Pyrazine-2-carboxylic acid

PBT assessment

PBT assessment: overall result

PBT status:

the substance is not PBT / vPvB

Justification:

Classification of Pyrazine-2-carboxylic acid for effects in the environment:

 

The chemical Pyrazine-2-carboxylic acid (CAS no. 6928-85-4) is used as intermediate in chemical synthesis and pharmaceutical industry. The aim was to assess whether the PBT criterion within Annex XIII was fulfilled for Pyrazine-2-carboxylic acid. The PBT criterion was herein assessed based on experimental data in conjunction with standardized environmental fate models. Here follows a description of the PBT assessment.

 

 

Persistence assessment

The tested substance does not fulfil the P criterion within Annex XIII based on the assessment that here follows:

 

Biotic degradation

In a weight of evidence study from review article (K. R. Girija et. al; 2002), biodegradation study of test substance pyrazine-2-carboxylic was performed in both aerobic and anaerobic conditions. Bacterial cultures were used as a test inoculum isolated from environmental samples like cow dung, domestic sewage sample, industrial effluents, effluents from common effluent treatment plant etc. The bacterial isolates were grown in mineral salt medium containing pyrazine-2-carboxylic acid (1 mM) as sole nitrogen source and glucose (3%) as sole source of carbon. Concentration of the test substance used for the study was 124.099 mg/l. Aerobic bioreactor is used as a test vessel in the study when Pseudomonas aeruginosa strain “Cd” is taken for cultivation. 0.5 ml of various bacterial cultures were inoculated into 5 ml of pyrazine-2-carboxylic acid (nitrogen source) and glucose (carbon source) in both test tubes and screw cap tubes. It is then incubated under both aerobic and anaerobic conditions for 24 hrs. Test vessel containing the test substance pyrazine-2-carboxylic acid, glucose but not the bacterial culture was used as a control. Biodegradation was determined by measuring the U.V absorption maxima at 268 nm and decrease in O.D at that lambda i.e; at 268 nm and at 540 nm. Among the tested bacterial strains, only Pseudomonas aeruginosa strain “Cd” was able to degrade the test substance pyrazine-2-carboxylic acid in aerobic conditions. The percent degradation was determined to be 90% within 5 days after which no further degradation was observed. Thus, based on percentage degradation, test chemical pyrazine-2-carboxylic acid was considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for evaluating the percentage biodegradability of test substance Pyrazine-2-carboxylic acid (CAS no. 98-97-5) (K. S. Rajini, Ch. Sasikala, Ch. V. Ramana, 2010). Stenotrophomonas sp. HCU1 was used as a test inoculum. Strain HCU1 was serendipitously identified from a pyrazine-2-carboxylate contaminated broth growing in lab. Conc. of test chemical used for the study was24.81 mg/l (200µM).Pyrazine-2-carboxylate (used as an additional supplement) adapted strain HCU1 was harvested by centrifugation (16,0009gfor 10 min) and the pellet was washed (twice) with 0.05 M Tris–HCl buffer (pH 7.4) and resuspended in 1 ml of the same buffer. Cells were sonicated with MS-72 probe (Bandelin, model-UW 2070) to complete cell lysis after 8–9 cycles. The sonicated cell lysate was used for assay of pyrazine-2-carboxylate reduction and degradation. The assay mixture (1 ml) contained; 200µM of pyrazine-2-carboxylate, 200µM of NADPH, 5 mM MgCl2 and 40µl (130–140µg ml-1 protein) of the cell free extract in Tris HCl (pH 7.4). The reaction was stopped at an appropriate time by adding 10µl of 5 N HCl and filtered through 0.22µm polyvinyldienefluoride membrane. The samples were stored at 4°C and were analyzed in HPLC. Ring reduction of pyrazine-2-carboxylate was enzymatically assayed using 1 mM PCA, 200µM NADPH and increase in absorbance of PCA was calculated in terms of % peak height increase/microgram gram protein/minute. Km and Vmax of the enzyme were calculated using the software graph pad prism 5.0.

Mid-logarithmic phase culture (8 h) of strain HCU1 was harvested by centrifugation (12,0009gfor 20 min) and the supernatant was directly concentrated in flash evaporator to dryness. The concentrate was extracted with 50 ml methanol and the precipitated impurities were removed by centrifugation (12,0009gfor 15 min). The methanolic supernatant was diluted with methanol, water (1:0.2) where further precipitation occurred, which was separated by centrifugation (12,0009gfor 15 min). The precipitate was redissolved in methanol (fraction A) and the methanolic water supernatant was concentrated to dryness (fraction B). Both fractions were used for further metabolite purification using semi-preparative HPLC. Analysis of substrates and products was performed in HPLC at room temperature using a Shimadzu SPD-10AT isocratic or gradient system. HPLC instrument details are: solvent flow rate—1.5 ml min-1, Luna 5µC18 100A column (250×4.6 mm) and the heterocyclic compounds were detected using PDA detector at 268 nm. Pyrazine-2-carboxylate, (tR=1.77 min) were identified using standards. Increase in the absorbance (m AU) at 268 nm in HPLC was attributed to aromatic ring reduction of the compounds and loss as its utilization. Purity of the metabolites were confirmed using HPLC with three different solvent systems (methanol: water [1:1]; methanol: water: acetonitrile [1:1: 0.25]; water: acetonitrile [7:3]) at 230, 268, 280 and 350 nm for independent analysis. In addition to this, LC-MS, NMR and Gel exclusion chromatography in FPLC (Fast Protein Liquid Chromatography) was also carried out.  

