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Carcinogenicity

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Description of key information

No evidence of a carcinogenic potential was observed in a combined chronic toxicity/carcinogenicity study with rats following closely the requirements of OECD TG 453. Data on purity of the test substance are lacking; however, since no adverse effects were observed, this is not considered to affect the evaluation of the carcinogenic potential of ammonium sulfate in an adverse manner.
Similarly to other salts, high doses of ammonium sulfate may have the capability of tumor promotion in the rat stomach; it is, however, much less potent than sodium chloride when tested under identical conditions. However, these data need careful evaluation as high salt concentrations were given as a bolus dose directly into the stomach. It is known that high salt concentrations can denature proteins leading to cell injury or cell death. Subsequently cell proliferation might occur as a repair mechanism causing an increase in ornithine decarboxylase in the glandular stomach.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Principles of method if other than guideline:
In this study, the chronic toxicity (52-week oral feeding) and carcinogenicity (104-week oral feeding) was investigated.
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Yoneyama Kagaku Kogyo (Osaka Japan)
- Age at study initiation: 5 weeks
- Housing: three or four rats per plastic cage
- Diet: ad libitum
- Water: Tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 °C
- Humidity (%): 55 ± 5 %
- Air changes (per hr): 18
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Recapture rates for ammonium sulfate from the admixed diet at each concentration level were confirmed to be 95.4-98.7%.
Duration of treatment / exposure:
52 and 104 weeks
Frequency of treatment:
continuously in the diet
Post exposure period:
no
Dose / conc.:
42 mg/kg bw/day (actual dose received)
Remarks:
males (52-week chronic toxicity study) 0.1% nominal in diet
Dose / conc.:
256 mg/kg bw/day (actual dose received)
Remarks:
males (52-week chronic toxicity study), 0.6% nominal in diet
Dose / conc.:
1 527 mg/kg bw/day (actual dose received)
Remarks:
males (52-week chronic toxicity study), 3% nominal in diet
Dose / conc.:
48 mg/kg bw/day (actual dose received)
Remarks:
females (52-week chronic toxicity study), 0.1% nominal in diet
Dose / conc.:
284 mg/kg bw/day (actual dose received)
Remarks:
females (52-week chronic toxicity study), 0.6% nominal in diet
Dose / conc.:
1 490 mg/kg bw/day (actual dose received)
Remarks:
females (52-week chronic toxicity study), 3% nominal in diet
Dose / conc.:
564.1 mg/kg bw/day (actual dose received)
Remarks:
males (2-year carcinogenicity study), 1.5% nominal in diet
Dose / conc.:
1 288.2 mg/kg bw/day (actual dose received)
Remarks:
males (2-year carcinogenicity study), 3% nominal in diet
Dose / conc.:
649.9 mg/kg bw/day (actual dose received)
Remarks:
females (2-year carcinogenicity study), 1.5% nominal in diet
Dose / conc.:
1 371.4 mg/kg bw/day (actual dose received)
Remarks:
females (2-year carcinogenicity study), 3% nominal in diet
No. of animals per sex per dose:
Chronic toxicity study: 10/sex
Carcinogenicity study: 50/sex
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: every 2 weeks until week 10 and every 5 weeks thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 52 weeks
- Anaesthetic used for blood collection: Yes: ether
- Animals fasted: Yes: overnight
- How many animals: 10
- Parameters checked: red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (Plt) and white blood cell count (WBC). Differential leukocyte counts and the reticulocyte count (Ebl).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 52 weeks
- Animals fasted: Yes: overnight
- How many animals: 10
- Parameters checked: total protein (TP), albumin (Alb), albumin/globulin ratio (AIG), total bilirubin (T-bil), total cholesterol (T-Cho), triglyceride (TG), blood urea nitrogen (BUN), creatinine (Cre), calcium (Ca), inorganic phosphorus (IP), sodium (Na), potassium (K), chloride Cl), aspartate transaminase (AsT), alanine transaminase (AlT), alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (gamma-GTP)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Brain, lungs, heart, spleen, liver, adrenals, kidneys and testes were weighed. As for adrenals, kidneys and testes, weights of each side were separately recorded and the total of both sides was used for calculation of group mean and SD values.
In addition to these organs, the nasal cavity, trachea, aorta, pituitary, thyroids, parathyroids, salivary glands, tongue, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, pancreas, urinary bladder, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, mammary glands, skin, mesenteric and submandibular lymph nodes, thymus, sternum, femur including bone marrow, sciatic nerve, trigeminal nerve, spinal cord (cervical, thoracic and lumber cords), eye, Harderian gland and thigh muscle. All organs and tissues in the control and 3.0% 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.
Statistics:
Variance in data for body weights, hematology, serum biochemistry and organ weights were checked for homogeneity by Bartlett test. When the data were homogeneous, one-way analysis of variance (ANOVA) was used. In the heterogeneous cases, the Kruskal-Wallis test was applied. When statistically significant differences were indicated, Dunnett's multiple test was employed for comparison between control and treated groups. Final survival rates and the incidences of tumor and non-neoplastic lesions were compared with the Fisher's exact probability test or the Mann-Whitney's U-test.
Details on results:
CLINICAL SIGNS AND MORTALITY
In the chronic toxicity study, no mortality was found in any groups throughout the treatment period.
In the carcinogenicity study, the survival rate of control, 1.5% and 3.0% groups were 88%, 78% and 76%, respectively, for males, and 76%, 80% and 80%, respectively, for females, and no significant differences were observed among the groups.

