Hydroxylamine

Endpoint summary

Description of key information

For hydroxylamine (free base) no data is available. However, valid studies of the corresponding salt "hydroxylamine sulfate" are available:
CAS No. 10039-54-0:
oral
- 90 d, rat, oral: NOAEL = > 0.9 mg/kg bw/d (nominal) for males and females (BASF AG 1989, Val. 2)
- 28 d, rat, oral: NOAEL = 25 ppm for males and 100 ppm for females (BASF AG 1989, Val. 2)
- 12 months, rat, oral: NOAEL = 0.2/0.3 mg/kg bw/d in males and 0.4 mg/kg bw/d in females for systemic effects (BASF 2001, Val. 1)
- 12 weeks, mice, oral: LOAEL = 100 mg/kg bw/d in males and 0.4 mg/kg bw/d in females for systemic effects (Yamamoto 1967, Val. 3)
- 7 and 28 d, rat, oral: no NOAEL determined: mechanistical study and no guideline study, therefore no effect levels determined (BASF 2003, Val. 1)

Additional information

For hydroxylamine (free base) no data is available. However, valid studies of the corresponding salt "hydroxylamine sulfate" are available:

CAS No. 10039-54-0:

Valid experimental data were available to assess the toxicity of the tested material after repeated administration.

oral

In a subchronic oral toxicity study similar according to OECD TG 408 (no recovery period), groups of 10 male and 10 female Wistar rats received hydroxylammonium sulfate (purity ≥99%) in the drinking water at concentrations of 0, 10, 50, or 250 ppm for 90 consecutive days. The doses administered corresponded to a mean daily hydroxylammonium sulfate intake of about 0, 0.9, 4 or 21 mg/kg bw. Regarding mortality, general appearance and behaviour of the animals, there was no difference between treated and untreated animals. No toxicologically relevant differences in mean feed consumption as well as body weight and body weight development were detected in male and female rats. The following findings were obtained and assessed as substance-induced: At 250 ppm, rats of both sexes showed dark coloration of the urine. This was considered to be due to the substance-related effects on the blood. The hematological examination revealed indications of an increased destruction of red blood cells. At 250 ppm in both sexes and at 50 ppm in females there was a reduction of the erythrocytes and haemoglobin values. In addition there was an increase of the MCH values in both sexes at 250 ppm and in females at 50 ppm, furthermore reduced values of hematocrit in females and of the MCHC in males at 250 ppm. In males receiving 50 ppm, decreased counts of red blood cells and decreased values of haemoglobin were also apparent during the treatment period, although these did not attain statistical significance. These findings were considered to be related also to an increased decay of erythrocytes. The increase of the MCV, and reticulocyte counts at 250 ppm in males and females were assessed as sign of compensate increased erythropoiesis. As a consequence of an increased leaving of juvenile erythrocytes out of the bone marrow a reinforced polychromasia was seen dose-dependent at 250 and 50 ppm in both sexes. At 50 ppm, a slight increase of reticulocytes in male and female rats was noted, and moreover in females a marginal increase of the MCV values. Furthermore, an increase of bilirubin concentration in both sexes at 250 ppm was observed. In males and females receiving 50 ppm, increased values of bilirubin were also apparent during the treatment period, although these did not attain statistical significance. This finding appeared to be due to the increased decay of erythrocytes. The elevated methaemoglobin concentration and the reinforced evidence of Heinz bodies in both sexes at 250 ppm were indicative for methaemoglobinemia. Increased absolute and relative spleen weights were seen at 250 ppm in male and female rats. Increase of relative liver weights were noted only in males. In males and females receiving 50 ppm, increased absolute and relative adrenal weights were noted. Histopathological findings representing secondary effects to the anemia included increased hemosiderin deposits in the spleen and liver of both males and females given 250 ppm. At 50 ppm, moderate increased hemosiderin deposits in the spleen were revealed in both sexes. In addition, sinus dilatation together with congestion of the spleen were observed dosedependent in both sexes at 50 and 250 ppm. 10 ppm did not alter the blood parameters in rats of both sexes.

In conclusion, repeated administration of 50 and 250 ppm hydroxylammonium sulfate (equivalent to about 4 and 21 mg/kg bw/d respectively) to rats via the drinking water for 3 months led to toxicity in male and female rats at both dose levels. In the present study it has been demonstrated that hydroxylammonium sulfate has a hematotoxic potential. In the males and females of the 50 and 250 ppm groups the administration of hydroxylammonium sulfate led to hemolytic anemia (dose-related) with methaemoglobinemia and to organ weight increases in the spleen and liver together with the specific histopathological findings in the liver and spleen seen as increased hemosiderin deposits in both sexes. The NOAEL for all adverse effects of this rat study was 10 ppm (equivalent to about 0.9 mg/kg bw/d) for both sexes. No local toxic effects were seen in male and female rats treated with the highest tested dose level of 250 ppm, equivalent to about 21 mg/kg bw/d (BASFAG 1989, Val. 2).

