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Genetic toxicity in vitro

Bacterial reverse mutation assay (Ames test)

Harlan Laboratories Ltd (2010) performed a bacterial reverse mutation assay (Ames test, the method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No.471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines) in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- with and without metabolic activation (rat liver homogenate metabolising system: 10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Vehicle and positive controls without metabolic activation (N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate for WP2uvrA-, 3 µg/plate for TA100 and 5 µg/plate for TA1535; 9 -aminoacridine (9AA): 80 µg/plate for TA1537 and 4 -Nitroquinoline-1 -oxide (4NQO): 0.2 µg/plate for TA98) and with metabolic activation (2 -Aminoanthracene (2AA) and Benzo(a)pyrene (BP)) were tested simultaneously and were observed to be valid. Phosphoric acid tested negative with and without metabolic activation.

In vitro mammalian chromosome aberration test The National Institute of Environmental Research (2005) performed a chromosome aberration test with phosphoric acid in Chinese Hamster Lung (CHL/IU) cell line with and without metabolic activation (according to OECD Guideline 473). Concentrations of 112.5, 225.0 and 450.0 µg/mL were tested. Vehicle (water for injection), negative and positive controls (Mitomycin C without metabolic activation and benzo(a)pyrene with metabolic activation) were tested simultaneously. Phosphoric acid tested negative with and without metabolic activation. Cytotoxicity was observed: the IC50 (inhibition concentration 50%) were calculated as 450.0 µg/mL, in the short time treatment and continuous treatement system without and with metabolic activation system. Positive and negative controls were valid. Mammalian cell gene mutation assay (Mouse lymphoma assay): Harlan Laboratories Ltd (2010) performed a mouse lymphoma assay according to the UK Environmental Mutagen Society guidance which is equivalent/similar to OECD Guideance 476 and EU method B.17. Concentrations of 0, 61.25, 112.5, 245, 490, 735 and 980 µg/mL were tested on the L5178Y TK+/- 3.7.2 c mouse lymphoma cell line. Solvent control and positive control (ethylmethanesulphonate without metabolic activation and cyclophosphamide with metabolic activation) were tested simultaneously. Phosphoric acid was found to be negative with (4 hours of exposure) and without metabolic activation (4 and 24 hours of exposure). Cytotoxicity was very modest: no evidence of any reductions in viability was observed. Therefore, no residual toxicity occured. Genetic toxicity in vivo No reliable data were available on the genetic toxicity in vivo endpoint. Phosphoric acid tested negative in in vitro toxicity tests (Ames test, mammalian chromosome aberration test, mouse lymphoma assay) and therefore no in vivo mutagenicity tests should be performed with this substance.

Short description of key information:
Bacterial reverse mutation assay (Ames test):
Phosphoric acid was found to be negative with and without metabolic activation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA-. The test was performed according to OECD Guideline 471.
In vitro mammalian chromosome aberration test:
Phosphoric acid was found to be negative with and without metabolic activation in Chinese Hamster Lung (CHL/IU) cell line. The test was performed according to OECD Guideline 473.
Mammalian cell gene mutation assay (mouse lymphoma assay):
Phosphoric acid was found to be negative with and without metabolic activation in L5178Y TK+/-3.7.2c mouse lymphoma cell line with and without metabolic activation. The test was performed according to the UK Environmental Mutagen Society guidance which is equivalent/similar to OECD Guideline 476 and EU method B.17.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Phosphoric acid was found to be negative in all available in vitro gene mutation tests and therefore, the substance should not be classified for mutagenicity.