematological examinations, blood chemistry and the histopathological evaluation confirmed the absence of any deleterious effects.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards with acceptable restrictions.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male sprague-Dawley rats were fed diets containing 0.06 % 3'methyl-4-dimethylaminoazobenzene (3'MeDAB) plus 0.84 % Na2SO4 or a diet containing Na2SO4 for 41 or 27 weeks, with intermittent breaks on basal diet.
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Mean weight: 211.6 g
Source: random-bred departmental stock
Housing: 5 to a cage
Water: ad libitum
Diet: pair-fed by a modified method whereby the total food consumption of each cage of rats was restricted to that of the cage of rats consuming the least.
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
Rats from each experimental group were started on test diets at two different times (Series A and B). The rats were pair fed one of the test diets for 16 consecutive weeks, then returned to the basal diet for 8 weeks. At this time 2 rats from each of the groups on the 3'MeDAB-containing diets and one each from the control or Na2SO4-containing diets in Series A were killed and the livers examined. The remaining rats were then returned to the various experimental diets for several 4-week periods, with a week between each period, during which they were fed basal diets.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
Series A - 41 weeks
Series B - 27 weeks
Frequency of treatment:
Daily for 16 consecutive weeks, followed by basal diet for 8 weeks after which rats were returned to the experimental diet for several 4-week periods with a week in between each during which they returned to basal diet.
Dose / conc.:
0.84 other: %
Remarks:
0.84 % (8.4 g/kg) NA2SO4


No. of animals per sex per dose:
Series A:
Na2SO4 = 5 males/dose
3'MeDAB = 15 males/dose
3'MeDAB with Na2SO4 = 15 males/dose

Series B:
Na2SO4 = 5 males/dose
3'MeDAB = 5 males/dose
3'MeDAB with Na2SO4 = 5 males/dose
Control animals:
other: plain diet with maize oil
Details on study design:
The progression with time of the cumulative probability of observing liver tumours of 1 cm or more in diameter, evidence of metastatis spread or multiple tumours at death was calculated by actuarial method. The significance of differences between the numbers of rats bearing tumours of at least 1 cm diameter or with metastases in the groups fed 3'MeDAB-containing diets was compared by Fisher's exact test as the expected frequencies in any one cell were less than 5 in each instance. The correlation coefficient was computed in the standard manner.
Positive control:
No data
Observations and examinations performed and frequency:
Body weights were noted at the conclusion of the test (see table).
Sacrifice and pathology:
Liver lesions greater than 0.2 cm in diameter were fixed in acetic acid:formalin:ethanol (5:10:85) for 24 h, then transferred to 70 % ethanol (v/v) prior to processing to paraffin.
Liver and spleen weights were recorded. In some instances haemoglobin estimates were performed. Samples of the livers and any macroscopic lesions in other organs were fixed and processed. Liver and lung sections were routinely stained with haematoxylin and eosin and by the periodic acid-Schiff (PAS) method. In some instances alcian blue or toluidine blue stains were also used.
Other examinations:
No data
Statistics:
The progression with time of the cumulative probability of observing liver tumours of 1 cm or more in diameter, evidence of metastatic spread or multiple tumours at death was calculated by an actuarial method. The significance of differences between the numbers of rats bearing tumours of at least 1 cm diameter or with metastases in the groups fed 3'MeDAB-containing diets was compared by Fisher's exact test as the expected frequencies in any one cell were less than 5 in each instance. The correlation coefficient was computed in the standard manner.
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):
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
No significant differences in overall body weight gain or in liver weight were noted between the various experimental groups at the conclusion of the experiment, with the exception of livers bearing tumours which on occassion weighed as much as 82.1 g. The livers of many rats which survived at least 16 weeks on diets containing 3'MeDAB or 3'MeDAB plus Na2SO4 were finely granular in appearance and showed microscopic evidence of mild cirrhosis; local areas of hyperplasia or dyplasia were also frequently observed. These comprised discrete localised alterations in the cells and could take the form of groups of deeply basiphilic cells which sometimes showed a tubular appearance, cells with vacuolated cytoplasm, large cells with a normal nucleocytoplasmic ratio and hyperchromatic pleomorphic nuclei and cells which showed pleomorphism and variations in the nucleocytoplasmic ratio. These areas often contained less PAS positive material than the surrounding liver but in some instances PAS staining was increased. Areas of cells which were basiphilic when stained with haematoxylin and eosin were hyperbasiphilic when stained with toluidine blue. It was shown that the addition of Na2SO4 to the diet shortened the latent period but did not affect the rate of tumour development. The relative risk of developing multiple metastatic tumours respectively were also increased if the 3'MeDAB diet was supplemented with Na2SO4. Tumours attaining 1 cm or more in diameter were usualy either hepatocarcinomas or cholangiocarcinomas with some tumours showing areas of both hepatocarcinoma and cholangiocarcinoma within the one nodule.
Because of variations in the microscopic appearance in sections made from different areas of the same tumour, no attempt was made to classify tumours exclusively as either hepatocarcinoma or cholangiocarcinoma. Heptaocarcinomas frequently show areas with either a trabecular or an acinar pattern and if this observation is taken into account the majority of liver tumours induced in this experiment should probably be regarded as being in this category, which is in agreement with the relatively low incidence of cholangiofibrosis noted in this study, which has previously been indicated as a precursor of chloangiocarcinoma.
Tumours were found in all lobes of the liver, but a detailed analysis of the relative frequency of tumours in the various lobes was not undertaken. All carcinomas observed contained much less PAS positive material than the surrounding liver, and were hyperbasiphilic when stained with toluidine blue.
Metastases within the peritoneal cavity were observed in 7 of the 11 rats fed 3'MeDAB plus Na2SO4 which had liver carcinomas 1 cm or more in diameter, and in 4 of these rats multiple foci of secondary tumour were present in the lungs. In one rat fed 3'MeDAB plus Na2SO4, the entire contents of the peritoneal cavity had to be removed en bloc because of diffuse infiltration by tumour tissue. In contrast peritoneal metastases was only observed in one of the six rats fed 3'MeDAB which had liver carcinomas of this size, and pulmonary metastases were not present.
Many nodules smaller than 1 cm diameter were observed in the livers of rats fed either 3'MeDAB or 3'MeDAB plus Na2SO4 diets. No abnormalities were noted in the livers of rats on the control or Na2SO4 diets.
Microscopic analysis of nodules greater than 0.2 cm diameter were present in the livers. Many of the lesions less than 1 cm diameter, particularly in the group receiving 3'MeDAB alone were hyperplastic liver nodules. In many instances, both hyperplastic nodules and carcinoma were present in the same liver but there were relatively more rats with carcinoma and no hyperplasia nodules in the group which had been fed 3'MeDAB plus Na2SO4. In contrast there were more rats in which hyperplastic liver nodules represented the most advanced lesion in the group fed 3'MeDAB. This overlap in the incidence of hyperplastic nodules and carcinoma and the lower frquency of hyperplastic nodules relative to carcinoma in rats receiving 3'MeDAB plus Na2SO4, which had a significantly greater risk of developing metastasizing carcinomas supports the idea that hyperplastic nodules are precursors of carcinoma.
There were no significant differences in the mean number of tumours/tumour-bearing rats between groups which were fed either 3'MeDAB alone, or 3'MeDAB plus Na2SO4. This observation was true regardless of whether all those tumours ≥ 1 cm or ≥ 0.2 cm (3'MeDAB-fed group: 6.077 ± 3.904 (S.D.); 3'MeDAB plus Na2SO4-fed group: 7.33 ± 4.470) in diameter were considered. It therefore seems likely that the enhancement of 3'MeDAB carcinogenesis by dietary Na2SO4 is due to a decrease in the latent period rather than an increase in the number of transformed foci.
Dose descriptor:
other: increased probability of developing liver tumours
Effect level:
8.4 other: g/kg
Sex:
male
Basis for effect level:
other: Increased liver tumours in rats fed diets containing 0.84 % test material
Critical effects observed:
not specified

Groups used, initial body weights and body weights and liver weights of rats killed at the conclusion of the experiment:

Dietary groups

Series*

No. of rats/

group

Effective

no. of rats/

group†

Starting weight

(g)

mean ± S.D.

No. of rats

surviving the

duration of

the experiment

Terminal body weight (g)

mean ± S.D. or range ‡

Terminal liver weight (g)

mean ± S.D. or range‡

Control

A

5

5

230.8 ± 18.6

2

358 (345 - 372)

10.11 (9.94 – 10.27)

B

5

5

193.4 ± 33.6

5

358.4 ± 16.0

10.20 ± 0.49

Na2SO4

A

5

5

252.0 ± 30.2

3

414.7 ± 39.5

14.30 ± 1.42

B

5

5

194.2 ± 35.7

5

332.0 ± 53.7

10.03 ± 0.99

3’MeDAB

A

15

14

234.0 ± 29.4

6

351.3 ± 46.1

29.55 ± 21.17

B

5

5

200.8 ± 20.7

2

334.5 (328 - 341)

40.97 (20.76 – 61.18)

3’MeDAB and Na2SO4

A

15

14

231.0 ± 32.7

3

346.3 ± 37.5

26.06 ± 1.87

B

5

5

181.8 ± 26.6

1

3610.

28.23

* rats in Series and Series B were started on the dietary regimens at different times; all rats remaining in series A were killed after 41 weeks and those remaining in Series B were killed after 27 weeks.

† animals dying during the experiment which could not be autopsied because of cannibalism or advanced autolysis were excluded from the effective number of rats/group.

‡ these data were derived from rats which survived for the duration of the experiment (either 27 weeks for Series B or 41 weeks for Series A).

Showing incidence of hyperplastic and/or dyplastic change, cholangiofibrosis and mild cirrhosis in livers of rats surviving at least 16 weeks on the various regimens and either found dead or killed at subsequent stages of the experiment.*

Dietary

groups

No. of rats

Surviving for

16 weeks

No

change

Mild

cirrhosis†

Areas of

 hyperplasia‡

and dyplasia

Areas of

cholangiofibrosis§

Control

10

10

-

-

-

Na2SO4

10

10

-

-

-

3’MeDAB

16

-

10 (62.5 %)

15 (93.8 %)

2 (12.5 %)

3’MeDAB

and Na2SO4

18

-

17 (94.4 %)

17 (94.4 %)

5 (27.8 %)

* some rats receiving diets containing 3'MeDAB or 3'MeDAB and Na2SO4 which died in the first weeks of the experiment showed a periportal round cell infiltrate and in a few instances, fatty change.

† the external surface of these livers was observed to be finely granular and an increase in periportal connective tissue was observed microscopically; this occassionally connected adjacent portal triads.

areas of basiphilic cells discrete collections of vacuolated cells or areas of large cells with large nuclei.

§ based on microscopic findings.

Effect of dietary suplementation wtih sodium sulphate on tumour induction by 3'MeDAB in the liver of male Sprague-Dawley rats*

Dietary

groups

No. of rats

Surviving for

16 weeks

Weeks to

first

tumour†

Other rats alive

at time first

tumour found

No. of rats developing

tumours by conclusion

of experiment

Mean no. of

tumours/tumour-

bearing rats

(± S.D.)

Total tumours

in tumour-

bearing rats

Microscopic

findings in

tumour-

bearing

rats‡

Rats with metastases

Hyperplastic nodule

Carcinoma

Control

10

-

-

-

-

-

-

-

-

Na2SO4

10

-

-

-

-

-

-

-

-

3’MeDAB

16

27

11

6

3.83 ± 2.14

23

-

6

1

3’MeDAB

and Na2SO4

18

17

17

12

3.25 ± 2.05

39

1

11

7

* the data presented represents pooled results from Series A and Series B and includes data from all rats either found dead or killed subsequent to the first 16 weeks of the experiment.

† the term tumour refers to a macroscopic nodule ≥ 1 cm diameter.

‡ the results represent the most advanced lesion present.

§ a subcutaneous fibrosarcoma was found in a rat in this group which was killed at 33 weeks. The inhibitionof liver tumour induction when acetanilide was added to the 3'MeDAB-containing diet was significant (P < 0.01, Fisher's exact test). There were metastases present in the peritoneal cavity in all of these rats and in addition there were metastases present in the lungs in 4 rats in the group receiving 3'MeDAB and Na2SO4. There was a significant increase in the number of rats with metastases when the diet containing both 3'MeDAB and Na2SO4 was fed rather than the 3'MeDAB diet ( P < 0.05, Fisher's exact test).