 

In a 28-day range-finding oral toxicity study mostly according to OECD TG 407 groups of five male and five female Wistar rats received hydroxylammonium sulfate (purity ≥99%) in the drinking water in doses of 0, 25, 100, 400 or 1600 ppm for 4 consecutive weeks. The analytical investigation of the test substance preparations are performed at the end of the study. But, the estimation of compound consumption based on water consumption by male and female rats could not definitely be done in all dose groups because of some study restrictions. There were some technical shortcomings concerning definite calculated achieved intake of hydroxylammonium sulfate from the drinking water in the 25, 100 and 400 ppm dose groups because of the instability of hydroxylammonium sulfate in water. No test substance or only minor amounts were verifiable especially for the low dose groups. Therefore, the hydroxylammonium sulfate dose is given in ppm instead of mg/kg bw. Stability tests for hydroxylammonium sulfate at the highest dose of 1600 ppm in the drinking water for a storage of 4 days resulted in an average of 1437 ppm. So the average hydroxylammonium sulfate concentration in drinking water of the 1600 ppm dose group was approximate 142 mg/kg bw/d for males and 149 mg/kg bw/d for females.No deaths occurred during the study. Food intake of rats was similar to that of the controls. At 1600 ppm, rats of both sexes showed decreased water consumption, cyanosis and discoloration (yellow-red) of the urine in the last treatment week.

In summary, hemolytic anemia and splenomegaly were the prominent manifestations of hydroxylammonium sulfate toxicity, esp. make in the 1600 and 400 ppm dose groups corresponding with cyanosis, changes in red blood parameters (enhanced levels of methaemoglobin, Heinz bodies and a shift in blood cell pattern, e.g. increase in immature forms of red blood cells - reticulocytes -, and a rise in leukocytes: neutrophile and eosinophile granulocytes, lymphocytes, and monocytes), changes in the biochemical composition of the plasma and relevant toxic effects in spleen, liver and kidneys. Increased decomposition of erythrocytes was seen as hemosiderin deposits and iron pigment deposition in these organs. Further, in Kupffer cells erythrophagocytosis were observed. Compensating effects were revealed in the spleen and liver as extramedullary hematopoiesis. Congestion of the spleen, enlargement of spleen sinus, splenomegaly and increased organ weight of the spleen were caused by immatureerythrocytes. Reticuloid hyperplasia and necrosis in bone marrow which were seen only in males at 1600 ppm, may be a possibly indication of bone marrow damage.No treatment related effects were observed in animals of both sexes at 25 ppm. The NOAEL for systemic effects was 25 ppm in males and 100 ppm in females (BASF AG 1989, Val. 2).

 

