Hydroxylamine

Endpoint summary

Additional information

For hydroxylamine (free base) no data is available. However, valid studies of the corresponding salts hydroxylamine sulfate (CAS 10039-54-0) and hydroxylamine chloride (CAS 5470-11-1) are available: CAS No. 10039-54-0:

GENOTOXICITY IN-VITRO:

no data available

GENOTOXICITY IN-VIVO:

Hydroxylammonium sulfate showed no mutagenic activity in in-vivo assays with rodents. However, a positive response was found in an in-vivo test with insects with limited validity.

In a mouse micronucleus assay on polychromatic erythrocytes (BASF AG, 1992) hydroxylammonium sulfate led to a negative result after single oral administrations of 300, 600 and 1200 mg/kg bw. Sampling times were 16, 24 and 48 h. All doses led to clinical signs of toxicity. An inhibition of erythropoiesis (decrease of PCE:NCE ratio) was observed at 1200 mg/kg at sacrifice intervals of 16 and 24 hours. Five male and five female mice per dose group were used. In a pre-test lethality was observed at 1400 mg/kg bw.

Another in vivo micronucleus test was also negative after oral administration of hydroxylammonium sulfate (Litton Bionetics 1980). In this test, however, only low doses of 15.6 and 125 mg/kg were used (no clear statement whether the tested doses were given in two parts or twice). Sampling time was 6 h after last administration. Four male and four female mice per dose group were used. Data about toxic effects were not given. No cytotoxicity was induced.

A rodent germ cell test is available for hydroxylammonium sulfate (Epstein et al. 1972). In a dominant lethal assay with mice hydroxylammonium sulfate led to a negative result after single intraperitoneal injection of 102 and 112 mg/kg bw with respect to early fetal deaths and preimplantation loss. The findings were not described in detail. Seven to nine males were used per group, each treated male was caged with three untreated virgin females which were replaced weakly for eight consecutive weeks. There were no concurrent positive or negative control groups. The tested doses were equivalent to the LD50 and the LD25.

A test with Drosophila melanogaster with limited validity is also available for hydroxylammonium sulfate (Parkash and Miglani 1978). According to the authors, hydroxylammonium sulfate induced chromosomal inversions in cells of salivary glands of Drosophila. This result was considered as not relevant due to methodological insufficiencies.

 

CAS No. 5470-11-1:

GENOTOXICITY IN-VITRO:

Valid experimental data were available to assess the genetic toxicity in-vitro. Tests were available investigating gene mutation in bacteria and in mammalian cells, as well as cytogenicity in mammalian cells and DNA damage and/or repair.

Gene mutation in bacteria

An Ames test was conducted in S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2uvrA strains, with and without metabolic activation (Dunkel et al. 1985). The test gave no indication of mutagenicity: There was no dose-related increase found with at least two doses being greater or equal to twofold background. The maximum revertant factor was in some single cases increased > 2-fold but not in a dose-related and reproducible manner. As the test material was tested up to 333.3 µg/per plate only, no prediction in terms of genotoxic effects of the test material at higher doses (up to 5000 µg/plate) is possible. A slight decrease in the number of back-revertants was sometimes seen in the highest concentration tested.

In another Ames test (Zeiger et al. 1992), conducted in S. typhimuriumTA 97, TA 98, TA 100, TA 1535, with and without metabolic activation, weakly positive mutagenic responses were observed at doses > 100 µg/plate in S. typhimurium TA 100 in the presence of rat liver S9 mix only. The mutation rate was not doubled at any dose, however a dose-related increase of the mutation rate was seen in some cases. In the absence of metabolic activation, and in none of the other strains tested, a positive response was seen. At doses >= 750 µg per plate a slight clearing of the background lawn was sometimes seen in all strains tested.

Information on two further Ames tests was available - however, due to limited documentation, these reports were not considered sufficient for assessment: Kier et al. (1986) described negative results in Salmonella typhimurium TA 100 with and without S-9 mix, and Simmon (1979) reported negative results in S. typhimurium TA 1535, 1536, 1537, 1538, 98, 100 with and without metabolic activation. Sledziewska-Gojska et al. (1992) published results of bacterial mutagenicity assay in E. coli K12. In this assay, hydroxylamine hydrochloride was positive with respect to induction of bacterial gene mutations at an extremely high dose of 1 mol/l (69500 μg/ml) without S-9 mix after incubation for 20 minutes. The study is deemed unreliable because the method is not validated, only one very high concentration was tested, no metabolic activation system was used, and data on toxicity were not provided.

An E. coli DNA repair test with hydroxylamine hydrochloride (Rosenkranz et al. 1980) was negative in strain polA without S-9 mix at 500 μg/ml - however, this study was also considered unreliable because only one concentration was tested and no data on toxicity were given.

Gene mutation in mammalian cells

Two mouse lymphoma assays with hydroxylamine hydrochloride (Myhr and Caspary 1988; Mitchell et al. 1988) led both to weakly positive results with and without S-9 mix. These effects were reproducible and dose-dependent. In both investigations the treatment time was 4 h. With S-9 mix doses ranging from 12.5 to 583 μg/ml were tested; the lowest observed effect dose (LOED) was in the range of 200 to 410 μg/ml. Without S-9 mix doses from 3.9 to 205 μg/ml were tested; the LOEDs varied from 31 to 67 μg/ml. The maximum mutation frequencies were 2- to 4-fold that of the corresponding negative controls. In one of the two assays toxic effects were observed without S-9 mix.