Conclusions:
No abnormalities were noted in the livers of rats fed a diet of 0.84% Na2SO4. Also no significant difference in overall body weight gain or in liver weight were noted between the treatment and control.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Japnese paper .The data is from a literature abstract and results tables which are in English
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 13-week subchronic oral toxicity study of ammonium sulfate was performed on F344 rats by feeding them a CRF-1 powder diet containing concentrations of 0 %, 0.38 %, 0.75 %, 1.5 % and 3.0 % of the susbstance. rats were randomly divided into 5 groups consisting of 10 males and 10 females.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
None specified
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
A 13-week subchronic oral toxicity study of ammonium sulfate was performed on F344 rats by feeding them a CRF-1 powder diet containing concentrations of 0 %, 0.38 %, 0.75 %, 1.5 % and 3.0 % of the susbstance.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Not specified
Dose / conc.:
0 other: % nominal in diet
Dose / conc.:
0.38 other: % nominal in diet
Dose / conc.:
0.75 other: % nominal in diet
Dose / conc.:
1.5 other: % nominal in diet
Dose / conc.:
3 other: % nominal in diet
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Not specified
Positive control:
None specified
Observations and examinations performed and frequency:
Not specified
Sacrifice and pathology:
Not specified
Other examinations:
Serum biochemistry, histopathology, body weight, organ weight and haematology were examined.
Statistics:
None specified
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Male animals in the 3 % group exhibited diarrhoea during the administration period. No changes indicating obvious ammonium sulfate toxicity were observed in the body weights, organ weights, haematological , serum biochemical or histopathological examinations.
Dose descriptor:
NOEL
Effect level:
886 mg/kg bw/day (nominal)
Sex:
male
Dose descriptor:
NOEL
Effect level:
1 975 mg/kg bw/day (nominal)
Sex:
female
Dose descriptor:
other: Maximally tolerated dose
Effect level:
3 other: %
Sex:
not specified
Remarks on result:
other: MTD for 2-year carcinogenicity studies
Critical effects observed:
no

Body weight, food intake and chemical intake in rats containing ammonium sulfate for 13 weeks:

Group ( % )

Final body

weight (g)

Food intake

(g/rat/day)

Total chemical Intake

(mg/kg/day)

Male

0

297.8 ± 13.0

14.2

0

0.38

2763.4 ± 14.8

14.0

222

0.75

266.7 ± 21.4

14.36

441

1.5

282.0 ± 17.7

14.1

836

3.0

283.9 ± 15.2

13.8

1792

Female

0

150.8 ± 7.5

9.2

0

0.38

157.0 ± 9.6

9.1

239

0.75

151.6 ± 7.7

9.3

484

1.5

161.2 ± 8.0

9.3

961

3.0

158.1 ± 10.4

8.4

1975

a) Mean ± S.D.

* Significantly different from the control at p < 0.05.

Haematological changes in male rats fed diet containing ammonium sulfate for 13 weeks:

Dose level

0

0.38

0.75

1.5

3.0

No. of animals

10

10

10

10

10

RBC

1012/L

8.87 ± 0.20a)

9.36 ± 2.4*

9.09 ± 0.41

9.13 ± 0.51

8.93 ± 0.29

Hb

g/dl

14.8 ± 0.3

15.6 ± 0.5*

15.2 ± 0.7

15.2 ± 0.8

15.0 ± 0.5

PCV

%

43.1 ± 1.0

45.5 ± 1.3*

44.4 ± 2.0

44.5 ± 2.4

43.6 ± 1.3

MCV

Fl

48.5 ± 0.3

48.6 ± 0.5

48.9 ± 0.4*

48.7 ± 0.2

48.9 ± 0.3

MCH

Pg

16.7 ± 0.2

16.4 ± 0.3*

16.7 ± 0.2

16.7 ± 0.1

16.8 ± 0.2

MCHC

g/dl

34.4 ± 0.4

34.2 ± 0.8

34.1 ± 0.4

34.2 ± 0.3

34.4 ± 0.3

Plt

1012/L

0.73 ± 0.03

0.80 ± 0.05*

0.77 ± 0.05*

0.74 ± 0.03

0.73 ± 0.04

Ebl

Count/200 WBC

1.2 ± 1.1

0.8 ± 0.9

1.92 ± 2.0

1.2 ± 1.4

0.8 ± 0.9

WBC

109/L

3.85 ± 0.3

3.16 ± 0.63*

2.94 ± 0.47*

3.00 ± 0.37*

3.36 ± 0.20*

Differential cell count (%)

Eosino

0.8 ± 0.5

1.1 ± 0.3

1.0 ± 1.0

0.9 ± 0.5

1.0 ± 0.7

Neut-S

22.2 ± 2.2

22.2 ± 5.2

26.6 ± 5.2*

24.7 ± 5.4

25.3 ± 5.4

Lympho

74.6 ± 2.4

74.6 ± 5.7

71.9 ± 5.3

71.0 ± 10.4

72.4 ± 4.9

Mono

1.2 ± 1.1

1.2 ± 0.9

0.9 ± 0.7

0.8 ± 0.4

1.3 ± 0.9

Haematological changes in female rats fed diet containing ammonium sulfate for 13 weeks:

Dose level

0

0.38

0.75

1.5

3.0

No. of animals

10

10

10

10

10

RBC

1012/L

9.34 ± 0.32a)

8.64 ± 0.18*

8.6 ± 0.31*

8.90 ± 0.27*

8.72 ± 0.34*

Hb

g/dl

16.3 ± 0.5

15.2 ± 0.4*

15.3 ± 0.5*

15.8 ± 0.5*

15.5 ± 0.6*

PCV

%

47.7 ± 1.4

44.5 ± 0.7*

44.2 ± 1.5*

45.3 ± 1.5*

44.1 ± 1.6*

MCV

Fl

51.1 ± 0.9

51.5 ± 0.4

51.3 ± 0.5*

52.8 ± 0.4*

50.6 ± 0.2

MCH

pg

17.5 ± 0.2

17.5 ± 0.1

17.7 ± 0.3*

17.7 ± 0.2*

17.7 ± 0.2*

MCHC

g/dl

34.2 ± 0.4

34.1 ± 0.5

34.7 ± 0.5*

34.9 ± 0.3*

35.1± 0.3*

Plt

1012/L

0.95 ± 0.05

0.84 ± 0.05*

0.77 ± 0.03*

0.83 ± 0.04*

0.81 ± 0.03*

Ebl

Count/200 WBC

2.5 ± 1.3

2.5 ± 2.0

2.8 ± 1.5

3.0 ± 2.4

4.6 ± 2.8

WBC

109/L

3.85 ± 0.36

3.71 ± 0.32

3.27 ± 0.44*

3.35 ± 0.51*

3.45 ± 0.51

Differential cell count (%)

Eosino

0.9 ± 0.8

0.8 ± 0.8

1.0 ± 0.8

1.1 ± 0.6

0.8 ± 0.3

Neut-S

16.3 ± 4.5

13.8 ± 5.2

17.7 ± 5.1

16.0 ± 3.7

14.4 ± 2.2

Lympho

81.1 ± 5.4

74.6 ± 5.7

79.3 ± 6.1

81.2 ± 3.6

82.8 ± 2.0

Mono

1.6 ± 1.6

1.2 ± 0.9

1.8 ± 1.2

2.1 ± 1.7

1.7 ± 1.4

a) Mean ± S.D.

* Significantly different from the control at p < 0.05.

Serum biochemistry in male rats trated with ammonium sulfate for 13 weeks:

Dose level (%)

0

0.38

0.75

1.5

3.0

No. of animals

10

10

10

10

10

TP

g/dl

6.4 ± 0.14a)

6.28 ± 0.11*

6.35 ± 0.17

6.34 ± 0.20

6.48 ± 0.18

A/G

 

2.4 ± 0.18

2.68 ± 0.27*

2.48 ± 0.25

2.52 ± 0.28

2.36 ± 0.11

Alb

g/dl

4.52 ± 0.18

4.56 ± 0.14

4.51 ± 0.10

4.53 ± 0.08

4.54 ± 0.11

TCho

mg/dl

61 ± 6

69 ± 5*

73 ± 11*

73 ± 11*

66 ± 7

BUN

mg/dl

18.9 ± 2.58

17.22 ± 2.15*

16.61 ±1.35*

16.61 ±1.18*

17.4 ±1.01

Na

mEQ/L

145 ± 1

146 ± 1*

146 ± 1*

146 ± 1*

146 ± 1*

Cl

mEQ/L

103 ± 0.8

107 ± 1.0*

106 ± 0.7*

105 ± 0.8*

103 ± 0.8

K

mU/dl

4.42 ± 0.17

4.56 ± 0.18

4.73 ±0.19*

4.79 ± 0.14*

4.60 ± 0.25

Ca

mg/dl

10.1 ± 0.1

10.1 ± 0.3

10.1 ± 0.2

9.9 ± 0.2*

10.1 ± 0.2

P

mg/dl

5.9 ± 0.4

6.0 ± 0.4

5.7 ± 0.6

5.6 ± 0.3*

5.3 ± 0.4*

GOT

IU/L

83 ± 11

68 ± 8*

75 ± 6

83 ± 6

77 ± 5

GPT

IU/L

48 ± 8

46 ± 7

61 ± 9*

67 ± 10*

50 ± 4*

ALP

IU/L

410 ± 28

294 ± 59*

295 ± 76*

288 ± 81*

390 ± 21

a) Mean ± S.D.

* significantly different from the control at P < 0.05.

Serum biochemistry in male rats trated with ammonium sulfate for 13 weeks:

Dose level (%)

0

0.38

0.75

1.5

3.0

No. of animals

10

10

10

10

10

TP

g/dl

6.81 ± 0.15a)

6.13 ± 0.11

6.12 ± 0.19

6.14 ± 0.14

6.11 ± 0.17*

A/G

 

3.05 ± 0.20

2.83 ± 0.27

2.87 ± 0.19

2.70 ± 0.18*

2.70 ± 0.22*

Alb

g/dl

4.65 ± 0.11

4.52 ± 0.14*

4.52 ± 0.12*

4.48 ± 0.09*

4.45 ± 0.14*

TCho

mg/dl

93 ± 8

94 ± 5

94 ± 8

87 ± 6

87 ± 8

BUN

mg/dl

21.14 ± 1.32

19.45 ± 1.90*

21.44 ± 1.70

19.65 ±1.20

19.87 ± 2.64

Na

mEQ/L

146 ± 0.9

145 ± 0.8*

146 ± 1

145 ± 0.5

146 ± 1

Cl

mEQ/L

108 ± 0.9

107 ± 1.2

107 ± 0.5*

107 ± 0.5*

106 ± 1.2*

K

mU/dl

4.14 ± 0.18

4.15 ± 0.31

4.20 ±0.22

4.20 ± 0.17

4.17 ± 0.26

Ca

mg/dl

9.9 ± 0.2

10.1 ± 0.2

9.9 ± 0.2

9.9 ± 0.2

10.1 ± 0.3

P

mg/dl

5.6 ± 0.3

5.8 ± 0.4

5.6 ± 0.3

5.8 ± 0.3

6.0 ± 0.5*

GOT

IU/L

86 ± 11

68 ± 5*

74 ± 4*

76 ± 3*

69 ± 5*

GPT

IU/L

45 ± 7

36 ± 4*

37 ± 4*

39 ± 2*

39 ± 3*

ALP

IU/L

240 ± 14

255 ± 35

227 ± 16

287 ± 25*

278 ± 27*

a) Mean ± S.D.

* significantly different from the control at P < 0.05.