BODY WEIGHT AND WEIGHT GAIN
No test substance-related change in the body weights was found.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A tendency for increase of food intake in the male 3.0% group in the chronic toxicity study was noted.

HAEMATOLOGY
No significant variation was found.

CLINICAL CHEMISTRY
No dose related alteration was found.

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.

GROSS PATHOLOGY
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.

HISTOPATHOLOGY:
In the carcinogenicity study, non-neoplastic and neoplastic lesions were noted in the control and treatment groups, with no significant inter-group difference in their incidences or severity.


Key result
Dose descriptor:
NOAEL
Effect level:
256 mg/kg bw/day
Sex:
male
Basis for effect level:
other: estimated from the chronic toxicity study
Key result
Dose descriptor:
NOAEL
Effect level:
284 mg/kg bw/day
Sex:
female
Basis for effect level:
other: estimated from the chronic toxicity study

Organ weight of male rats fed diet containing ammonium sulfate for 52 weeks (Chronic toxicity study).

   control  0.1%  0.6%  3.0%
 Body weight (g)  410.9 ± 12.3  428.6 ± 17.6 416.7 ± 23.7  400.5 ± 15.1
 Brain (g) 2.04 ± 0.05 2.03 ± 0.07  2.05 ± 0.05  2.04 ± 0.05
Lungs (g)   1.20 ± 0.09 1.23 ± 0.21   1.16 ± 0.07  1.13 ± 0.06
 Heart (g) 1.09 ± 0.08  1.10 ± 0.07 1.08 ± 0.05  1.08 ± 0.07
 Spleen (g) 0.73 ± 0.05  0.72 ± 0.04  0.83 ± 0.36  0.68 ± 0.04 *
 Liver (g)  9.62 ± 0.58  9.92 ± 0.73  10.26 ± 0.63  10.0 ± 0.85
 Adrenals (g)  0.03 ± 0.01  0.04 ± 0.01  0.04 ± 0.00  0.04 ± 0.00
 Kidneys (g) 2.35 ± 0.25 2.32 ± 0.11  2.42 ± 0.11  2.51 ± 0.11 *
Testes (g)  3.38 ± 0.17  3.27 ± 0.11  3.25 ± 0.25  3.29 ± 0.14

Organ weight of female rats fed diet containing ammonium sulfate for 52 weeks (Chronic toxicity study).