One metabolite was isolated through semi-preparative HPLC from fraction A. This metabolite had a tR of 1.55 min. The metabolite was characterized based on IR, 1H, 13C NMR and mass spectroscopic analyses. The metabolite is a white amorphous solid and has absorption maximum at 268 nm. Based on the spectral data the metabolite is proposed as 1,2,5,6-tetrahydropyrazine-2-carboxylate. Eight chromatographically (HPLC) distinct peaks were observed from fraction B. This metabolite had a tR of 1.55 min. The metabolite is an orangish sticky compound and has an absorption maximum at 400 nm. The metabolite was characterized based on IR, 1H, 13C NMR and mass spectroscopic analyses. Based on the spectral data analyses, the metabolite is proposed as 2-amino-2-hydroxy-3-(methylamino)propanoic acid. The percentage degradation of test substance Pyrazine-2-carboxylic acid was determined to be 86% by HPLC parameter. Thus, based on percentage degradation, Pyrazine-2-carboxylic acid is considered to be readily biodegradable in nature.

 

In a prediction using the Estimation Programs Interface Suite (EPI suite, 2017), the biodegradation potential of the test compoundPyrazine-2-carboxylic acid(CAS No. 98-97-5) in the presence of mixed populations of environmental microorganisms was estimated. The biodegradability of the substance was calculated using seven different models such as Linear Model, Non-Linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI Linear Model, MITI Non-Linear Model and Anaerobic Model (called as Biowin 1-7, respectively) of the BIOWIN v4.10 software. The results indicate that chemical Pyrazine-2-carboxylic acid is expected to be readily biodegradable.

 

Further, experimental studies for the read across substances (CAS: 108-99-6 and 65-85-0) also indicate that they are readily biodegradable.

 

Environmental fate

According to the fugacity model levels III, the most likely environmental fate for this test chemical is soil (i.e.estimated to 65.4%). In soil, Pyrazine-2-carboxylic acid was expected to have rapid mobility based upon an estimated Log KOCin the range 0.0 – 1.008. Thehalf-life in soil (30 days estimated by EPI suite) indicates that the chemical is not persistent in soil and the exposure risk to soil dwelling animals is moderate to low.

 

If released in to the environment, 34.5 % of the chemical will partition into water according to the Mackay fugacity model level III in EPI suite version 4.1 (2017). However, the half-life (15 days estimated by EPI suite) indicates that the exposure risk to aquatic animals is moderate to low.

 

Moreover, its persistent characteristic is only observed in the sediment compartment but Fugacity modelling shows that sediment is not an important environmental fate (less than 1% when estimated by EPI Suite version 4.1).

 

Hence it has been concluded that Pyrazine-2-carboxylic acid is not persistent in nature.

 

 

Bioaccumulation assessment

The tested substance does not fulfil the B criterion within Annex XIII based on the assessment that here follows:

 

Theestimated BCF value from various databases was determined to be in the range 1.0 – 4.28 and theoctanol water partition coefficient of the test chemical is -0.18 which is less than the threshold of 4.5. If this chemical is released into the aquatic environment, there should be a low risk for the chemical to bioaccumulate in fish and food chains.

 

Toxicity assessment

The tested substance does not fulfil the T criterion within Annex XIII based on the assessment that here follows:

 

Mammals

The tested chemical is regarded to be not classified for carcinogenicity, mutagenicity and reprotoxicity, Further, there is no evidence of chronic toxicity, as identified by the classifications STOT (repeated exposure), category 1(oral, dermal, inhalation of gases/vapours, inhalation of dust/mist/fume) or category 2 (oral, dermal, inhalation of gases/vapours, inhalation of dust/mist/fume).

 

Aquatic organisms

All of the available short-term eco-toxicity estimations for fish, invertebrates and algae for the substance indicates the LC50/EC50 value to be >100 mg/L. These value suggest that the substance is likely to be non-hazardous to Aquatic organisms at environmentally relevant concentrations and can be considered to be not classified as per the CLP regulation.

 

There are no available long-term toxicity evaluations for Pyrazine-2-carboxylic acid. By speculation, long-term NOEC for aquatic organisms were not expected for the substance at concentration below 0.01 mg/L based on the data mentioned above.

 

The chemical was therefore not considered as hazardous to aquatic environments as per the criteria set out in Annex XIII.

 

Conclusion

Based on critical, independent and collective evaluation of information summarized herein, the tested compound does not fulfil the P, B and T criterion and has therefore not been classified as a PBT compound within Annex XIII.