Organ weight of male rats treated with ammonium sulfate for 13 weeks:

Parameter

Dose Level (%)

0

0.38

0.75

1.5

3.0

Body weight (g)

297.8 ± 13.0

273 ± 14.8*

286 ± 21.4

282.0 ± 17.7*

283.9 ± 15.2*

Absolute (g)

Brain

1.932 ± 0.061a)

1.903 ± 0.034

1.907 ± 0.062

1.932 ± 0.034

1.905 ± 0.040

Lung

0.930 ± 0.078

0.921 ± 0.056

0.942 ± 0.093

0.905 ± 0.056

0.893 ± 0.076

Heart

0.847 ± 0.049

0.806 ± 0.046

0.855 ± 0.080

0.848 ± 0.061

0.838 ± 0.059

Spleen

0.561 ± 0.034

0.522 ± 0.023*

0.554 ± 0.044

0.540 ± 0.052

0.516 ± 0.044*

Liver

7.051 ± 0.037

5.702 ± 0.497*

6.039 ± 0.871*

6.082 ± 0.879*

6.834 ± 0.393

Adrenal

0.038 ± 0.006

0.040 ± 0.002

0.036 ± 0.003

0.041 ± 0.004

0.036 ± 0.004

Kidneys

1.804 ± 0.063

1.716 ± 0.088*

1.819 ± 0.155

1.873 ± 0.139

1.956 ± 0.121*

Testis

2.952 ± 0.116

3.043 ± 0.107

3.083 ± 0.086

3.019 ± 0.075

3.052 ± 0.127

Relative (g/100g b.w.)

Brain

0.649 ± 0.025

0.698 ± 0.035*

0.668 ± 0.046

0.687 ± 0.038*

0.673 ± 0.042

Lung

0.312 ± 0.022

0.338 ± 0.030*

0.329 ± 0.022

0.322 ± 0.023

0.314 ± 0.016

Heart

0.284 ± 0.012

0.296 ± 0.023

0.298 ± 0.021

0.301 ± 0.011*

0.295 ± 0.011

Spleen

0.188 ± 0.007

0.192 ± 0.016

0.193 ± 0.008

0.191 ± 0.010

0.182 ± 0.011*

Liver

2.367 ± 0.070

2.088 ± 0.187*

2.097 ± 0.160*

2.147 ± 0.187*

2.408 ± 0.078

Adrenal

0.013 ± 0.002

0.014 ± 0.001

0.013 ± 0.001

0.014 ± 0.002

0.013 ± 0.002

Kidneys

0.606 ± 0.024

0.629 ± 0.037

0.634 ± 0.015*

0.664 ± 0.025*

0.689 ± 0.025*

Testis

0.962 ± 0.039

1.115 ± 0.057*

1.079 ± 0.062*

1.074 ± 0.069*

1.077 ± 0.058*

a) mean ± S.D.

* Significantly different from the control at P < 0.05

Organ weight of female rats treated with ammonium sulfate for 13 weeks:

Parameter

Dose Level (%)

0

0.38

0.75

1.5

3.0

Body weight (g)

150.8 ± 75

157.0 ± 96

151.6 ± 7.7

161.2 ± 8.0

158.1 ± 10.4

Absolute (g)

Brain

1.770 ± 0.051a)

1.771 ± 0.044

1.766 ± 0.052

1.776 ± 0.029

1.773 ± 0.039

Lung

0.708 ± 0.066

0.683 ± 0050

0.717 ± 0.121

0.699 ± 0.037

0.692 ± 0.051

Heart

0.515 ± 0.045

0.542 ± 0.037

0.515 ± 0.032

0.528 ± 0.021

0.545 ± 0.042

Spleen

0.348 ± 0.020

0.361 ± 0.032*

0.351 ± 0.018

0.369 ± 0.015

0.370 ± 0.027

Liver

3.043 ± 0.215

3.366 ± 0.254*

3.189 ± 0.157

3.460 ± 0.139

3.357 ± 0.301

Adrenal

0.040 ± 0.005

0.039 ± 0.005

0.040 ± 0.004

0.039 ± 0.004

0.039 ± 0.004

Kidneys

1.000 ± 0.057

1.041 ± 0.078

1.015 ± 0.050

1.081 ± 0.030

1.126 ± 0.065*

Relative (g/100g b.w.)

Brain

1.175 ± 0.046

1.129 ± 0.047*

1.166 ± 0.063

1.104 ± 0.050*

1.125 ± 0.066

Lung

0.469 ± 0.054

0.435 ± 0.034

0.476 ± 0.095

0.435 ± 0.030

0.438 ± 0.031

Heart

0.341 ± 0.017

0.346 ± 0.022

0.340 ± 0.020

0.328 ± 0.019

0.345 ± 0.020

Spleen

0.231 ± 0.009

0.230 ± 0013

0.232 ± 0.020

0.229 ± 0.009

0.235 ± 0.020

Liver

2.016 ± 0.075

2.142 ± 0.095*

2.104 ± 0.081*

2.149 ± 0.073*

2.237 ± 0.096*

Adrenal

0.027 ± 0.004

0.025 ± 0.003

0.027 ± 0.002

0.035 ± 0.002*

0.025 ± 0.002

Kidneys

0.663 ± 0.020

0.663 ± 0.033

0.670 ± 0.033

0.672 ± 0.030

0.713 ± 0.017*

a) Mean ± S.D.

* Significantly different from the control at P < 0.05.

Conclusions:
Based on these results, the NOEL of ammonium sulfate for F344 rats was judged to be 1.5 % in males (886 mg/kg/day) and 3 % in females (1975 mg/kg/day) and the maximally tolerated dose for 2-year carcinogenicity studies in F344 rats was concluded to be 3.0% or more in the diet.
Executive summary:

A 13-week subchronic oral toxicity study of ammonium sulfate was performed on F344 rats by feeding them a CRF-1 powder diet containing concentrations of 0 %, 0.38 %, 0.75 %, 1.5 % and 3.0 % of the susbstance. Rats were randomly divided into 5 groups each consisting of 10 males and 10 females. Male animals in the 3 % group exhibited diarrhoea during the administration period. No changes indicating obvious ammonium sulfate toxicity were observed in the body weights, organ weights, haematological , serum biochemical or histopathological examinations. Based on these results, the NOEL of ammonium sulfate for F344 rats was judged to be 1.5 % in males (886 mg/kg/day) and 3 % in females (1975 mg/kg/day) and the maximally tolerated dose for 2-year carcinogenicity studies in F344 rats was concluded to be 3.0% or more in the diet.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was not performed according to GLP and while the test parameters are well documented and scientifically acceptable, no guideline is specified.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Chronic toxicity studies of ammonium sulfate were performed in male and female rats in a 52-week toxicity study.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals and environmental conditions:
A total of 190 male and 190 female Fischer 344/DuCrj rats were purchased at 5 weeks of age from Charles River Japan and acclimated for 1 week prior to the commencement of the experiment. Rats were allocated 10 animals/sex/group for the chronic toxicity study and 50 animals/sex/group for assessment of carcinogenicity and maintained in a room with a barrier system under constant conditions: temperature 24 ± 1ºC, relative humidity 55 ± 5 %, ventilation frequency of 18 times/h and 12-h light/dark cycle. The animals were housed 3 or 4 rats per plastic cage on sterilized soft wood chips and were given free access to tap water and diet.
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
Ammonium sulfate was mixed at the specified concentrations into powdered basal diet, CRF-1, and then pelletted. Recapture rates for ammonium sulfate from the admixed diet at each concentration level were confirmed to be 95.4 - 98.7 %. Stability of ammonium sulfate in the solid pellet diet was evaluated and no decomposition was confirmed after storage.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not specified
Duration of treatment / exposure:
52 weeks and 104 weeks
Frequency of treatment:
Daily
Dose / conc.:
0 other: % nominal in diet
Remarks:
52 week chronic toxicity
Dose / conc.:
0.1 other: % nominal in diet
Remarks:
52 week chronic toxicity
Dose / conc.:
0.6 other: % nominal in diet
Remarks:
52 week chronic toxicity
Dose / conc.:
3 other: % nominal in diet
Remarks:
52 week chronic toxicity
Dose / conc.:
0 other: % nominal in diet
Remarks:
104 week carcinogenicity test.
Dose / conc.:
1.5 other: % nominal in diet
Remarks:
104 week carcinogenicity test.
Dose / conc.:
3 other: % nominal in diet
Remarks:
104 week carcinogenicity test.
No. of animals per sex per dose:
10 animals/sex/group for the chronic toxicity and 50 animals/sex/group for the assessment of carcinogenicity.
Control animals:
yes, plain diet
Details on study design:
Doses were selected on the basis of a previous 13-week dietary study during which rats were administered diets containing 0 %, 0.38 %, 0.75 % and 3.0 % ammonium sulfate during which the NOAEL was estimated to be 1.5 % in males and 3.0 % in females.
Positive control:
None specified
Observations and examinations performed and frequency:
Clinical signs and mortality were observed daily
Body weight and food consumption were recorded every 2 weeks until week 10 and every 5 weeks thereafter.
Sacrifice and pathology:
All survivors were euthanized. In the chronic toxicity study animals were fasted over night prior to the terminal necrscopy and blood samples were collected from the abdominal aorta at the time of sacrifice. Haematological examinations were performed and the following parameters determined: red blood cell count, haemaglobin concentration, hematocrit, MCV, MCH, MCHC, platelet count and WBC. Differential leukocyte counts and the reticulocyte count were also obtained.
Serum biochemistry was performed for: total protein, albumin, albumin/globulin ration, total bilirubin, total cholesterol, triglyceride, blood urea nitrogen, creatinine, calcium, inorganic phsophorus, sodium, potassium, chloride, aspartate transaminase, alanin transaminase, alkaline phosphatase and γ-glutamyl transpeptidase.
All animals were subjected to complete necroscopy. Those that died or became moribund during the treatment period were also subjected to a complete necroscopy as soon as they were found. Brain, lungs, heart, spleen, liver, adrenals, kidneys and testes were weighed. As for adrenals, kidneys and testes, weights of each side were recorded separately and the total of both sides was used for calculation of group mean and SD values. In addition to these organs, the nasal cavity, trachaea, aorta, pituitary, thyroids, parathyroids, salivary glands, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, urinary bladder, epididymides, prostate, seminal vesicels, ovaries, uterus, vagina, mammary glands, skin, mesenteric and submandibular lymph nodes, thymus, sternum, femur including bone marrow, sciatic nerve, trigeminal nerve, spinal cord, eye, Hardarian gland and thigh muscle were excised and specimens from all these organs or tissues were fixed in 10 % buffered formalin or paraffin-embedding sectioning and staining with hematoxylin and eosin for histopathological examination.
All organs and tissues in the theological examination. All organs and tissues in the control and 3 % group animals were histopathologically examined. Additionally, macroscopically abnormal sites in the 0.1 % and 0.6 % group animals in the chronic study and all organs and tissues of the 1.5 % animals in the carcinogenicity study were also histopathologically examined.
Other examinations:
None specified
Statistics:
Variance in data for body weighs, haematology, serum biochemistry and organ weights was checked for homogeneity by the Bartlett test. When the data were homogenous, one-wy ANOVA was used. In the heterogenous cases, the Kruskal-Wallis test was applied. When statistically significant indices were indicated, Dunnett's multiple test was employed for comparison between control and treated groups. Final survival rates and the incidences of tumors and non-neoplastic lesions were compared with the Fisher's exact probability test or the Mann-Whitney's U-test.
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):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Mortality:
In the chronic toxicity study, no mortality was found in any group throughout the treatment period. In the carcinogenicity study, the survival rate of control, 1.5 % and 3.0 % groups were 88 % , 78 % & 76 % respectively for males and 76 %, 80 % & 80 % respectively for females and no significant difference was observed among the groups.

General conditions, body weight, food consumption and chemical intake:
No obvious findings, including diarrhoea were observed in any group in either the chronic toxicity or carcinogenicity studies. No test substance-related change in the body weights or food intake was found, except for a tendency for increase of food intake in the male 3.0 % group in the chronic toxicity study. The actual daily intake of the test substance showed a good correlation with expected doses.

Haematology and serum biochemistry:
No significant variation was found in erythrocytic parameters and Plt among the groups in the chronic toxicity study. Some slight changes were found in WBC paramters but there was no dose-relation. No dose-related alteration was found in any of the serum biochemical parameters in the chronic toxicity study.

Organ weights:
Absolute and relative kidney weights were increased or showed a tendency for increase at 3.0 % in both sexes in the chronic toxicity study. Absolute spleen weights were decreased and relative liver weights were increased in the 3.0 % male dose group. No dose-related changes were found in the other organs.