   control  0.1%  0.6%  3.0%
 Body weight (g)  207.4 ± 13.49  220.3 ± 8.68  219.2 ± 13.62  212.7 ± 24.39
 Brain (g)  1.86 ± 0.04  1.83 ± 0.04  1.83 ± 0.05  1.82 ± 0.05
 Lungs (g)  0.82 ± 0.06 0.79 ± 0.10 0.83 ± 0.12  0.79 ± 0.05
 Heart (g)  0.65 ± 0.05  0.67 ± 0.05  0.70 ± 0.03  0.67 ± 0.05
 Spleen (g)  0.44 ± 0.04  0.44 ± 0.02  0.45 ± 0.03  0.45 ± 0.07
 Liver (g)  4.44 ± 0.26  4.66 ± 0.35  4.69 ± 0.40  4.89 ± 0.42
 Adrenals (g)  0.04 ± 0.00  0.04 ± 0.01 0.04 ± 0.01  0.04 ± 0.01
 Kidneys (g)  1.25 ± 0.07  1.35 ± 0.08 *  1.35 ± 0.09   1.39 ± 0.08 **

* Significantly different from the control at p<0.05. **Significantly different from the control at p<0.01.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
284 mg/kg bw/day
Quality of whole database:
OECD TG 453

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the substance is not considered to be classified for carcinogenicity under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179

Additional information

A chronic oral toxicity and carcinogenicity study was conducted in rats, similar to the requirements of OECD TG 453. In the subchronic part of the study, groups of 10 rats/sex were fed a diet containing the test substance (purity not given) at concentrations of 0, 0.1, 0.6, or 3% for 1 year. These concentrations corresponded to average daily intakes of 0, 42, 256, and 1527 mg/kg bw/d for males and 0, 48, 284, and 1490 mg/kg bw/d for females, respectively. For investigation of the carcinogenic potential, groups of 50 rats/sex were fed a diet containing the test substance (purity not given) at concentrations of 0, 1.5, or 3% for 2 years. These concentrations corresponded to average daily intakes of 0, 564.1, and 1288.2 mg/kg bw/d for males and 0, 4649.9, and 1371.4 mg/kg bw/d for females respectively.

Absolute and relative kidney weights were increased at the high dose level for both sexes. Absolute spleen weights were decreased and relative liver weights were increased in high dose males. No macroscopic changes were recorded by gross pathology, except for massive nodular or focal lesions suggesting neoplastic changes. At histopathological examination, non-neoplastic and neoplastic lesions were noted in the control and treatment groups, with no significant inter-group difference in their incidences or severity.

The authors concluded that the no observed adverse effect level of ammonium sulfate was the 0.6% diet, which is equivalent to 256 and 284 mg/kg bw/d in males and females, respectively, and the compound is noncarcinogenic under the conditions of the study. There was no evidence of a long-term carcinogenic activity of the test substance. (Ota et al., 2006)

  Groups of 4 male Fischer rats were fasted overnight and were then given of an aqueous solution of the test substance (reagent grade; no further data on purity) by gastric intubation at doses of 500, 1000, 1500, 2660 mg/kg bw. Control rats were administered the vehicle (Furihata et al. 1989).

The effect of various salts including ammonium sulfate on ornithine decarboxylase (ODC) as a marker for tumor promotion in rat stomach mucosa was reported. A 73-fold increase in enzyme activity was measured with a maximum at 16 hours after a single oral gavage (500 - 2660 mg/kg bw). In comparison, an equimolar dose of NaCl (25.7 mmol = 1500 mg/kg bw) induced a 248 fold increase in enzyme activity. The authors concluded that the various tested salts may have the capability of tumor promotion in the glandular stomach of rats. In this study, very high salt concentrations were given in a bolus directly into the stomach. High salt concentrations can denature proteins resulting in cell injury or cell death with subsequent cell proliferation as a repair mechanisms causing the increase in ODC activity which is a normal, secondary physiological response to cellular injury and death. Therefore, the study did not provide supporting evidence for carcinogenicity of ammonium sulfate.