In a combined chronic toxicity/carcinogenicity study according to OECD TG 453, hydroxylammonium sulfate (purity commercial grade) was administered to groups of 50 male and 50 female Wistar rats in the drinking water at concentrations of 0, 5, 20 and 80 ppm for 24 months (main groups). In order to define the hematotoxic potential of the test substance, groups of 10 animals per sex and dose were treated for 12 months (satellite groups). In these satellite animals, assays of blood parameters were performed every three months. The doses administered corresponded to a mean daily hydroxylammonium sulfate intake in the main groups of about 0, 0.2, 1.0, and 3.7 mg/kg bw/d in males and 0, 0.4, 1.6, and 6.2 mg/kg bw/d in females; and in the satellite groups of about 0, 0.3, 1.1, and 4.5 mg/kg bw/d in males and 0, 0.4, 1.6, and 6.2 mg/kg bw/d in females. Food consumption, water consumption and body weight were determined once a week during the first 13 weeks. Thereafter water consumption and body weight were determined in 4-week intervals, and food consumption was ascertained in 3-months intervals. The animals were examined for signs of toxicity or mortality at least once a day. Moreover, comprehensive clinical examinations and palpation of the animals were performed once a week. Hematology was carried out in the satellite groups after 3, 6, 9 and 12 months. In the main groups, hematology was carried out after 12, 18 and 24 months. Complete necropsy and microscopy were performed on all animals. In satellite groups and main groups, no treatment-related clinical signs were observed in animals of both sexes, and no statistically significant differences in survival were noted between dose groups and control groups and sexes, respectively. The average body weights and body weight gains of treated males and females were comparable to those of the matched controls throughout the study. Results of hematology indicated dose-related anemia that was characterized as hemolytic and regenerative in rats observed at 3, 6, 9, 12 and 24 months. Signs of anemia were more pronounced in males. In satellite groups administration of 80 ppm caused hemolytic anemia in male and female rats expressed as statistically significantly decreases in red blood cell counts, haemoglobin concentrations and hematocrit values. Furthermore, there were statistically significant increases in MCV values and MCH values, and in platelet counts in females. A slightly elevated number of Heinz bodies, Howell-Jolly bodies and reticulocytes were noted in both males and females. The mean absolute and relative spleen weights were statistically significant increased in rats of both sexes at 80 ppm. At microscopy of the spleen, congested vessels, characterized by dilated, blood-filled vascular spaces were observed in males and females at 80 ppm. Hemosiderin storage in the spleen occurred in nearly all control and hydroxylammonium sulfate-treated animals, however with higher degree in males receiving 20 and 80 ppm, and in females at 80 ppm. In main groups, examination of erythrocyte morphology revealed at 80 ppm mild anisocytosis and microcytosis in males, increased polychromasia in females, and an elevated number of Howell-Jolly bodies in both sexes of these groups. Statistically significant increases in mean absolute and relative spleen weights were noted in females receiving 80 ppm. Microscopically, the following non-neoplastic findings were noted in the spleen: an increased number of congested vessels in animals of both sexes at 80 ppm, and hemosiderin storage in nearly all control and hydroxylammonium sulfate-treated animals, however with higher degree of severity in males given 80 ppm, and in females dosed with 20 and 80 ppm. Extramedullary hematopoiesis was also observed in the spleen in most of the animals of the main groups. In these animals the degree of severity was higher in males and females given 80 ppm, while the incidence was slightly increased in females. Comparable to the increased incidence of hematopoiesis in the spleen, an increased hematopoiesis was noted in the bone marrow in the 80 ppm dosed animals. Furthermore, a multifocal or diffuse pigment storage was noted in the liver. Most of the stored pigment turned out to be hemosiderin after iron staining. Diffuse pigment storage in the liver was noted in higher incidences and higher degrees in the 80 ppm dosed males and females.

In conclusion, the prolonged oral administration of 80 ppm hydroxylammonium sulfate via the drinking water to male and female rats caused hemolytic anemia, characterized by significant reduced counts of erythrocytes, haemoglobin concentrations and hematocrit values, increases in MCV, MCH, and furthermore, increased number of Heinz bodies, Howell-Jolly bodies, and reticulocytes in the peripheral blood. These adverse effects were associated with increases of spleen weights, increased red blood cell regeneration by the bone marrow and increased extramedullary hematopoiesis in the spleen and the liver. At 20 ppm (equivalent to about 1.0 mg/kg bw/d in males and 1.6 mg/kg bw/d in females), hemosiderin storage in the spleen, sign of hemolysis, were significantly increased when compared with controls in male rats after 12 months of treatment and in female rats after 24 months of treatment, respectively. No hematotoxic effects were detected in animals given 5 ppm. Therefore, the NOAEL for systemic effects was 5 ppm in males, corresponding to a mean daily hydroxylammonium sulfate intake of about 0.2/0.3 mg/kg bw/d, and 20 ppm in females corresponding to a mean daily hydroxylammonium sulfate intake of about 1.6 mg/kg bw/d.

No local toxic effects were noted in male and female rats treated with the highest tested dose level of 80 ppm, equivalent to about 3.7 mg/kg bw/d in males and 6.2 mg/kg bw/d in females (BASF AG 2001, Val. 1).

 

In an early subchronic toxicity study with limited validity, the repeated administration of hydroxylammonium sulfate in the drinking water to Swiss-Webster mice (males and females) at a concentration of 200 mg/kg bw/d for 52 weeks caused splenomegaly and anemia, and at 100 mg/kg bw/d bone formation in the spleen. A NOAEL for systemic effects in male and female mice could not be established. No local toxic effects were noted in male and female mice treated with the highest tested dose level of 200 mg/kg bw/d (Yamamoto et al. 1967, Val. 3; see chapter Carcinogenicity).