Necropsy and histopathology:
There were no obvious macroscopic findings in any group in either the chronic toxicity or carcinogenicity studies, except for massive, nodular or focal lesions suggesting neoplastic change in the carcinogenicity study. Histopathologically, in the chronic toxicity study, several non-neoplastic lesions, such as bile duct proliferation in the liver and focal myocarditis in the heart were noted in the control and 3.0 % group, and there were no significant differences in their incidences between the groups in either sex. As neoplastic lesions, malignant pheochromocytoma of the adrenal in the male 3.0 % group, two adenomas in the anterior pituitary in females of the 3.0 % group and uterine endometrial stromal polyp in a female control rat were noted. In the carcinogenicity study, as a non-neoplastic lesion, incidence of chronic nephropathy in the kidney was significantly increased in the male 1.5 % group. As for other lesions, there were no significant inter-group differences in their incidences or severity. Neoplastic lesions are known to occur in this strain and neither increases in their incidences nor specific types of lesions were observed in the test-substance-adminsitered groups.
Dose descriptor:
NOAEL
Effect level:
256 mg/kg bw/day (nominal)
Sex:
male
Remarks on result:
other: result basedon on NOAEL at 0.6% diet
Dose descriptor:
NOAEL
Effect level:
284 mg/kg bw/day (nominal)
Sex:
female
Remarks on result:
other: result basedon on NOAEL at 0.6% diet
Critical effects observed:
no

Final body weight, food consumption and the amount of ammonium sulfate intake in rats fed diet containing ammonium sulfate for 52 weeks.

 

Dose level (%)

Final body weight (g)

Food consumption

(g/rat/day)

Intakes of ammonium sulfate

(mg/kg b.w./day)

Male

0

410.9 ± 12.3

13.9

-

0.1

428.6 ± 17.6

13.6

42

0.6

416.7 ± 23.7

13.4

256

3.0

400.5 ± 15.1

15.7

1527

Female

0

207.4 ± 13.5

8.4

-

0.1

220.3 ± 8.7

8.6

48

0.6

219.2 ± 13.6

8.4

284

3.0

212.7 ± 24.4

8.6

1490

Each value represents the mean throughout the administration period.

Final body weight, food consumption and the amount of ammonium sulfate intake in rats fed diets containing ammonium sulfate for 104 weeks:

 

Dose level (%)

Final body

weight (g)

Food consumption

(g/rat/day)

Intakes of ammonium sulfate

(mg/kg b.w./day)

Male

0

409.6 ± 37.3

13.7

-

1.5

412.0 ± 49.6

13.6

564.1

3.0

395.1 ± 24.1

14.8

1288.2

Female

0

266.0 ± 30.0

8.8

-

1.5

279.7 ± 22.3

9.2

649.9

3.0

258.1 ± 27.0

9.1

1371.4

Each value represents the mean throughout the experimental period.

Haematology in male rats fed diet containing ammonium sulfate for 52 weeks (No. of animals = 10)

Parameter

Dose level (%)

0 (control)

0.1

0.6

3.0

RBC 1010/L

841.0 ± 39.3

875.0 ± 35.5

842.0 ± 22.3

868.0 ± 40.2

Hb g/dL

13.6 ± 1.0

14.3 ± 0.7

13.9 ± 0.4

14.1 ± 0.5

Ht %

43.6 ± 1.9

45.7 ± 2.0

44.1 ± 1.0

44.9 ± 2.1

MCV fL

51.8 ± 0.6

52.2 ± 0.6

52.4 ± 0.5

51.7 ± 1.0

MCH pg

16.2 ± 0.7

16.4 ± 0.3

16.5 ± 0.3

16.2 ± 0.4

MCHC g/dL

31.3 ± 1.5

31.3 ± 0.6

31.6 ± 0.5

31.3 ± 0.6

Plt 1010/L

65.6 ± 5.7

70.3 ± 6.1

66.5 ± 4.9

65.4 ± 11.6

Ebl counts/200 WBC

2.4 ± 2.0

3.9 ± 2.2

3.1 ± 1.7

2.4 ± 1.6

WBC 108/L

51.8 ± 16.6

44.7 ± 13.5

46.1 ± 8.1

49.6 ± 13.5

Differential cell count (%)

Band

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

Seg

36.4 ± 4.7

44.5 ± 9.0

43.6 ± 4.1

39.9 ± 8.2

Eosino

2.14 ± 1.3

1.9 ± 0.8

2.7 ± 2.0

1.9 ± 0.8

Baso

0.1.9 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.1 ± 0.2

Lympho

61.1 ± 3.8

53.2 ± 9.0

53.4 ± 4.0

57.8 ± 8.5

Mono

0.4 ± 0.7

0.4 ± 0.6

0.4 ± 0.5

0.5 ± 0.6

Each value represents the mean ± S.D.

* significantly different from the control at P < 0.05

Haematology in female rats fed diet containing ammonium sulfate for 52 weeks (No of animals = 10)

 

Dose level (%)

0 (control)

0.1

0.6

3.0

RBC 1010/L

770.0 ± 83.8

792.0 ±44.3

742.0 ± 29.1

744.0 ±40.2

Hb g/dL

13.4 ± 1.5

14.0 ± 0.8

13.0 ± 0.4

13.0 ± 1.0

Ht %

43.6 ± 4.5

44.7 ± 2.2

42.2 ± 1.5

42.3 ± 2.1

MCV fL

56.6 ± 0.3

56.5 ± 0.4

56.8 ± 0.3

56.9 ± 0.4

MCH pg

17.4 ± 0.6

17.7 ± 0.4

17.5 ± 0.6

17.5 ± 0.7

MCHC g/dL

30.7 ± 1.1

31.4 ± 0.7

30.8 ± 1.0

30.7 ± 1.3

Plt 1010/L

61.3 ± 4.3

62.6 ± 3.7

60.0 ± 2.8

60.1 ± 5.1

Ebl counts/200 WBC

7.3 ± 4.2

8.3 ± 3.0

6.5 ± 3.1

8.5 ± 3.4

WBC 108/L

31.1 ± 6.5

28.8 ± 3.0

24.0 ± 2.9

28.5 ± 9.7

Differential cell count (%)

Band

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

Seg

32.4 ± 16.6

26.3 ± 6.6

27.0 ± 3.6

26.0 ± 4.0

Eosino

1.1 ± 1.0

1.5 ± 0.9

1.8 ± 1.3

1.0 ± 0.7

Baso

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

Lympho

66.0 ± 16.4

71.5 ± 6.3

70.4 ± 4.7

72.4 ± 4.2

Mono

0.5 ± 0.5

0.7 ± 0.6

0.8 ± 0.5

0.6 ± 0.6

Each value represents the mean ± S.D.

* significantly different from the control at P < 0.05

Serum biochemistry in male rats fed diets containing ammonium sulfate for 52 weeks (No. of animal = 10)

Parameter

Dose Level (%)

0 (control)

0.1

0.6

3.0

TP g/dL

7.3 ± 0.3

7.3 ± 0.3

7.5 ± 0.3

7.1 ± 0.3

Alb g/dL

4.8 ± 0.2

4.8 ± 0.2

4.9 ± 0.20

4.7 ± 0.3

A/G

1.9 ± 0.1

2.0 ± 0.1*

1.9 ± 0.1

2.0 ± .01*

T-Bil mg/dL

0.1 ± 0.1

0.2 ± 0.1

0.3 ± 0.1**

0.1 ± 0.0

T-Cho mg/dL

119 ± 12

127 ± 13

133 ± 15

115 ± 16

TG mg/dL

129 ± 28

155 ± 50

174 ± 57

123 ± 44

BUN mg/dL

18.9 ± 1.7

20.1 ± 1.6

18.4 ± 1.0

18.5 ± 1.5

Cre mg/dL

0.36 ± 0.03

0.33 ± 0.02*

0.32 ± 0.02**

0.36 ± 0.02

Ca mg/dL

10.6 ± 0.2

10.8 ± 0.2

10.9 ± 0.4

10.8 ± 0.5*

IP mg/dL

5.1 ± 0.4

5.0 ± 0.5

4.5 ± 0.3**

4.9 ± 0.4

Na mEQ/L

142 ± 3

143 ± 2

143 ± 4

143 ± 5

K mEQ/L

4.3 ± 0.3

4.5 ± 0.3

4.6 ± 0.3

4.4 ± 0.3

Cl mEQ/L

102 ± 3

104 ± 2

104 ± 2

102 ± 3

AsT IU/L

88 ± 4

109 ± 20*

134 ± 45**

88 ± 21

AlT IU/L

55 ± 7

79 ± 19*

97 ± 39**

57 ± 18

ALP IU/L

354 ± 75

391 ± 43

418 ± 46

375 ± 48

Γ-GTP IU/L

5.0 ± 3.0

5.0 ± 1.0

7.0 ± 2.0

7.0 ± 2.0

Each value represents the mean ± SD

* Significantly different from the contorl at P < 0.05

** significantly different from the control at P <0.01

Serum biochemistry in female rats fed diet containing ammonium sulfate for 52 weeks (No. of animals = 10)

Parameter

Dose Level (%)

0 (control)

0.1

0.6

3.0

TP g/dL

7.5 ± 0.3

7.7 ± 0.5

7.8 ± 0.4

7.6 ± 0.7

Alb g/dL

5.4 ± 0.3

5.5 ± 0.4

5.6 ± 0.4

5.5 ± 0.4

A/G

2.5 ± 0.2

2.6 ± 0.1

2.6 ± 0.2

2.6 ± 0.2

T-Bil mg/dL

0.2 ± 0.1

0.3 ± 0.1

0.3 ± 0.2

0.4 ± 0.3

T-Cho mg/dL

123 ± 6

129 ± 8

129 ± 16

134 ± 13

TG mg/dL

84 ± 24

126 ± 36

121 ± 40

126 ± 67

BUN mg/dL

21.8 ± 6.7

18.6 ± 1.2

17.1 ± 1.6*

19.1 ± 2.2

Cre mg/dL

0.36 ± 0.04

0.33 ± 0.02*

0.34 ± 0.03

0.33 ± 0.02*

Ca mg/dL

10.5 ± 0.3

10.6 ± 0.4

10.7 ± 0.2

10.7 ± 0.4

IP mg/dL

4.0 ± 0.7

4.4 ± 0.5

4.3 ± 0.5

4.1 ± 0.6

Na mEQ/L

142 ± 1

144 ± 5

142 ± 1

142 ± 1

K mEQ/L

4.0 ± 0.4

4.2 ± 0.2

4.1 ± 0.2

4.0 ± 0.2

Cl mEQ/L

103 ± 3

104 ± 4

104 ± 1

101 ± 2

AsT IU/L

68 ± 7

63 ± 5

64 ± 5

64 ± 6

AlT IU/L

35 ± 6

36 ± 4

33 ± 2

33 ± 4

ALP IU/L

165 ± 35

150 ± 9

134 ± 19*

141 ± 21

Γ-GTP IU/L

< 2

< 2

< 2

< 2

Each value represents the mean ± SD

* Significantly different from the control at P < 0.05

Organ weights of male rats fed diet containing ammonium sulfate for 52 weeks (No. of animals = 10).

Parameter

Dose level (%)

0 (control)

0.1

0.6

3.0

Body weight

410.9 ± 12.3

428.6 ± 17.6

416.7 ± 23.7

400.5 ± 15.1

Absolute (g)

Brain

2.04 ± 0.05

2.03 ± 0.07

2.05 ± 0.05

2.04 ± 0.05

Lungs

1.20 ± 0.09

1.23 ± 0.21

1.16 ± 0.07

1.13 ± 0.06

Heart

1.09 ± 0.08

1.10 ± 0.07

1.08 ± 0.05

1.08 ± 0.07

Spleen

0.73 ± 0.05

0.72 ± 0.04

0.83 ± 0.36

0.68 ± 0.04*

Liver

9.62 ± 0.58

9.92 ± 0.73

10.26 ± 0.63

10.0 ± 0.85

Adrenals

0.03 ± 0.01

0.04 ± 0.01

0.04 ± 0.00

0.04 ± 0.00

Kidneys

2.35 ± 0.25

2.32 ± 0.11

2.42 ± 0.11

2.51 ± 0.11*

Testes

3.38 ± 0.17

3.27 ± 0.11

3.25 ± 0.25

3.29 ± 0.14

Relative (g/100g b.w.)

