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Appendices 1-18 to: Report on the Health Effects of Selected Pesticide Coformulants

11   Animal toxicity

11.1   Single dose toxicity

11.1.1   Inhalation

No mortality or signs of gross toxicity was observed in 6 young adult male rats that were exposed to diammonium sulphate aerosols at a concentration of 1000-1200 mg/m³ (particle size averaged 2-3 mm) 8 hours/day for 3 consecutive days. Eight of 20 guinea pigs exposed to 800-900 mg/m³, 1/6 exposed to 600-700 mg/m³, and 0/6 exposed to 500-600 mg/m³ for 8 hours died during exposure. The survivors recovered with no noticeable after effects. (Pepelko et al. 1980).  

No effect on pulmonary macrophage numbers and shape was found in hamsters exposed to 0.86 mg/m³ of diammonium sulphate with a MMAD of 0.3 mm for 12 hours (US Department of Commerce - quoted from IUCLID 2000).

Groups of 6-8 adult sheep were exposed by inhalation to diammonium sulphate aerosols in a concentration of 0.1 or 4 mg/m³ (MMAD = 0.5 or 1.5 mm) or to other sulphates (ammonium bisulphate, sulphuric acid) for 4 hours. The sheep first inhaled physiologic buffer, then the known bronchoconstrictor carbachol, and finally half an hour later sulphate aerosols. Some sheep were exposed to more than one salt. These exposures were separated by at least 1 week. The mean pulmonary flow resistance was not altered following exposure to diammonium sulphate. The airway responsiveness to inhaled carbachol was enhanced at some time within the 24-hour period immediately following exposure to diammonium sulphate with a MMAD of 1.5 mm. (Abraham et al. 1983).

A group of five male, mixed breed rabbits were exposed by oral inhalation to clean air, diammonium sulphate aerosols in a concentration of 2.0 mg/m³ (MMAD = 0.4 mm) and to other sulphates (ammonium bisulphate, sodium sulphate). Exposures for individual rabbits were separated by at least 1 week and lasted for 1 hour. The bronchiolar mucociliary clearance was not affected by the treatment with diammonium sulphate. (Schlesinger 1984).

The bronchiolar or tracheal mucociliary clearance was not affected either in donkeys, rats or sheep inhaling diammonium sulphate with a MMAD of 0.1-0.4 for 1-4 hours in doses of 0.3-3.0, 3.6 or 1.1 mg/m³, respectively (Schlesinger et al. 1978, Phalen et al. 1980, Sackner et al. 1981 – all quoted from Schlesinger 1984).

11.1.2   Oral intake

The oral LD₅₀-values reported for diammonium sulphate range from 2840 to 4250 mg/kg b.w. for rats (3 values reported) and from 610 to 640 mg/kg b.w. for mice (2 values reported) (IUCLID 2000).

Groups of 2-3 Japanese white rabbits were anaesthetised and dosed with 0 or 1500 mg/kg b.w. of diammonium sulphate in saline through a gastric probe. The three rabbits dosed with diammonium sulphate showed similar symptoms such as mydriasis, irregular respiratory rhythms, local and general convulsions, until they fell into respiratory failure with cardiac arrest about an hour after the dosing. Electroencephalogram showed slow, suppressive waves initially and 15-20 minutes later a high-amplitude slowing wave pattern in correspondence to local convulsions. Electrocardiogram did not show any change of wave pattern except for bradycardia immediately after the general convulsions and until cardiac arrest. The blood pressure fell down rapidly after the general convulsions. The concentration of ammonium and sulphate ions in serum increased remarkable and blood gas analysis showed severe metabolic acidosis. Biochemical analysis of blood and histological examination of brain, heart, lung, spleen, kidney, liver and stomach revealed no significant changes except for a moderate increase in K⁺ just before cardiac arrest. (Sato et al. 1999). 

11.1.3   Dermal contact

Diammonium sulphate was not irritating to intact or abraded rabbit skin that was tested for 20 or 8 hours, respectively. No further details were given. (BASF 1969 – quoted from IUCLID 2000).

11.1.4   Other routes

11.1.4.1     Ocular irritation

Diammonium sulphate was not irritating to rabbit eyes. No further details were given. (BASF 1969 – quoted from IUCLID 2000).

