Bis(hydroxylammonium) sulphate

Endpoint summary

Additional information

GENOTOXICITY IN VITRO:

CAS No. 10039-54-0:

Only limited data is available with very limited validity. In a poorly reported Ames screening study, the substance was tested in Salmonella TA 98, 100, 97 and 1535. No mutagenicity was observed. In another Ames screening study, the substance was tested in Salmonella TA 98 and 100, no mutagenicity was observed. In both tests, the test concentrations used were unclear.

In a non-guideline and non-validated cell transformation assay, the substance was tested on Balb/3T3 cells. The report is well documented, however the interpretation of results and biological relevance of the findings are unclear, therefore this study cannot be used for assessment.

CAS No. 5470-11-1:

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. The results obtained were ambiguous: There was no dose-related increase found with at least two doses being greater or equal to twofold background. However, the maximum revertant factor was in some single cases increased >2-fold but not in a repeated manner as defined in the criteria for a positive response. 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. The test was considered as ambiguous.

Also a second Ames test, conducted in S. typhimurium TA 97, TA 98, TA 100, TA 1535, with and without metabolic activation also provided ambiguous results: A weak positive trend at doses >100 µg/plate was seen, but not below this concentration with S. typhimurium TA 100 strain only. The mutation rate was not doubled at any dose, however a dose-related increase of the mutation rate was seen in some cases. The spontaneous mutation rates over all tests within the TA 100 strain ranged from ca. 100 to 170, where the majority of single results with HA were located >100 and <230 µg/plate, either. At doses >= 750 µg per plate a slight clearing of the background lawn was sometimes seen in all strains tested. The test was considered as ambiguous.

A third test of limited validity described negative results for hydroxylamine hydrochloride in Salmonella typhimurium TA 100 with and without S-9 mix and a fourth Ames test (also of limited validity) also described negative results for hydroxylamine hydrochloride in S. typhimurium TA 1535, 1536, 1537, 1538, 98, 100 with and without metabolic activation.

Two further studies were disregarded due to major methodological deficiencies: The substance was positive with respect to induction of bacterial gene mutations in E.coli K12 at an extremely high dose of 1 mol/l (69500 μg/ml) without S-9 mix after incubation for 20 minutes. Data on cytotoxicity were not given, the test system was not validated. In the second disregarded study, an E. coli DNA repair test was negative in strain polA without S-9 mix at the only tested dose of 500 μg/ml. Data on cytotoxicity were not given and also this test method is not validated.

Gene mutation in mammalian cells

Two mouse lymphoma assays 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 cytotoxic 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. Therefore, all four publications are considered of limited validity and were partly disregarded for assessment. In three publications, results were described as positive and in one as negative.

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.

Two further studies were disregarded due to significant methodological deficiencies: One study 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. This method was not validated and no information was given on controls, therefore it was considered unreliable.

A negative effect of hydroxylamine hydrochloride on chromosome damage in human leukocytes was described 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 totally toxic. The DNA repair synthesis was determined by autoradiography.

Further studies on DNA damage/repair were disregarded due to major methodological deficiencies: Hydroxylamine hydrochloride was marginally positive in lymphocytes from the Indian muntjac. The tested dose of 25 μg/ml induced a ca. 1.5-fold increase in the SCE frequency after treatment for 1h. Data on cytotoxicity were not given.

A weak effect of hydroxylamine hydrochloride in an SCE test in V79 cells was reported 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:

CAS No. 10039-54-0:

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 which was disregarded due to major methodological deficiencies.

In a mouse micronucleus assay conducted according to OECD Testing Guideline and GLP, hydroxylammonium sulfate led to a negative result after single oral administration of 300, 600 and 1200 mg/kg bw. Sampling times were 16, 24 and 48 h. All doses led to toxic signs. There was no clear effect on local cytotoxicity (PCE/NCE ratio), however due to the effects of the test substance on the blood as reported in the acute and repeated dose toxicity, exposure of the blood can be expected. Five male and five female mice per dose group were used. In a pre-test lethal effects were observed at 1400 mg/kg bw.

Another in vivo micronucleus test was also negative after oral administration of hydroxylammonium sulfate. 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. 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 is also available for hydroxylammonium sulfate. According to the authors, hydroxylammonium sulfate induced chromosomal inversions in cells of salivary glands of Drosophila. However, this study was disregarded for assessment due to major methodological insufficiencies.

 

Several studies investigating the genotoxic potential of hydroxylamine hydrochloride in vivo are available, however, all studies are suffering from severe methodological insufficiencies.

An in vivo chromosomal aberration test with hydroxylamine hydrochloride in mice 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.

Several additional studies were disregarded for assessment due to severe methodological deficiencies: Hydroxylamine hydrochloride was tested in a somatic mutation and recombination test with Drosophila in a SMART assay. 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) a positive result was reported after feeding with hydroxylamine hydrochloride in a dose of 0.03 mol/l (2070 μg/ml). Another study 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.

 

Justification for classification or non-classification

Mainly negative results were obtained in bacterial genotoxicity tests. In two gene mutation tests in bacteria ambiguous effects were seen. Hydroxylammonium chloride was weakly positive in mouse lymphoma assays and seems to express a genotoxic potential in insects.

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

Overall it may be concluded that the tested material had 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.