Brain

0.50 ± 0.02

0.47 ± 0.02

0.49 ± 0.04

0.51 ± 0.02

Lungs

0.29 ± 0.02

0.29 ± 0.04

0.28 ± 0.02

0.28 ± 0.01

Heart

0.26 ± 0.01

0.26 ± 0.02

0.26 ± 0.02

0.27 ± 0.01

Spleen

0.18 ± 0.01

0.17 ± 0.01

0.20 ± 0.08

0.17 ± 0.01

Liver

2.34 ± 0.13

2.31 ± 0.09

2.46 ± 0.10

2.50 ± 0.016*

Adrenals

0.01 ± 0.00

0.01 ± 0.00

0.01 ± 0.00

0.01 ± 0.00

Kidneys

0.57 ± 0.07

0.54 ± 0.02

0.58 ± 0.04

0.63 ± 0.04*

Testes

0.83 ± 0.04

0.76 ± 0.03*

0.78 ± 0.07

0.82 ± 0.04

Each value represents the mean ± S.D.

* Significantly different from the contorl at P < 0.05

Organ weights of female rats fed diet containing ammonium sulfate for 52 weeks (No. of animals = 10)

Parameter

Dose level (%)

0 (control)

0.1

0.6

3.0

Body weight

207.4±13.49

220.3 ± 8.68

219.2 ± 13.62

212.7 ± 24.39

Absolute (g)

Brain

1.86 ± 0.04

1.83 ± 0.04

1.83 ± 0.05

1.82 ± 0.05

Lungs

0.82 ± 0.06

0.79 ± 0.10

0.83 ± 0.12

0.79 ± 0.05

Heart

0.65 ± 0.05

0.67 ± 0.05

0.70 ± 0.03

0.67 ± 0.05

Spleen

0.44 ± 0.04

0.44 ± 0.02

0.45 ± 0.03

0.45 ± 0.07

Liver

4.44 ± 0.26

4.66 ± 0.35

4.69 ± 0.40

4.89 ± 0.42

Adrenals

0.04 ± 0.00

0.04 ± 0.01

0.04 ± 0.01

0.04 ± 0.01

Kidneys

1.25 ± 0.07

1.35 ± 0.08*

1.35 ± 0.09

1.39 ± 0.08**

Relative (g/100g b.w.)

Brain

0.90 ± 0.05

0.83 ± 0.04

0.84 ± 0.05

0.86 ± 0.09

Lungs

0.39 ± 0.04

0.36 ± 0.05

0.38 ± 0.06

0.37 ± 0.04

Heart

0.31 ± 0.02

0.31 ± 0.03

0.32 ± 0.03

0.32 ± 0.02

Spleen

0.21 ± 0.03

0.20 ± 0.01

0.21 ± 0.02

0.21 ± 0.03

Liver

2.15 ± 0.17

2.11 ± 0.13

2.14 ± 0.18

2.31 ± 0.18

Adrenals

0.02 ± 0.00

0.02 ± 0.00

0.02 ± 0.00

0.02 ± 0.00

Kidneys

0.60 ± 0.01

0.61 ± 0.04

0.61 ± 0.06

0.66 ± 0.05

Each value represents the mean ± S.D.

* Significantly different from the control at P < 0.05

** Significantly different from the contorl at P < 0.01

Histopathological findings in male rats fed diet containing ammonium sulfate for 52 weeks (No. of animals = 10)

Organs

Lesions

Grade

Dose Level

0 (control)

3.0

Liver

Bile duct proliferation

+

9

8

 

++

1

2

Necrosis, focal

+

1

4

Pancreas

Acinar cell atrophy, focal

+

4

2

Kidney

Basophilic tubules

+

1

2

Chronic nephropathy

+

0

1

Heart

Myocarditis, focal

+

6

6

Tongue

Mononuclear cell infiltration

+

1

0

Rectum

Erosion

+

1

0

Pituitary

Basophilic cell hyperplasia, diffuse

+

1

0

Adrenal

Pheochromocytoma, malignant

 

0

1

Prostate

Luminal dilatation

+

5

6

Spinal cord

Calcification

+

1

1

Adipose tissue in

abdominal cavity

Necrosis, focal

+

1

1

Histopathological findings in female rats fed diets containing ammonium sulfate for 52 weeks (No. of animals = 10)


Organs

Lesions

Grade

Dose Level

0 (control)

3.0

Liver

Bile duct proliferation

+

0

3

Granuloma

+

3

2

Altered hepatocellular foci

+

7

9

Spleen

Increased extramedullary hematopoiesis

+

1

0

Pancreas

Acinar cell atrophy, focal

+

1

0

Heart

Myocarditis, focal

+

1

2

Tongue

Mononuclear cell infiltration

+

1

2

Pituitary

Cyst

+

1

2

Adenoma, anterior lobe

+

0

2

Thyroid

C-cell hyperplasia

 

1

0

Uterus

Endometrial stromal polyp

 

1

0

Harderian gland

Mononuclear cell infiltration

+

3

0

Conclusions:
The observed no effect level of ammonium sulfate was the 0.6 % diet, which is equivalent to 256 and 284 mg/kg b.w./day in males and females, respectively; and the compound is non-carcinogenic under the conditions of the study.
Executive summary:

Chronic toxicity and carcinogenicity studies of ammonium sulfate were performed in male and female Fisher 344 rats at dietary concentrations of 0 %, 0,1 %, 0.6 % and 3.0 % in a 52-week toxicity study; and 0 %, 1.5 % and 3.0 % in in a 104-week carcinogenicity study. Treatment with ammonium sulfate caused significant increase in kidney and /or liver weights in males and females of the 3.0 % diet group, but no effets were found on survival rates, body weights and hematological, serum biochemical or histopathological parameters at any dose level in the chronic toxicity study. Regarding carcinogenicity, ammonium sulfate did not exert any significant influence on the incidences of tumors in any of the organs and tissues examined. It was concluded that the observed no effect level of ammonium sulfate was the 0.6 % diet, which is equivalent to 256 and 284 mg/kg b.w./day in males and females, respectively; and the compound is non-carcinogenic under the conditions of the study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study lacks sufficient experimental details and does not assess parameters of a guideline study.
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vivo studies: rats were exposed in a nose-only facility to an aerosol of calcium sulfate at 100 mg m-3 for 6h/day, 5 days/week for 3 weeks.
In vitro studies: Type II cells from un-exposed and calcium-exposed animals were stained for alkaline phosphatase activity. In addition, type II cells were exposed in vitro to calcium sulfate over night and then stained for alkaline phosphatase activity.
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac.
- Age at study initiation: no data
- Weight at study initiation: 130 - 150 g


Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: No data
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 weeks
Frequency of treatment:
6 h/day for 5 d/week
Dose / conc.:
100 other: mg/m3
No. of animals per sex per dose:
36 males/group
Control animals:
not specified
Positive control:
No data
Observations and examinations performed and frequency:
Biochemical analyses: protein and non-protein sulphydryl concentrations and γ-GT activity.
Macrophages/alveoli were counted in the H & E sections.
Sacrifice and pathology:
Lungs from 5 % of the animals were formalin fixed, sectioned and stained (H & E). Other lungs were lavaged (saline 8 mL, 0.15 M) the lavage centrifuged (300 g, 20 min) and the supernatant used for biochemical analyses.
Other examinations:
No data
Statistics:
No data
Details on results:
Histopathology: no significant difference was found between groups in the number of macrophages per alveolus in exposed animals.
BAL: exposure did not alter the protein concentration or γ-GT activity. NPSH levels were increased in animals exposed to calcium sulfate.
Type II cells: the viability of type II pneumocytes from rats pre-exposed to calclium sulfate was unchanged when compared to cells from unexposed animals. Cells from animals incubated with calcium sulfate showed no change in alkaline phosphatase staining at any concentration.
Remarks on result:
other: based on the number of alveoar macrophages there was no inflammatory response, although inflammation may have been localised to the acini region.
Critical effects observed:
no
Conclusions:
In vivo: based on the number of alveoar macrophages there was no inflammatory response, although inflammation may have been localised to the acini region.
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study lacks sufficient experimental details and does not assess parameters of a guideline study.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were exposed to an aerosol of anhydrous calcium sulfate fibres for 3 weeks for 6 h/day, 5 days/ wk at a concentration of approximately 10 mg m-3 in a nose-only inhalation system.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac., Bicester, Oxon.
- Age at study initiation: no data
- Weight at study initiation: 130 - 150 g
- Fasting period before study: no data
- Housing: felxible film isolator
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: No data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only system
- Method of holding animals in test chamber: no data
- Source and rate of air: no data
- Method of conditioning air: no data
- System of generating particulates/aerosols: no data
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: air was drawn through a filter system during exposure at 1 L/min for up to 6 h and the fibre concentration in the aerosol was measured gravimetrically.
- Treatment of exhaust air: no data


TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes


VEHICLE
- Justification for use and choice of vehicle: no data
- Composition of vehicle: no data
- Type and concentration of dispersant aid: no data
- Concentration of test material in vehicle: no data
- Lot/batch no. of vehicle: no data
- Purity of vehicle: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
During inhalation, fibre samples were taken from the aerosol, both isoaxially and isokinetically under conditions in which the sampling probe had little effect on the flow before it entered the exposure tower. Twice a day the aerosol breathed by the animals was sampled by replacing one animal at random with a filter system during the exposure period. Air was drawn through the filter at 1 L/min for up to 5 h and the fibre concentration was measured gravimetrically.
Duration of treatment / exposure:
3 weeks
Frequency of treatment:
6 h/day for 5 days/week
Dose / conc.:
10 other: mg/m3
No. of animals per sex per dose:
36 males only
Control animals:
not specified
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment: no data
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: no data
- Section schedule rationale: no data
Positive control:
No data
Observations and examinations performed and frequency:
Alveolar macrophages and "concentrated" bronchoalveolar lavage fluid (cBALF) from 12 animals were prepared by filling the lungs, via the trachaea, with 0.15 M saline (8 mL) by means of syringe attached to the Luer port cannula. The fluid was withdrawn (usually about 4 mL) and made up to 8 mL) with fresh saline. This was then reintroduced into the lung and withdrawn. The resulting fluid was centrifuged at 300 x g for 20 min to remove the free cell population (mainly macrophages - data not provided). The supernatant ("concentrated" acellular BAL, cBALF) was removed and the macrophages resuspended in phosphate-buffered saline. Aliquots of cBALF and macrophages were stored at -80 ºC. Prior to assay, cell samples were thawed and sonicated for 2 min to ensure lysis and the following biochemical measurements were made on the samples: protein, non-protein sulfhydryl (mainly glutathione) and γ-glutamyl transpeptidase (γ-GT). The numbers of macrophages per alveolus were counted in haematoxylin and eosin (H & E) stained fixed sections and in the lavage fluid using a haemocytometer.
Sacrifice and pathology:
The lungs from 6 animals in each group were removed and fixed with 10 % buffered formalin. Lungs from the remaining 12 animls in each group were perfused with saline before removal. Paraffin blocks were made for histological study. The post caval lobes from a minimum of 2 animals in each group were tied off and kept for fibre analysis. The samples were dried with acetone and plasma ashed.
Calcium levels in lung tissue from gypsum-treated animals were measured in the ashed postcaval lobe by inductively coupled plasma spectroscopy (ICP at a wavelength of 393.366 nm.
Other examinations:
No data
Statistics:
Data was normalised to control values and the significance of findings were determined using a two-tailed Student's t-test.
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):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Details on results:
No gysum fibres were found in any of the samples analyzed desite measuring airborne levels of, on average, 15 mg•m-3. Analysis of calcium sulfate levels in ashed lung, in an attempt to estimate lung burden, was made but calcium levels in animals from all groups, including controls were not significantly different from each other. Even animals that were killed immediately post-exposure did not have raised calcium levels. However, hiostological sections showed the presence of fibres in the lungs, proving that there were enough respirable fibres in the aerosol to reach the alveoli (data not shown). It is possible that the method of solubilizing the lung may have interfered with the calcium assay or that the amount of calcium remaining in the lung was below detection limits.
The number of macrophages per alveolus did not increase overall following any treatment (see table) although there were some localised areas containing a high number of macrophages, notably around the acinar region. The number of macrophages isolated by lavage was on average 0.5 - 0.6E06 and this did not alter significantly, regardless of treatment.
There were no significant changes in protein concentration in any of the samples following treatment.
There was a significant decrease in extracellular GGT after recovery following calcium sulfate exposure. Under all conditions the γ-GT levels did not differ significantly from the controls. An increase in glutathione (NPSH) levels was seen in cBALF after calcium sulfate exposure. Macrophages from the calcium sulfate-treated animals lost almost all of their NPSH.
Remarks on result:
other: The number of macrophages per alveolus did not increase overall following any treatment.
Critical effects observed:
not specified