11.2   Repeated dose toxicity

11.2.1   Inhalation

Young male Sprague-Dawley rats were pre-treated intratracheally with either physiologic saline (to model normal lungs) or porcine pancreatic elastase (to model chronic pulmonary impairment). Groups of 30 rats from each pre-treatment group were exposed to filtered air (control), to 1 ppm sulphur dioxide, to wet diammonium sulphate aerosols in concentrations of 0.5 mg/m³ (MMAD = 0.44 mm), or to combined sulphur dioxide and diammonium sulphate for 5 hours/day, 5 days/week for 4 or 8 months. Half of the rats exposed for 8 months were held for an additional 3-month recovery period.
At 4 months, bronchial epithelial hyperplasia and alveolar mean and median cord (a long flexible structure) length significantly increased in the saline/diammonium sulphate and saline/diammonium sulphate/sulphur dioxide groups but decreased in the elastase/diammonium sulphate and elastase/ diammonium sulphate/sulphur dioxide groups compared to air controls. The number of non-ciliated epithelial cells in the bronchioles was significantly increased in the saline/diammonium sulphate group and focal haemosiderosis was significantly increased in the elastase/diammonium sulphate/sulphur dioxide group.
At 8 months, alveolar interstitial fibrosis tended to be greater in aerosol-exposed rats with either normal or impaired lungs compared to air controls, but was significant only in the saline/diammonium sulphate exposed group. Other significant effects observed at 8 months following exposure to diammonium sulphate and compared to air controls included increased lung volume, emphysema and focal haemosiderosis in elastase treated rats and an increased count of non-ciliated epithelial cells in the bronchioles in saline treated rats.

Effects of elastase treatment (e.g. greater lung volumes, emphysema, and alveolar interstitial fibrosis) were the only effects that persisted throughout the recovery period. In addition, a significant increase in alveolar mean and median cord length in saline/diammonium sulphate treated rats was observed.
No significant pathological changes were observed in the nasal cavities of any group at any time point. The pulmonary function of rats exposed for 4 months to diammonium sulphate alone was significantly decreased compared to air control for a few measured parameters (residual volume/total lung capacity, quasistatic compliance, nitrogen-slope) in either saline or elastase treated rats. No significant changes in the pulmonary function were observed in rats exposed to both diammonium sulphate and sulphur dioxide. Immunological studies of peripheral lymphocytes and spleen cells of rats exposed to diammonium sulphate for 4 months revealed no significant depressive effects on the immune system.
The authors concluded that changes related to exposure to sulphur dioxide and/or diammonium sulphate were minimal and transient. No additive effect appeared to occur between diammonium sulphate and sulphur dioxide. The authors were mainly concerned about the increased alveolar fibrosis (observed after 8 months of exposure but not after 4 months of exposure or after 3 months of recovery) associated with diammonium sulphate exposure and the implications it might have in progressive fibrotic disease in human lungs. (Smith et al. 1989).

Male Sprague-Dawley rats and Hartley strain guinea pigs were instilled intratracheally with either sterile saline or porcine pancreatic elastase dissolved in saline. Groups of 15-29 animals from each pre-treatment group were exposed to filtered air (control), to diammonium sulphate aerosols in concentrations of 1.0 mg/m³ (MMAD = 0.4 mm), or to ammonium nitrate for 6 hours/day, 5 days/week for 5 or 20 days. Pulmonary function evaluations conducted in guinea pigs showed no significant effects of diammonium sulphate (or ammonium nitrate) exposure. Compared with air-exposed animals, rats exposed to diammonium sulphate aerosols had increased values of residual volume and functional residual capacity and a decreased nitrogen-slope in saline as well as elastase treated rats.
Diammonium sulphate exposure aggravated elastase-induced emphysema in the rat. Guinea pigs (but not rats) exposed to diammonium sulphate had hypertrophy and hyperplasia of non-ciliated epithelial cells in the alveoli and bronchioles. Alveolar septa of saline/diammonium sulphate exposed animals were affected by interstitial thickening caused by an increased collagen content and an increased number of interstitial cells. The alveolar cord length was increased in saline/diammonium sulphate exposed animals. (Loscutoff et al. 1985, Busch et al. 1984).

Groups of 6 male Sprague-Dawley rats were exposed to diammonium sulphate aerosols in concentrations of 0, 1 or 5 mg/m³ (MMAD = 0.5-0.6 mm), ozone in concentrations of 0, 0.20, 0.64 or 0.96 ppm or mixtures of both for 23.5 hours per day for 2-7 days. Exposure to diammonium sulphate alone showed no effect in several biochemical and morphometric assays for lung injury (e.g. protein content of lung lavage fluid, collagen synthesis rates, soluble proline content, fibroblast content, lung lesions). Ozone alone caused lung injury as evidenced by significantly differences between the control rats and the rats exposed to ozone in the assays for lung injury. A synergistic effect of the combination of ozone and diammonium sulphate (at a dose of 5 mg/m³, but not at 1 mg/m³) was observed in the form of significantly greater increases in the measured parameters as compared with results observed in rats exposed to ozone alone. Based on these and other studies with combinations of ozone or nitrogen dioxide with different aerosols, the authors conclude that acidity and size of the particles in the aerosol is responsible for the synergy. (Last 1991).