 

The aim of another study was to determine a possible mechanism of tumor induction in the spleen after oral administration of hydroxylammonium sulfate. Hydroxylammoniumsulfate was administered to groups of 10 male and 10 female Wistar rats in drinking water at concentrations of 0 and 400 ppm for 7 and 28 days. One week prior to necropsy, osmotic minipumps containing bromodeoxyuridine (BrdU) were implanted subcutaneously. Cell proliferation (S-Phase response) was evaluated semiquantitatively in the spleen. Heinz bodies and methemoglobin were determined, kidneys and spleen were weighed. Additional groups of 10 male and 10 female Wistar were treated with 0 and 400 ppm for 28 days, and 8-HO-deoxyguanosine and lipid peroxidation (measured as the amount of TBA-reactive material found in tissue) were determined in kidneys and spleen. This was done in order to investigate the hypothesis of induction of oxidative stress by the test substance in spleen . Food consumption, water consumption and body weights were determined weekly in all animals, and the animals were examined for clinical signs of toxicity or mortality at least once a day. Clinical examinations revealed no substance-related findings. Heinz bodies and methemoglobin formation were not increased by treatment. In both sexes, lipidperoxidation as well as 8-HO-deoxyguanosine levels in DNA were increased indicating that HAS induces oxidative stress in spleen. Histopathologically, hemosiderosis, extramedullar hematopoiesis, increased cellularity of red pulp cells, and sinusoidal congestion were seen all of which are indicative for the anemia induced by HAS. Endothelial cells did not proliferate within the time when osmotic minipumps were implanted (1 week). Semiquantitative assessment of cell proliferation in the other compartments of the spleen (white pulp, hematopoietic cells, and red pulp) did not reveal any substance-related effects. In conclusion, the present study gives indications that HAS induces oxidative stress in spleen, but does not lead to cell proliferation of endothelial cells or other compartments of the spleen under the conditions of this study (BASF AG 2001, Val. 1).

 

dermal

no data available

inhalation

no data available

Justification for classification or non-classification

For the classification of hydroylamine the same justification as of hydroxylamin sulfate was used. This is warranted because the relative amounts of hydroxylamine in a ≤55% aqueous solution and in hydroxylamine sulfate crystals are comparable.

Based on hydroxylamine sulphate data:

There have been discussions in the Technical Committee on Classification and Labelling (TC C&L) and the Specialised Experts group whether the data suggests a classification as R48/22 or whether a classification as R48/25 is warranted. The experts came to the conclusion that the available data does not warrant a classification as R48/25 for the following reasons (only non-neoplastic lesions and relevant concentrations are shown):

- 90 day / 3 month study

10 ppm (ca. 0.9 mg/kg bw/day): no substance-related effects.

50 ppm (ca. 4 mg/kg bw/day): minor effects on clinical chemistry and hematology parameters. Moderately increased hemosiderin deposits in the spleen of the males and females. This was not considered an effect which warrants a R48/25 classification. The high dose (250 ppm / 21 mg/kg bw/day) is beyond the limit concentration of 5 mg/kg bw/day and thus not relevant for the decision.

 

- 12 month study:

5 ppm (males: 0.3 / females: 0.4 mg/kg bw/day): no substance-related effects.

20 ppm group (males: 1.1 / females: 1.6 mg/kg bw/day): no hematotoxic effects; the hemosiderin deposits were observed in males and this effect had no dose response relationship.

80 ppm group (males: 4.5 / females: 6.2 mg/kg bw/day): only compensatory effects could be found. Histopathology showed signs of an anemia to an extent that can not be considered as “serious”. This concentration is considered to be higher than the limit concentration of 1.25 mg/kg bw/day (5 mg/kg bw/day divided by 4 as it was a 12 month study) and thus not relevant for the decision.

 

- 24 month study:

5 ppm (males: 0.3 / females: 0.4 mg/kg bw/day): no substance-related effects.

20 ppm group (males: 1.0 / females: 1.6 mg/kg bw/day): higher degree of hemosiderin storage in the spleen of females. Because hemosiderin storage was seen in the 12 month study in males only, and in the 24 month study to a higher extent only in females, this is considered to be a very week effect. This concentration is higher than 0.625 mg/kg bw/day (5 mg /kg bw/day divided by 8 as this was a 24 month study) and thus not relevant for the decision.

The same is true for GHS STOT RE classification, because when the guidance value for STOT RE 1 is taken into account (10 mg/kg bw/day), the data suggest a STOT RE 2 classification when the same evaluation of the effects is made.

Furthermore, it should be considered that hydroxylamine sulfate has a cancer classification (DSD: R40, Cat. 3 / CLP: Cat. 2).

According to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, Annex VI, the substance is classified:

- STOT RE 2

- R48/22