Number of macrophages per alveolus counted from formalin-fixed sections

Fibre

Number of macrophages per alveolus (± SD)

Sacrificed

immediately

(n = 10)

Sacrificed after

4 week recovery

(n = 10)

Air controls

0.22 (± 0.13)

0.18 (± 0.06)

CaSO4

0.33 (± 0.10)

0.2 (± 0.02)

Protein levels in cBALF and macrogphages from animals exposed to calcium sulfate or air

Fibre

Protein levels

Sacrificed immediately

Sacrificed after

4 week recovery

cBALF

(mg/lung)

Macrophages

(mg/106cells)

cBALF

(mg/lung)

Macrophages

(mg/106cells)

Air controls

0.92 (± 0.2)

0.21 (± 0.07)

0.51 (± 0.2)

1.27 (± 0.4)

CaSO4

1.39 (± 0.4)

0.53 (± 0.2)

0.59 (± 0.3)

0.36 (± 0.1)

Conclusions:
The number of macrophages per alveolus did not increase overall following any treatment.
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study lacks sufficient experimental details and does not assess parameters of a guideline study.
Qualifier:
according to
Guideline:
other: no data
Deviations:
not applicable
Principles of method if other than guideline:
Rats were exposed to an aerosol of anhydrous calcium sulfate for 3 weeks, 6h/.day, 5 days/week at a concentration of approximately 60 mgm-3 in a nose only inhalation system. Control animals were treated similarly, but exposed to air only.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac, Bicester, Oxon.
- Age at study initiation: no data
- Weight at study initiation: 130 - 150 g
- Fasting period before study: no data
- Housing: flexible ilm isolator
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: No data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:nose-only inhalation system
- Method of holding animals in test chamber: no data
- Source and rate of air: no data
- Method of conditioning air: no data
- System of generating particulates/aerosols: no data
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: no data
- Treatment of exhaust air: no data


TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of particulate in the aerosol was measured gravimetrically.
- Samples taken from breathing zone: yes


VEHICLE
- Justification for use and choice of vehicle: no data
- Composition of vehicle: no data
- Type and concentration of dispersant aid: no data
- Concentration of test material in vehicle: no data
- Lot/batch no. of vehicle: no data
- Purity of vehicle: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Air was drawn through a filter at 1 L/min for about 1.5 h and the concentration of particulate in the aerosol was measured gravimetrically.
Duration of treatment / exposure:
3 weeks
Frequency of treatment:
6 h/day, 5 days/wk
Dose / conc.:
60 other: mg/m3
No. of animals per sex per dose:
36 males/experiment
Control animals:
other: treated similarly but exposed only to air
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment: no data
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: no data
- Section schedule rationale: no data
Positive control:
No data
Observations and examinations performed and frequency:
Fibre samples were taken from the aerosol, but isoaxially and isokinetically under conditions in which the sampling probe had little effect on the flow before it entered the exposure tower. Sampling the aerosol breathed by the animals was acheived by replacing one animal with a filter system twice a day during the exposure period. Air was drawn through the filter at 1 L/min for about 1.5 h and the concentration of the particulate in the aerosol was measured gravimetrically.
Sacrifice and pathology:
At the end of the exposure period, half of the animals were sacrificed immediately. The remaining animals were kept for 3 wks without further exposure before sacrifice. Each sub-group of 18 was treated as follows; the lungs from 6 were removed and fixed with 10 % buffered formalin. Paraffin blocks were made for histological study. The postcaval lobes from 2 animals (of the 12) in each group were tied off and kept for fibre analysis. This lobe is approximately 10 % of the total lung wet weight; therefore assuming even distribution throughout the lung the fibres in it are about 10 % of the total lung burden.
Statistics:
No data
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
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:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
The milled gypsum fibre was dosed at a concentration of approximately 60 mg m-3. A particle count of the milled and natural fibrous gypsim showed that the milled gypsum contained 38 times more particles per unit mass than the fibrous sample. Because of the low durability of this material in vivo, fibre counting in the lung post-exposure was not possible. Lung calcium levels were analyzed in an attempt to estimate lung burden, but calcium levels in animals from all groups, including controls were not significantly different from eachother.
Intact type II cells were successfully isolated from dosed and control animals and their viability was shown by the presence of alkaline phosphatase staining. The mean number of cells isolated was between 3 and 5E06 per animal with approximately 85 % of these staining alkaline phosphatase positive.
The number of macrophages per alveolus did not increase overall following any treatment, although there were some localised areas of inflammation. The number of macrophages isolated by lavage was on average around a milion cells. For this work, the lungs were only lavaged twice and consequently the yield of cells is lower than that commonly reported and probably represents only a few percent of the total macrophage content of the lungs. If the entire lung burden was engulfed by these cells, the volume of their individual burden would be less than 10 μm3, which is below the estimates for lung overload.
Exposure to both fibrous and milled calcium sulfate had effectively doubled NPSH levels by the end of exposure, an increase that was at least sustained to the end of the recovery period of the fibrous material but not for the milled. There was no change in levels in either macrophages or type II cells. There was no significant increase in γ-GT of type II cells following exposure. Levels had doubled for both milled and fibrous calcium sulfate by the end of the recovery period. Due to the variability of the data this was not shown to be significant.
Remarks on result:
other: see remarks
Remarks:
Exposure to both fibrous and milled calcium sulfate had effectively doubled NPSH levels by the end of exposure, an increase that was at least sustained to the end of the recovery period of the fibrous material but not for the milled. There was no change in levels in either macrophages or type II cells. There was no significant increase in γ-GT of type II cells following exposure. Levels had doubled for both milled and fibrous calcium sulfate by the end of the recovery period. Due to the variability of the data this was not shown to be significant.
Critical effects observed:
not specified

Calcium levels in postcaval lobes of the lung

Group

Mean (ppb)

± SD

Blank

170

16

Control

430

15

No recovery

464

59

3 weeks of recovery

452

47

Conclusions:
Exposure to both fibrous and milled calcium sulfate had effectively doubled NPSH levels by the end of exposure, an increase that was at least sustained to the end of the recovery period of the fibrous material but not for the milled. There was no change in levels in either macrophages or type II cells. There was no significant increase in γ-GT of type II cells following exposure. Levels had doubled for both milled and fibrous calcium sulfate by the end of the recovery period. Due to the variability of the data this was not shown to be significant.
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study not performed to guideline or GLP. Does not measure all parameters
Qualifier:
no guideline followed
Principles of method if other than guideline:
Guinea pigs were exposed to dust under various environmental conditions.
GLP compliance:
no
Limit test:
no
Species:
guinea pig
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Housing: cages along the sides of a 8ft cubical room.




Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: Not specified
Details on inhalation exposure:
The inhalation experiments were conducted in 8 ft. cubical rooms. The animals , kept in cages along the sides of the room, were exposed to the aerosol for 8 h a day for 5.5 days a week. During such periods a rotating paddle device created and maintained in the room an atmospheric suspension of the dust under study. For the rest of the time the air in the room was free of any substantial amount of dust. Periodically, in most cases intervals of a few months, animals were killed for study, so that the progression of any tissue reaction to the dust or to any infection given to the animals could be closely followed.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
During each experiment the dust concentration was checked frequently by the impinger method.
Duration of treatment / exposure:
The duration of the experiments were as followed:
Calcined gypsum dust: 24 months
Calcined gypsum dust for 24 months followed by normal air for 22 months
Calcined gypsum dust for 24 months and then quartz dust for 18 months
Calcined gypsum dust for t3 weeks and then alternately quartz dust for 1 week and calcined gypsum dust for the next week, with a continuation of this programme of alternating weekly exposures for 24 months.
Mixed dust composed of equal parts of calcined gypsum dust for 25 months.
Mixed dust composed of equal parts of calcined gypsum dust and quartz dust for 25 months and then normal air for 12 months.
Mixed dust composed of two parts of calcined gypsum dust and one part of quartz dust for 29 months
Calcined gypsum dust for 6 months, then infection with R1 tubercle bacilli, and then calcined gypsum dust for 18 months more.
Calcined gypsum dust for 25 months, then infection with R1 tubercle bacilli, and then normal air for 12 months.
Mixed dust composed of equal parts of calcined gypsum dust and quartz dust for 3 months then infection with R1 tubercle bacilli and then the mixed dust for 27 months more.
Infection control R1 tubercle bacilli for 22 months.
Frequency of treatment:
8 h a day for 5.5 days a week.
Remarks:
Experiment 1: 448,000,000 particles per cubic foot of air
Remarks:
Doses / Concentrations:
Experiment 2: 448,000,000 particles per cubic foot of air for calcined gypsum dust; 120,000,000 particles per cubic foot of air for quartz dust.
Basis:
no data
Remarks:
Doses / Concentrations:
Experiment 3: 448,000,000 particles per cubic foot of air for calcined gypsum dust; 128,000,000 particles per cubic foot of air for quartz dust.
Basis:
no data
Remarks:
Doses / Concentrations:
Experiment 4: 318,000,000 particles per cubic foot mixed dust concentration and the average concentration of quartz particles was 124,000,000.
Basis:
no data
Remarks:
Doses / Concentrations:
Experiment 5: The average dust concentration was 245,000,000 particles per cubic foot of air.
Basis:
no data
No. of animals per sex per dose:
Experiment 1: Exposure to calcined gypsum dust alone: 21 in total, not specified per sex per dose.
Experiment 2: Exposure to calcined gypsum dust for 24 months, followed by prolonged exposure to quartz dust: 17 in total, not specified per sex per dose.
Experiment 3: Alternating weekly exposure to quartz dust and to calcined gypsum dust: 10 in total, not specified per sex per dose.
Experiment 4: Exposure to a mixture of equal parts of calcined gypsum dust and quartz dust: 35 in total, not specified per sex per dose.
Experiment 5: Exposure to a mixture of two parts of calcined gypsum dust and one part of quartz dust: 64 in total, not specified per sex per dose.
Experiment 6: Exposure to calcined gypsum dust, then infection with tubercle bacilli, and either continuation of the dust exposure or removal to normal air: 20 in total, during the first part, and 15 in the second; not specified per sex per dose.
Experiment 7: Alternative weekly exposures to calcined gypsum dust and to quartz dust and infection with R1 tubercle bacilli: 5 in total, not specified per sex per dose.
Control animals:
yes
Details on study design:
None specified.
Positive control:
None specified.
Observations and examinations performed and frequency:
Mortality: time interval not specified.
Chemical analyses to estimate the amount of dust in the lungs were carried out on some animals.

Sacrifice and pathology:
Necropsy was performed but further details were not specified.
Other examinations:
None specified
Statistics:
None specified
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Experiment 1: some of this group lived as long as 22 months after termination of dust exposure. 12 died of pneumonia or other pulmonary lesions. This mortality trend was widely dispersed over the whole experimental period. Deaths were slightly commoner in the earlier months but not significantly so. The death rate of 28.5 % per annum seemed slightly high. The fact that all deaths in the experiment were from respiratory causes may be meaningful. No significant gross signs of pulmonary diseased manifested and no nodular or diffuse pneumoconiosis ensued. In isoalted animals, pigmentation commenced to appear towards the end of 10 months and minute foci of atelectasis could be seen. After a year of dust exposure a minimal amount of pigmentation became an almost constant sign and atelectasis was more frequently observed. Although a diffuse cellular reaction ultimately ensued, no fibrosis attended this process. The lymph nodes showed irregular enlargement of a moderate degree, characterised by growth of the follicles more particularly. Only rarely could pigmentation be seen. There were no signs of degeneration, necrosis or fibrosis. Of the 10 animals removed to normal air, only 4 died naturally and in only two of them was pneumonia was the cause. Pigmentation persisted in the majority, but atelectasis soon disappeared though diffuse cellular proliferation could be seen. In the lymph nodes a low-grade chronic inflammatory reaction reaction developed during the first two months the animals were in normal air, but during subsequent months this sign receded.