Groups of 10 adult male rats were exposed to diammonium sulphate aerosols in concentrations of 0 or 300 mg/m³ (particle size averaged 1-2 mm) for 8 hours/day for 1, 3, 7 or 14 days. No significant differences could be detected between control and exposed rats in arterial blood gases, pH and standard bicarbonate, in body weight and wet lung weight, in vital capacity and residual volume of the lungs, or in histological examination of the trachea, bronchial lymph nodes and lungs. (Pepelko et al. 1980).

Groups of 7-12 male guinea pigs were exposed to diammonium sulphate aerosols in concentrations of 0, 0.2, 0.4 or 2.0 mg/m³ (MMAD = 0.7-0.9 mm) for 2 hours/day, 5 days/week for 7½ weeks. The animals were successively 3 times a week for 2½-3 weeks exposed to aerosols for 2 hours, and after 30 minutes, to an albumin spray for 30 minutes. Breathing curves were continuously recorded during the sensitisation periods (periods with exposure to albumin) and for 5 minutes before exposure to albumin. The experiments showed that the degree of asthmatic dyspnoea (immediate type induced by inhalation of albumin) was dose-dependently increased by exposure to diammonium sulphate aerosols (only significant at 2 mg/m³). The aerosols alone did not alter the breathing curves at the concentrations studied. (Kitabatake et al. 1991).

11.2.2   Oral intake

Groups of 10 F344 rats of each sex were fed 0, 0.38, 0.75, 1.5 or 3% of diammonium sulphate in their CRF-1 powder diet for 13 weeks. The only effect observed was diarrhoea during the administration period in the males dosed with 3% diammonium sulphate. No changes indicating toxicity were observed in body and organ weights, in blood and biochemical parameters, or in the histopathological examination. According to the authors, the NOEL in this study was 1.5% (equal to 886 mg/kg b.w. per day) in males but 3% (equal to 1975 mg/kg b.w. per day) in females. (Takagi H et al. 1999 – quoted from Toxline Plus).

11.2.3   Dermal contact

No data have been found.

11.2.4   Other routes

No data have been found.

11.3   Toxicity to reproduction

No data have been found.

11.4   Mutagenic and genotoxic effects

11.4.1   In vitro studies

Diammonium sulphate was not mutagenic when tested (in accordance with OECD guideline 471) in 4 strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium in a concentration of 20 – 5000 mg/plate in Ames tests with and without metabolic activation systems (BASF 1983 – quoted from IUCLID 2000).

In a cytogenetic assay without metabolic activation systems, an isotonic solution of diammonium sulphate did not cause chromosomal aberrations in V79 hamster cells. However, cells treated with a hypotonic diammonium sulphate solution (or other hypotonic solutions) had an increase in aberration frequencies. Post-treatment with a hypertonic solution of diammonium sulphate increased the mutagenicity of ethylmethanesulfonate, a known mutagen. (Nowak 1987, 1988 - both quoted from IUCLID 2000).

In two other cytogenetic assays performed in human lymphocytes and CHO cells, respectively, diammonium sulphate did not cause chromosomal aberrations (Obe & Kamra 1986, Obe et al. 1986 - both quoted from IUCLID 2000).

Diammonium sulphate tested negative in the yeast gene mutation assay in Saccharomyces cerevisiae D4 with and without metabolic activation systems (US Department of Commerce 1975 - quoted from IUCLID 2000).

11.4.2   In vivo studies

No data have been found.

11.5   Carcinogenic effects

Groups of Syrian hamsters received 5 mg of a known carcinogen (benzo(a)pyrene) by intratracheal intubation once a week for 15 weeks. One group of the animals was simultaneously exposed to 0.2 mg/m³ of diammonium sulphate 6 hours/day for 5 days/week for the 15 weeks. Other groups received benzo(a)pyrene or diammonium sulphate alone. There was no effect of diammonium sulphate inhalation on benzo(a)pyrene carcinogenesis. (US Department of Commerce - quoted from IUCLID 2000).

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