Ammonia, anhydrous

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Anhydrous ammonia is not considered to be genotoxic based on the negative results of in vitro testing (negative bacterial mutation with anhydrous ammonia and diammonium sulphate).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published, guideline-comparable study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine (S. Typhimurium); tryptophan (E.coli)

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Deficiency/Proficiency: histidine

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Deficiency/Proficiency: histidine

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Deficiency/Proficiency: histidine

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Deficiency/Proficiency: histidine

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: Deficiency/Proficiency: tryptophan

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix (induced with polychlorinated biphenyl, KC500)

Test concentrations with justification for top dose:

500, 1000, 2500, 5000, 10000 and 25000 ppm

Vehicle / solvent:

Air

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

sterilised air

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA100, TA98, TA1538 without S9.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

sterilised air

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

TA1535 and WP2uvrA without S9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

sterilised air

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

sterilised air

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA100, TA98, TA1537, TA1538 with S9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

sterilised air

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA1535 and WP2uvrA with S9

Details on test system and experimental conditions:

Exposure duration: 48 hours. Salmonella typhimurium strains and E. coli were deficient/proficient in histidine and tryptophan. The metabolic activation system was rat liver S9 mix (induced with polychlorinated biphenyl, KC500). Tests were performed in duplicate. The agar plates were exposed without a lid in a glass chamber.

Evaluation criteria:

No information

Statistics:

No information

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Additional information on results:

No further information

Remarks on result:

other: all strains/cell types tested

Ammonia was negative for genotoxicity in S. typhimurium and E. coli with and without metabolic activation.

Conclusions:

Interpretation of results: negative with and without metabolic activation

Ammonia was negative in an Ames test performed similar to OECD 471 with and without metabolic activation.

Executive summary:

The mutagenicity of anydrous ammonia was investigated in a Ames test in S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538) and in E. coli WP2 uvrA. The test method was modified appropriately to investigate a volatile test substance. Studies were performed in duplicate in the presence and absence of an exogenous metabolic activation system (Aroclor 1254 -induced male Sprague-Dawley rat liver S9 fraction). No evidence of mutagenicity was seen under the conditions of this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Anhydrous ammonia is not considered to be genotoxic based on the negative results of in vivo testing (negative Micronucleus test in mice with ammonium chloride). 

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached read-across justification

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not specified

Additional information on results:

The occurrence of micronucleated erythrocytes after single injection was 0.12%, and after four injections was 0.17%.

The occurrence of micronucleated erythrocytes after single injection was 0.12%, and after four injections was 0.17%.

Conclusions:

Interpretation of results: negative
Read-across substance ammonium chloride was negative for genotoxicity in the mouse micronucleus assay.

Executive summary:

The potential for the genotoxicity of read-across substance ammonium chloride was investigated in a bone marrow micronucleus assay in mice. Male ddY mice were administered ammonium chloride by single intraperitoneal injection at dose levels of 0, 62.5, 125, 250 or 500 mg/kg bw or as four injections within 24 hours at dose levels of 31.3, 62.5, 125 or 250 mg/kg bw. The maximum dose of ammonium chloride was determined by pilot experiments using the multisampling at multi-dose levels method. Dose levels of up to the maximum tolerated dose were used. Mice were killed 24 h after administration and femoral bone marrow cells were harvested, fixed and stained. 1000 PCEs per animal were scored using a light microscope and the number of micronucleated erythrocytes (MnPCEs) recorded. No evidence of genotoxicity was seen under the conditions of this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genotoxicity in vitro

No evidence of mutagenicity was seen in a key guideline-comparable Ames test performed with anhydrous ammonia in S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538 and in E. coli WP2 uvrA with and without metabolic activation (Shimizu et al,1985) or in a supporting guideline-compliant Ames test with the read-across substance diammonium sulphate in S. typhimurium TA98, TA100, TA1535, TA1537 with and without metabolic activation (BASF, 1989). Similarly, there was no evidence of mutagenicity in a supporting non-standard study using E. coli without metabolic activation (Szybalski, 1958).

Further in vitro testing for clastogenicity and mammalian mutagenicity is waived.

 

Genotoxicity in vivo

The potential for the genotoxicity of ammonium chloride was investigated in a bone marrow micronucleus assay in mice, dosed by single intraperitoneal injection at dose levels of 0, 62.5, 125, 250 or 500 mg/kg bw or as four injections within 24 hours at dose levels of 31.3, 62.5, 125 or 250 mg/kg bw. The maximum dose of ammonium chloride was determined by pilot experiments using the multisampling at multi-dose levels method. Dose levels of up to the maximum tolerated dose were used. Mice were killed 24 h after administration and femoral bone marrow cells were harvested, fixed and stained. 1000 PCEs per animal were scored using a light microscope and the number of micronucleated erythrocytes (MnPCEs) recorded. No evidence of genotoxicity was seen under the conditions of this assay (Hayashi et al, 1988).

 

Ammonia is a simple molecule and does not possess any structural alerts for genotoxicity. Ammonia is present at relatively low levels in the systemic circulation as a consequence of protein catabolism (largely in the liver) and is also present at higher levels in the hepatic portal circulation due to the breakdown of urea by gastrointestinal bacteria. The ubiquitous presence of ammonia in the leads to the conclusion that it is unlikely to be genotoxic. The WHO evaluation (EHC 54, 1986) concludes that there is no evidence that ammonia is mutagenic in mammals. A UK Health Protection Agency (HPA) evaluation similarly concludes that ammonia does not have significant mutagenic potential.

Justification for classification or non-classification

No classification according to CLP Regulation 1272/2008/EC is proposed for anhydrous ammonia: there is no evidence for genotoxicity in studies performed in vitro or in vivo.