Experiment 2: only 4 of the animals died spontaneously. This rate of 23 % for an 18-month period is lower than the average mortality trend of 21 % per annum in our dusting experiments with an inert amorphous silicate, suggesting that gypsum exerts some measure of protection against the action of quartz. The protective influence can be seen in the pulmonary tissue reaction. While pigmentation increased progressively, eventually attaining a geometric pattern and finally producing mustard-coloured patches, it was nevertheless not as extreme as the pigmentation which occurrs in animals exposed to quartz dust only. Atelectasis was pressent in animals in the earlier phases of the quartz-dusting period and disappeared towards the end of the experiment. The atelectasis may have been a perpetuation of that seen in later phases of the experiment with calcined gypsum alone. Consolidation, dominantly cellular in type, appeared towards the latter part of the quartz-dusting phase. From about 10 months onwards areas of cellular infiltration could be found. Strands of fibrous tissue appeared among these areas toward the end of the 12th month. Except in the last guinea pig killed, fibrosis remained moderate in degree throughout. In this animals, and the one preceding it in the series, some abscess formation could be seen. Necrosis was absent in earlier phases. No hyaline changes occurred in the lungs. The pulmonary lymph nodes showed moderate enlargement only with no tendency to increase in size towards the end of the experiment. Pigmentation remained minimal but some hyaline fibrosis occurred towards the end of the 14th month and in some instances was preceded by isolated areas of necrosis. These changes may also be indicative of the protective action of gypsum when compared with the control group exposed to quartz dust only, cellular infiltrates are recorded as having appeared within 2 months and were quite marked by the 8th month. Fibrosis could be discerned within 4 months and it increased progressively, reaching a marked degree towards the 10th month. Hyaline changes and necrosis appeared first towards the 8t month and became increasingly more prevelant later. Associated with these parenchyal pulmonary changes there was a marked catarrhal bronchiolitis in the quartz-exposed animals which was not seen in those that had first been exposed to calcined dust. The lymph nodes likewise showed a considerably enhanced and early expansion of the silicotic control guinea pigs with progressive fibrosis and ultimate hyalisation. This comparison suggets that the prior inhalation of calcine gypsum dust not only retards the onset of the major effects of inhaled quartz dust by at least 6 months but also materially diminishes the degree to which they evolve and modifies the nature of the reaction to the quartz dust.

Experiment 3: 4 of the 10 animals died of pulmonary causes towards the end of the first year and the close grouping of these deaths suggest an intercurrent infection. The total pulmonary reaction was not unlike that seen in the animals exposed to quartz only. Marked bronchiolitis was a prominant feature near the end of the first 12 months. Cellular infiltrates appeared focally and diffusely and degenerative changes followed rapidly and reached a marked degree during the 2nd year of exposure. The pulmonary lymph nodes showed the same pattern of reaction as that which occurs in the silicotic guinea pig. It is possible that protection was exerted by gypsum. The degree of reaction seen in the animal accidentally killed after 3 months was less than that found in the quartz-exposed animal at 2 months and the 8th month the animal from the latter groups showed a considerably greater degree of pulmonary and hilar node rection than that exposed to calcined gypsum dust for a comparable period.

Experiment 4: 55 % of the animals died of pulmonary causes, the majority of these succumbing within the earlier phase of the experiment. Despite this there is little evidence of a pneumococoniotic response before the 15th month. From that stage on, the total reaction was that of a dominantly cellular diffuse reaction. Atelectasis was a conspicuous feature in one instance and bronchiolitis occurred in two later cases, but both fibrosis and necrosis were in abeyance until about the 19th month of expsure and even then remained limited in their extent or intensity. The pulmonary lymph nodes likewise showed but minimal reaction. Though they exhibited a uniform tendency towards moderate enlargement, degenerative changes such as fibrosis, hyalinization and necrosis ensued, but a few cases and to a minimal degree only. The concurrent exposure to gypsum and quartz appered to have retarded and modified the influence of the quartz on the guinea pig lung. When the 15 surviving animals were treturned to normal air, the high rate of pulmonary deaths continued. Over 8 months, 53 % of the animals died of pulmonary causes. At the time of their death, the majority suffered from a chronic pneumonia which was partly based on the diffuse cellular quartz-induced infiltrates in the pulmonary parenchyma. There was a very marked type of fibrotic response, with hyaline changes in some instances. Unlike the silicotic cases there was little tendency towards necrosis, though a dust -engendered abcess was found in at least 1 instance towards the end of the experiment. The pulmonary lymph nodes were moderately enlarged and the seat of moderate to marked diffuse hyalinization in the majority of cases. It appears therefore that the concurrent inhalation of calcined gypsum and quartz dust though retarding the mitigating efects of the latter as long as exposure is actively continued, ultimately leaves the lung tissues diffusely vulnerable to the effects of quartz dust. Apparently after the exposed animals have resided for a period in normal air the protective action of the gypsum is withdrawn and the quartz particles then exert their usual harmful effect. The quartz and gypsum also appear to have been carried to the lymph nodes at a different rate which favoured the hyalinizing action of the quartz at these sites.

Experiment 5: of the 64 animals, 42 % died of respiratory disease in most cases of chronic pneumonia. These deaths were however evenly distributed over the full 29 month period and therefore may be of reduced statistical significance. In the majority of animals that died of natural causes, distinct pigmentation had developed and bronchiolitis of a moderate degree was in evidence. The latter condition may have been linked with the pneumonic process, as bronchiolitis was less common and less marked in these animals. Focal cellular proliferation due to dust could be discerned from about the 10th month onward and diffuse cellular infiltrations commenced to appear after the 14th month. However, except in isolated cases , collagen deposition, hyalinization and necrosis rarely were observed. Such degenerative changes were slightly commoner and more advanced in those animals that died of pneumonia. The hilar lymph nodes underwent enlargement mainly from cellular proliferation with but moderate and considerably delayed firbous changes and necrosis in isolated instances only. To show that the animals had actually trapped quartz in their pulmonary tissues, the lungs of guinea pigs exposed to dust of the gypsum-quartz mixture and to quartz dust alone were anlysed for their silica content. Although considerably less dust was retained in the lungs of the gypsum-quartz animals than was trapped in the tissues of the control animals, sufficient quartz accumulated in the lungs to have had some fibrogenic influence. It shold be noted that at the 24th month there was but little more quartz in the lung ash of the animals exposed to the mixed dst than there was at 8 months. At the 22-month period, the animals that had breathed quartz dust only had nearly three times as much SiO2 in their lung ash as had the animals exposed to the mixed gypsum and quartz dust for the same period. Therefore there may not be a specific inhibiting action against the effect but with a diluting effect.

Experiment 6: before the influence of inhaled calcined gypsum dust or quartz dust on the course of experimental tuberculosis can be interpreted, the effect of R1 tubercule bacilli on the guinea pig lung was reviewed. Of the 12 animals in the control experiment, 6 died within a 22-month period of observation. In all cases the deaths were due to pulmonary causes and in at least one instance the cause was spreading cavity tuberculosis. However only in 1 case was there spreading tuberculosis. In all other instances there were either no signs of tuberculosis or one to three small tubercles. In only 4 instances did these tubercules show any activity. Tuberculosis was detectable in the pulmonary lymph nodes in one instance but it was inactive. In the guinea pig with spreading pulmonary tuberculosis active tuberculosis could be found not only in the hilar nodes but also in the spleen and liver. When the guinea pigs in one of the dusted groups had been exposed to calcined gypsum dust for 6 months, infected and then further exposed to the gypsum dust for an additional 18 months, only 9 of the 20 animals died spontaneously. Of these deaths, only 6 were ascribed to pulmonary causes. While signs of healing were in evidence in most instances in a few cases it was associated calcification, spreads occurred in 6 instances; 2 cases developed a cavitary tuberculosis and one died from this disease. Fibrosis was a prominant feature in a majority of instances but caseation persisted in 8 cases. The tuberculosis process had however spread to the pumonary lymph nodes, liver and spleen but in most instances was partly healed. Caseation occurred in isolated instances and fibrocaseous tuberculosis on one case. There was a higher proportion of nodules in the animals surviving longer and in these there was also associated hyaline fibrosis. The occurrence of 34 % spreads in the gypsum-dusted guinea pigs indicates a distinct excess over that which occurred in the control series (8.3 %). However it should be noted that the control group was relatively small. Added to this tendency to spread there were in the dusted animals the signs of caseation, cavitation and fibrosis and metastatic dissemination to the hilar nodes and abdominal organs. All these changes indicate some stimulatory effect of the calcined gypsum dust on the tuberculosius process. There was no indication howeve that tuberculosis promotes any pneumoconiotic process. When the infection by means of the R1 tubercle bacilli was initiated at the end of a 25 month period of exposure to the calcined gypsum dust, the influence of the dust on the course of infection was entirely benign. But 3 of 15 animals died of pneumonia within the first month after infection and none thereafter. Mild spreads were found in 6 of the animals killed before the 6th month and none in later stages. Caseation was found twice only in the lungs and also twice in pulmonary lymph nodes. Pulmonary fibrosis was quite common in the healing tubercles. No pneumoconiosis occurred. This study indicates that the prior two-year period of inhaled calcined gypsum dust modified the native susceptibility of the guinea pigs to the R1 tubercle bacillus adversely, although not materially so. The localization of the lesions was affected and for their normal tendency to heal by resolution there was substituted a process of healing by fibrosis with calcification in a few instances.

Experiment 7: spreading and cavitary tuberlulosis was rapidly induced in three specimens within the first year after infection, while tuberculosilicosiswithin the pulmonary tissues dominated, particularly in the later phases of the study. Foci of the tuberculosis could be discerned in the liver and silicotic nodules occurred in the hilar lymph nodes in all cases and once in the spleen. These results and their relatively prompt occurrence leave no doubt that alternating gypsum and quartz expoures influence the course of tuberculosis adversely. The result is greatly modified, however, by the degree to which silicosis develops concurrently. In the second part of the experiment, signs of pneumoconiosis were slow to develop but from the 13th motn after infection (16 months from the start of dusting) marked and diffuse cellular infiltration supervened, with hyaline changes in many of these lungs. Pulmonary tuberculosis of a confluent type and of moderate extent made an early appearance and persisted throughout the long period of study. Caseation, cavitation of calcification, however, was limited to isolated instances, especially in latter months of the study. The pulmonary lymph nodes showed an early tendency toward fibrosis and tuberculosilicosis. Necrosis became a rather constant finding at a later stage. Necrotic foci occurred also in the liver, occassionally with tuberculosis. Rather more commonly tuberculosis metastases were found in the spleen and a number of cases of tuberculosilicosis likewise occurred in that organ.
Sex:
male
Remarks on result:
other: The effect of calcined gypsum dust on the lungs of guinea pigs exposed to dust for 2 years was too insignificant to be classed as pneumocoiosis.
Critical effects observed:
no

Biological action of calcined gypsum dust Guinea pigs were continuously exposed by inhalation to the dust until death.

Guinea pig No.

Exposure to dust, days

Fate

Cause of death

Tissue reaction in

Lungs

Pulmonary Lymph Node

Focal

Pgmentation

Atelectasis

Focal

Pigmentation

Enlarged

Nodes

553 – 28

51

Died

Pneumonia

-

-

-

-

50

69

Died

Pneumonia

-

-

-

-

53

252

Killed

-

-

-

-

-

82

203

Died

Pneumonia

-

-

-

-

81

284

Died

Pneumonia

-

-

-

-

64

283

Died

Pneumonia

-

-

-

-

40

200

Killed

-

+

+

-

-

63

300

Died

Pneumonia

-

-

-

++

44

312

Died

Pneumonia

-

-

-

-

42

373

Killed

-

+

-

-

-

70

374

Died

Pneumonia

-

-

-

++

43

426

Killed

-

+

-

+

-

61

475

Died

Pleurisy

+

-

-

+

45

610

Killed

-

+

+

-

-

47

610

Killed

-

+

+

-

++

93

667

Killed

-

-

-

-

++

55

688

Died

Pulmonary abscess

-

+

-

+++

76

715

Died

Pneumonia

+

+

-

+

45

723

Died

Pneumonia

-

+

-

++

54

732

Killed

-

+

+

-

-

46

782

Killed

-

+

+

-

-

Symbols: +, slight reaction; ++, moderate reaction; +++, advanced reaction.

