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In vitro Gene Mutation

Bacteria

Several valid studies are available for the assessment of gene mutation in bacteria.

In a mutagenicity assay (OECD471) the test substance was tested to evaluate its mutagenic potential. The dose levels tested were 50, 150, 500, 1500 and 5000µg/plate. Neither precipitation nor toxicity was observed. No positive mutagenic response was observed with the tester strains (TA98, TA100, TA1535, TA1537, E. coli WP2 uvrA) in either the presence or absence of S9 activation (Eastman 2016).

Using a protocol comparable to OECD TG 471, propionic acid (99 % pure) was tesd in two replicate assays as a coded chemical in the Bacterial Reverse Mutation Test using the preincubation method and Salmonella typhimurium strains TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538. In each replicate assay, propionic acid was tested in triplicates at doses of 0, 100, 333, 1000, 3333, and 10000 µg/ plate (no data on vehicle) in the presence and absence of Aroclor-induced liver S-9 mix from Sprague Dawley rats and Syrian hamsters. Toxicity was determined by a thinning of the background lawn, and/or a reduction in the number of colonies per plate. There was no evidence of induced mutant colonies over background up to a maximum dose of 10000 μg/ plate both in the presence and absence of metabolic activation (Zeiger et al.1992).

 

In a bacteria gene mutation assay performed using the NTP standard protocols, the mutagenic potential of propionic acid (no data on purity) in bacteria was determined using the preincubation method in Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and 97 at the concentrations of 0, 100, 333, 1000, 3333, 6667 μg/ plate without metabolic activation and 0, 100, 333, 1000, 3333, 10000 μg/ plate with metabolic activation. S-9 mix from liver of Aroclor-treated Sprague Dawley rats and Syrian hamsters was employed for metabolic activation. Water was employed as vehicle. Toxicity was determined by thinning of the background lawn, and/or a reduction in the number of colonies per plate. Cytotoxicity was evident only at the highest concentrations. Positive controls induced the appropriate responses. There was no evidence of induced mutant colonies over background up to a maximum dose of 10,000μg/ plate both in the presence and absence of metabolic activation (NTP 1986).

 

Using a protocol comparable to OECD TG 471, Basler et al. (1987) demonstrated that propionic acid (99% pure) in water was not mutagenic to bacteria (Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100) in the Bacterial Reverse Mutation Test at concentrations of 0, 0.01, 0.03, 0.1, 0.3, 1.0, 3.3, 10.0 μl/ plate using the standard plate incorporation assay protocols, in the presence and absence of metabolic activation (S9 mix from liver Aroclor 1254-induced male Wistar-rats). No cytotoxity was observed and the positive controls induced the expected responses.

 

Mammalian Cells (weight of evidence approach)

In a mammalian cell gene mutation assay (HPRT locus) performed with a homologue carboxylic acid (Formic acid, CAS 64-18-6) according to the OECD Guideline No. 476 and under GLP conditions. Chinese Hamster ovary cells cultured in vitro were exposed to formic acid at concentrations of 0, 31.25, 62.5, 125, 250, and 500 μg/mL in the presence, and of 0, 25, 50, 100, 200, and 400 μg/mL in the absence of mammalian metabolic activation. Formic acid was tested up to cytotoxic concentrations (i.e., 200 to 400 µg/mL in the absence, and 400 to 500 µg/mL in the presence of metabolic activation) without increasing mutation frequency at any concentration.  The positive controls did induce the appropriate response as did the vehicle control. There was no evidence of induced mutant colonies over background in vitro in the CHO/HPRT assay, with or without metabolic activation.

 

The same result was obtained in a mammalian cell gene mutation assay (CHO cells; HPRT locus) performed with n-butyric acid (CAS 107-92-6) according to the OECD Guideline No. 476 and under GLP conditions. At concentrations of 0 3.44, 6.88, 13.75, 27.5, 55, 110, 220, 440, 660, 880 μg/mL there was no evidence of induced mutant colonies over background in vitro in the CHO/HPRT assay, with or without metabolic activation. The positive controls did induce the appropriate response as did the vehicle control.

 

In conclusion, the homologue carboxylic acids (formic acid, n-butyric acid) are negative in the mammalian cell gene mutation assay. Thus, it can be concluded that propionic acid will also give a negative result in a mammalian cell gene mutation assay.

In vitro Cytogenetics

In a mammalian sister chromatid exchange assay equivalent to OECD TG 479, V79 cells cultures were exposed to propionic acid (99% pure) in water at concentrations of 0.1 0.3, 1.0, 3.3, 10, 33.3 mM for 3 hours with metabolic activation (S9 mix from the livers of Aroclor 1254 treated male rats) and for 28 hours without metabolic activation. Duplicate trials with triplicate cultures per trial were performed. 25 metaphases per dose were scored. Cytotoxicity was evident without metabolic activation only at concentrations of equal to or greater than 10mM. Positive controls induced the appropriate response. There was no evidence of SCE induced over background (Basler et al. 1987).

 

In another SCE assay, propionic acid (99.5% pure) was tested for the ability to induce SCEs in cultured human lymphocytes (peripheral blood). The lymphocyte cells were exposed without metabolic activation to concentrations of 0.25, 1.25, 2.5, 5, 10, 20 mM propionic acid in RPMI 1640 growth medium. 24 hours post treatment the cells were harvested and fixed. Positive control caused the appropriate response. Only one trial was performed. Propionic acid slightly increased SCEs (less than two fold) with statistical significance at 2.5 mM. However this concentration caused cytotoxicty. The minor increase in SCEs - less than twice the control level- resulting from a single trial with a single sampling time, needs to be treated with caution, especially since the concentration giving a positive effect is sub-toxic. The RIs of the treated cells decreased at the concentration yielding a positive SCEs indicating a decrease in cell proliferation at this dose. In this study, propionic acid is thus ambiguous for causing SCE in human lymphocytes (Sipi et al 1992).

 

In an in vitro bacterial DNA damage and repair assay (SOS Chromotest), propionic acid (99% pure) was tested at concentrations of 0.01, 0.03, 0.3, 1, 3.3, 10, 33.3 mM in water with Escherichia coli PQ37 for the incubation duration of 2 hours. Cytotoxicity was evident at the concentrations of 10 mM and above. Propionic acid was negative in this test (Basler et al 1987).

 

In vivo Cytogenetics

In a Chinese hamster (Cricetulus griseus) bone marrow micronucleus assay, 6 animals/ sex/ dose were treated once with propionic acid (99 % pure) by means of intra-peritoneal injection at doses of 0 and 2.5% in physiological saline (approx 125 mg/kg bw). Bone marrow cells were harvested at 12, 24 and 48 hours post-treatment. Four hamsters died within a few hours of dosing. The positive control elicited the appropriate response. There was no significant increase in the incidence or frequency of micro-nucleated polychromatic erythrocytes in the bone marrow of male or female hamsters treated with propionic acid as compared to concurrent controls (Basler et al.1987). This study is acceptable for assessment. The study design is comparable to the recommendations of the OECD TG 474 for in vivo cytogenetic mutagenicity data.


Short description of key information:
- In vitro bacteria gene mutation: negative
- in vitro mammalian gene mutation: negative (weight of evidence)
- In vitro cytogenicity: negative (weight of evidence)
- In vivo cytogenetics: negative

Endpoint Conclusion:

Justification for classification or non-classification

Classification for genetic toxicity is not warranted according to the criteria of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.