Cytogenicity in mammalian cells

Tests for induction of chromosomal aberrations are available for hydroxylamine hydrochloride. All four studies suffer from severe methodological insufficiencies such as no use of S-9 mix and positive controls. Furthermore, no differentiation was made of chromosomal aberrations with and without gaps. In three publications results were described as positive (Borenfreund et al. 1964; Brogger 1971; Gupta et al. 1982) and in one as negative (Oppenheim et al. 1965).

Brogger analysed the effect of hydroxylamine hydrochloride on induction of chromosomal aberrations in human lymphocytes for a dose range of 25 to 100 μg/ml. Two of three experiments were not considered because of very high chromosomal aberration rates of 15.0% and 7.0% in the negative controls. In the third experiment there were positive results at the tested doses of 25, 50 and 100 μg/ml after exposure for 4, 6, 12 and 24 h. The effects were dose-dependent; the maximum aberration frequency was 15% (negative control, 1.0%). Toxic effects were induced by doses of 50 and 100 μg/ml. Borenfreund et al. described that hydroxylamine hydrochloride induced chromosomal aberrations in cells of a Chinese hamster cell line, originally derived from a methylcholanthrene-induced tumour. The only tested dose of 0.072 μmol/l (5.0 μg/ml) induced an aberration frequency of 13% (negative control, 5.0%) and decreased the mitotic activity by ca. 40%. Gupta and Sharma reported that hydroxylamine hydrochloride induced chromosomal aberrations in Indian muntjac lymphocytes. The frequencies of chromosomal aberrations were increased after 1 h exposure to 25 and 50 μg/ml. Toxicity data were not given. A negative effect of hydroxylamine hydrochloride on chromosome damage in human leukocytes was described by Oppenheim et al. at a dose-range of 47 to 500 μmol/l (4.6 to 34.5 μg/ml). The authors speculated that this effect was due to technical factors. Leukocytes were contaminated with red blood cells. Since hydroxylamine hydrochloride is rapidly destroyed on contact with haemoglobin 2 to 3% contamination would suffice to remove the doses of hydroxylamine hydrochloride.

DNA damage and/or repair

Hydroxylamine hydrochloride was negative for induction of unscheduled DNA synthesis (UDS) in primary rat hepatocytes for doses up to up 1000 μg/ml. Higher doses were toxic. The DNA repair synthesis was determined by autoradiography (Williams et al. 1982).

Two SCE assays were available, which were considered unreliable due to methodological insufficiencies:

Hydroxylamine hydrochloride was also marginally positive in lymphocytes from the Indian muntjac (Gupta et al. 1982). The tested dose of 25 μg/ml induced a ca. 1.5-fold increase in the SCE frequency after treatment for 1 h. Toxicity data were not given.

Speit et al (1980) reported on a weak effect of hydroxylamine hydrochloride in V79 cells in the dose-range 10-5 to 5x10-3 mol/l (0.7 to 345 μg/ml). At doses from 10-5 mol/l to 5x10-4 mol/l (0.7 to 34.5 μg/ml) SCE frequencies were marginally increased after continuous treatment for 27 h. At higher doses from 10-3 to 5x10-3 mol/l (69 to 345 μg/ml) the treatment time was limited to 1 h because of drastic toxic effects. Again the induced effect was weak; the maximum SCE frequency was 1.5-fold that of the negative control.

GENOTOXICITY IN-VIVO:

Several studies investigating the genotoxic potential of hydroxylamine hydrochloride in-vivo are available - however, all studies suffer from severe methodological shortcomings or are insuficciently documented.

An in vivo chromosomal aberration test with hydroxylamine hydrochloride in mice (Volgareva, 1991) led to a negative result after single intraperitoneal doses of 6.7 and 67 mg/kg bw. The highest tested dose was 1/3 of the LD50. Only five mice (sex was not specified) per dose group were used. Sampling times were 24 and 48 h. Data on toxic effects were not given. Hydroxylamine hydrochloride was positive in a somatic mutation and recombination test with Drosophila in a SMART assay (Graf, 1989). After feeding of 90 and 120 mmol/l (6219 and 8280 μg/ml) for 48 h significant increases of frequencies of small and large single spots and twin spots in the wings were induced. In a sex-linked recessive lethal test with Drosophila (SLRL test) (Vijaykumar et al., 1979) a positive result was reported after feeding with hydroxylamine hydrochloride in a dose of 0.03 mol/l (2070 μg/ml). Fahmy and Fahmy (1970) described that hydroxylamine hydrochloride was negative in a test for dominant lethals in Drosophila after feeding of 0.1 mol/l (6900 μg/ml). Induction of chromosomal effects in spermatocytes of grasshoppers (Spathosternum prasiniferum) was reported (Bhattacharya et al. 1986).

Short description of key information:
For hydroxylamine (free base) no data is available. However, multiple studies of the corresponding salts "hydroxylamine sulfate" and "hydroxylamine chloride" are available. For details see discussion.

Endpoint Conclusion:

Justification for classification or non-classification

Mainly negative results were obtained in bacterial genotoxicity tests. In one gene mutation test in bacteria, marginal effects were observed at high doses in S. typhimurium TA100 only, whereas another bacterial gene mutation assay was negative. Hydroxylammonium chloride was weakly positive in mouse lymphoma assays and seems to express a genotoxic potential in insects.

However, clearly negative results were obtained concerning UDS in rat hepatocytes and chromosomal aberrations in rodent bone marrow cells. Further data were of relatively low reliability or significance.

Overall it may be concluded that the tested material has no or a low genotoxic potential. In any case, it is unlikely that a mutagenic potential is expressed in mammals in vivo. Therefore, hydroxylamine and its salts (sulfate and chloride) will not be classified as mutagen.

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

not classified