Biological action of calcined gypsum dust Guinea pigs were continuously exposed by inhalation to the dust for 24 months and then were transferred to normal air where they remained until death.

Guineapig No.

Exposure to dust, days

Fate

Cause of

death

Tissue reaction in

Lungs

Pulmonary Lymph Node

Focal

Pigmentation

Atelectasis

Focal

Pigmentation

Enlarged

Nodes

553-54

22

Killed

-

-

+

-

+

46

22

Killed

-

-

+

-

++

91

34

Died

?

+

+

-

-

59

41

Died

?

+

-

-

+

86

46

Died

Pneumonia

-

-

-

-

77

47

Died

Pneumonia

-

-

-

+

96

588

Killed

-

+

-

-

-

78

666

Killed

-

+

-

-

-

79

666

Killed

-

+

-

-

++

80

666

Killed

-

++

-

+

++

Symbols: +, slight reaction; ++, moderate reaction; +++, advanced reaction.

Combined biological action of calcined gypsum dust and quartz dust:

Guinea

pig no.

Survival in

quartz dust

(days)

Fate

Cause of

death

Microscopic evidence of pneumoconiosis

Pigmentation

Atelectasis

Cellular

infiltration

Degeneration

Fibrotic

Necrotic

553-60

30

Died

?

+

+

-

-

-

51

63

Killed

 

+

+

-

-

-

52

63

Killed

 

++

+

-

-

-

56

123

Killed

 

+

+

-

-

-

57

123

Killed

 

++

-

-

-

-

63

134

Killed

 

-

+

-

-

-

62

184

Died

Pneumonia

++

-

-

-

-

58

215

Killed

 

+++

-

-

-

-

75

245

Killed

 

+++

+

-

-

-

67

301

Killed

 

++

-

+

-

-

66

306

Died

?

++

-

+

-

-

68

366

Killed

 

++

-

+

+

-

69

366

Killed

 

++

-

+

++

-

71

428

Killed

 

++

-

+

++

+

72

428

Killed

 

+

-

++

++

-

73

487

Died

Pneumonia

+

-

++

+

++

74

553

Killed

 

++

-

++

+++

++

Symbols: +, slight reaction; ++, moderate reaction; +++, advanced reaction.

Combined biological action of calcined gypsum dust and quartz dust continued:

Guinea

pig no.

Survival in

quartz dust

(days)

Fate

Reaction in pulmonary lymph nodes

Enlargement

Pigmentation

Degeneration

Hyaline

Necrosis

553-60

30

Died

-

-

-

-

51

63

Killed

+

-

-

-

52

63

Killed

-

+

-

-

56

123

Killed

++

++

-

-

57

123

Killed

++

+++

-

-

63

134

Killed

++

-

-

-

62

184

Died

-

-

-

-

58

215

Killed

++

++

-

-

75

245

Killed

-

-

-

-

67

301

Killed

++

-

-

-

66

306

Died

-

-

-

-

68

366

Killed

+

-

-

+

69

366

Killed

++

-

-

+++

71

428

Killed

++

-

+++

-

72

428

Killed

++

-

+

-

73

487

Died

+

-

++

-

74

553

Killed

++

-

+

-

Control study: uninfected guinea pigs exposed by inhalation to quartz dust alone until death:

Guinea

pig no.

Exposure

(days)

Fate

Gross evidence of silicosis

Microscopic evidence of silicosis

Pigmentation

Pleural adhesion

Pleural plaques

Bronchiolitis

Cellular infiltration

Degeneration

Fibrous

Hyaline

Necrotic

002-44

63

K

-

-

-

+

+

-

-

-

3

123

K

-

-

-

-

+

+

-

-

2

184

K

+

-

-

-

++

++

-

-

4

245

K

++

-

-

-

+++

+++

+

+

9

306

K

-

-

+

-

+++

+++

+

+

6

566

K

++

-

+

-

+++

+++

++

++

8

428

K

++

+

-

-

+++

+++

++

+++

22

553

K

++

-

+

-

+++

+++

+++

+++

Control study: uninfected guinea pigs exposed by inhalation to quartz dust alone until death, continued:

Guinea

pig no.

Exposure

(days)

Fate

Tissue reaction to pulmonary lymph node

Macrophage

infiltration

Degeneration

Fibrous

Hyaline

002-44

63

K

+

-

-

3

123

K

+

-

-

2

184

K

+++

-

-

4

245

K

+++

+

-

9

306

K

++

++

-

6

566

K

++

++

-

8

428

K

++

++

-

22

553

K

+

+++

+++

Conclusions:
The effect of calcined gypsum dust on the lungs of guinea pigs exposed to dust for 2 years was too insignificant to be classed as pneumocoiosis.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral

Currently, seven different animal studies are available that have evaluated the repeated dose toxicity of (calcium) sulfate. None of the studies describes any severe toxicological effects after oral administration of (calcium) sulfate. NIER 2002 reported decreased haematological parameters (e.g. BUN, AST, ALT, protein, cholesterol, and creatinine). The haematological findings were not confirmed by other studies; Wurzner 1979, Takaagi 1999 and Ota 2006 explicitly state that no changes on haematological parameters or blood chemistry were observed. Alterations in liver weight were observed in different studies but were not consistent, reporting increase (Ota 2006) as well as decrease in liver weights (NIER 2002, Takaagi 1999). In all cases, no treatment-related histopathologic changes were observed after oral administration of sulfate. Only one study (Takaagi 1999) observed diarrhoea as consequence of ammonium sulfate administration whereas another (Ota 2006) explicitly state that no diarrhoea was noticed using the same substance and concentration.

 

Besides the animal studies, Heizer et al. (1997) found that in normal adult subjects, sulfate in drinking water at a concentration of 1200 mg/L caused a measureable but clinically insignificant increase in stool mass and decrease in stool consistency and appearance time, but no change in stool frequency and no complaint of diarrhoea.

 

European Food Safety Authority EFSA evaluated the use of calcium sulfate as food ingredient in detail. Calcium sulphate is an approved food additive (E 516). It was evaluated by the Scientific Committee on Food in 1990 and was allocated an Acceptable Daily Intake (ADI) not specified (SCF 1991). In 2003, 2004, and 2008 the Panel evaluated the use of calcium sulphate added for specific nutritional purposes in foods and concluded that this use is not of concern from the safety point of view (EFSA 2003, 2004, and 2008). Also the Joint FAO/WHO Expert Committee on Food Additives (JECFA) has evaluated calcium sulphate and has allocated an ADI not specified. Furthermore, ADIs not specified were also allocated to the single calcium and sulphate ions and to sodium sulphate (JECFA 1986, 2000, 2002).

The Panel concluded that use of calcium sulphate as a mineral substance in foods intended for the general population is not of safety concern.

 

Taken together, at best slight effects were observed after oral administration of calcium sulphate to animals. These included (unspecified) alterations in liver weight and blood chemistry/haematology. It is likely that these alterations may be caused by a disturbance of water balance rather than by toxic effects. As calcium sulphate is furthermore used as food additive with an ADI not allocated, it can be concluded intake of calcium sulphate should not pose a need for concern regarding its toxicity via the oral route.

 

References:

 

EFSA, 2003: Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the Commission related to Calicum Sulphate for use in foods for particular nutritional uses. Adopted on 10 December 2003. The EFSA Journal, 20, 1-6

 

EFSA, 2004: Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the Commission related to Calicum Sulphate as a mineral substance in foods intended for the general population. Adopted on 7 October 2004.The EFSA Journal, 112, 1-10

 

EFSA, 2008: Calcium sulphate for use as a source of calcium in food supplements; Scientific Panel on Food Additives and Nutrient Sources added to food. Adopted on 24 September 2008. The EFSA Journal, 814, 1-9

 

JECFA, 1986 Evaluation of certain food additives and contaminants. Twenty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, No. 733, 1986, and corringendum.

 

JECFA, 2000, Sodium Sulphate. WHO Food Additives Series ; 44 Prepared by the fifty third meeting of the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) IPCS – International Programme on Chemical Safety, World Health Organization, Geneva

 

JECFA, 2002, Evaluation of certain food additives and contaminants. Fifty-seventh report f the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series, No 909

 

SCF 1991: Report of the Scientific Committee for Food on a First Series of Food Additives of Various Technological Functions. Opinion expressed on 18 May 1990

 

 

Repeated dose toxicity: dermal

Currently, no studies are available that have evaluated the repeated dose inhalation toxicity of (calcium) sulfate.Calcium sulfate is an inorganic ionic solid and therefore expected to partition strongly to water rather than organic media. While it is not possible to measure or accurately predict an octanol/water partition coefficient for inorganic ionic substance, such a value would be expected to be very low. Electrolytes are also known not to penetrate the skin in any significant quantity. Given the physico-chemical properties of calcium sulfate, it is not expected that the substance would penetrate the skin in any significant quantity and so would therefore not cause any toxic effects following acute dermal exposure.

 

Furthermore, plaster of Paris, containing mainly the hemihydrous calcium sulfate, has been used in the immobilization of broken bones and is not known to have been associated with any toxic effects, despite intimate skin contact, for longer periods of time, over considerable areas of skin. As this use simulates dermal toxicity, it can safely be inferred that an dermal exposure test would be unlikely to cause any toxic effects.

 

 

Repeated dose toxicity: inhalation

Currently, four different animal studies are available that have evaluated the repeated dose inhalation toxicity of (calcium) sulfate. In none of these studies, using calcium sulfate concentrations ranging from 10-100 mg/m³ any mortality was observed. The only concern was a decrease in glutathione concentrations observed by Clouter 1997 and 1998. No other study was available that could affirm this observation. Mild indications for inflammation were also observed but it is not clear if this was calcium sulphate-specific or caused by the general dust burden.

 

Besides the animal studies, several health surveillance data, mainly from gypsum miners were assessed. None of these five studies, conducted on 4 to 241 subjects which were exposed to (calcium) sulfate dust for up to 30+ years has detected any signs of systemic toxicity caused by repeated exposure to calcium sulfate dust (Riddell 1934, Schepers 1955, Kelada 1978, Oakes 1982, Burilkov 1990). The exposure concentrations were only reported in 3 studies, ranging from 2.8-6 mg/m³ (Oakes 1982) over 16-40 mg/m³ (Burilkov 1990) to <5-150 mg/m³. It was also found that calcium sulfate per se causes no pneumoconiosis (Riddell 1934, Schepers 1955, Burilkov 1990); if such an indication is given, it was mainly attributed to the quartz (silica) content, which is a current impurity in raw gypsum (Oakes 1982) and known to cause pneumoconiosis (“silicosis”). Nevertheless, it must be admitted that in one study (Schepers 1955) appearance of pneumonia was found after necropsy of four gypsum plant workers. None of the other four publications (all with significantly higher numbers of participants) has given any indication for gypsum-induced pneumonia. Furthermore, the publication of Schepers (1955) provides no information on composition of the gypsum dust (e.g. silica content), exposure concentrations, or other confounding factors such as smoking. Therefore study is deemed not reliable.

Other clinical signs reported, such as running nose, dyspnoea, coughing, or altered breath sounds may predominantly be attributed to effects of dust in general and pose no substance-specific symptom.

 

Taken together, the inhalation exposure to calcium sulphate is highly likely to pose no hazard to human health. The most severe observations after inhalation exposure included slight inflammation in some but not all individuals. It is not proven that this is a calcium sulphate specific effect or just caused by general dust overload. No indication for calcium sulphate-induced pneumoconiosis was found.

Justification for classification or non-classification

No systemic toxicological findings could be detected after repeated administration of calcium sulphate by either oral or inhalation route. The only concern that was found after oral uptake could be the occurrence of loose stool or diarrhoea which is caused by osmotic effect in the gastrointestinal lumen but forces no classification as STOT RE. Also the observed slight signs of inflammation observed after inhalation exposure cannot clearly be attributed to calcium sulphate as in different studies no increase of lung macrophages was observed. Therefore, a classification as STOT RE is not justified.