2-oxoglutaric acid

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
EPISUITE v4.11

2. MODEL (incl. version number)
BIOWIN v4.10

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
SMILES: O=C(O)C(=O)CCC(=O)O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
For more detailed information please refer to the attached model description

5. APPLICABILITY DOMAIN
- Descriptor domain: Aerobic and anaerobic biodegradation, molecular weight, structural features
- Structural and mechanistic domains: BIOWIN estimates the probability of rapid aerobic and anaerobic biodegradation of an organic compound in the presence of mixed populations of environmental microorganisms. BIOWIN contains seven separate models. (see also Boethling et al. 2003).
- Similarity with analogues in the training set: When a structure contained none of the fragments occurring in the training or validation sets of the 7 used models, for which coefficients were developed, the program could not estimate the probability. With the inclusion of the molecular weight parameter, estimates are possible for all structures. Thus, exceeding molecular weight reveales unreliable predictions. Since the substance contains fragments which are included in either the training set or the validation set predictions are considered to be reliable.
- Other considerations (as appropriate): Currently there is no universally accepted definition of model domain. However, users may wish to consider the possibility that biodegradability estimates are less accurate for compounds outside the MW range of the training set compounds, and/or that have more instances of a given fragment than the maximum for all training set compounds. It is also possible that a compound may have a functional group(s) or other structural features not represented in the training set, and for which no fragment coefficient was developed; and that a compound has none of the 36 or 37 (Biowin 7) fragments in the model’s fragment library. In the latter case, predictions are based on molecular weight alone. These points should be taken into consideration when interpreting model results. Since 2-oxoglutaric acid contains structural fragments which are present in the training data sets, the test item is considered to fall within the applicability domain of the model.


6. ADEQUACY OF THE RESULT
As explained in detail in the attached justification the substance falls within the range of reliable predictivity 2-oxoglutaric acid exhibits a molecular weight of 146.10 g/mol and consists of common functional groups, therefore the result of the estimation is considered to be sufficient to fulfil the information requirements for registration.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Software tool(s) used including version: EPISUITE v4.11
- Model(s) used: BIOWIN v4.10
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Key result

Parameter:

probability of ready biodegradability (QSAR/QSPR)

Remarks on result:

readily biodegradable based on QSAR/QSPR prediction

Validity criteria fulfilled:

not applicable

Interpretation of results:

readily biodegradable

Conclusions:

In the present report biodegradability was estimated by QSAR prediction: Using EPISUITE software and the BIOWIN models 2-oxoglutaric acid was considered to be readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Determination of the reversibility of α-ketoglutarate decarboxylation. E.coli cells were grown and subsequently harvested and grounded with powdered glass. The reaction as studied in the presence of malonate is a reversible oxidative decarboxylation involving a half mole of oxygen uptake for each mole of a-ketoglutaric acid utilized for the production of a mole of succinate and a mole of CO2. The exchange reactions were carried out according to Warburg.
- Short description of test conditions: E. coli was grown for 16-18 hrs. at 30°C. in a medium with an initial pH of 7.0. The cells were then harvested and ground with powdered glass and subsequently extracted with phosphate buffer solution. The exchange reactions were carried out in 125 mL Warburg-Barcroft reaction vessels with two side arms. The final concentration of the a-ketoglutarate was 0.01 M and that of malonate 0.05 M. Depending on the activity of the juice, a quantity varying between 5 and 10 ml. was added to each flask. The mixture was buffered at pH 6.6. Appropriate concentrations of the two buffers were mixed with the substrate and enzyme after temperature equilibration (30.4°C.) had been established. pH of the resulting mixture was approximately 7.2 and the volume 25 or 30 ml. The reaction was allowed to continue until approximately half of the substrate was utilized. The residual CO2 in the mixture was then determined by aeration. The C1302 was liberated by the addition of 6 N H2SO4 and collected in 8 ml. Malonate was removed by the addition of 10 mL of 0.025 M sodium bisulfite to the reaction mixture from which the residual CO2 had been removed. The deproteinated sample was extracted for 72 hrs. with ether. The extracted solution was further acidified, heated and aerated for 15 minutes. α-Ketoglutaric acid was degraded to succinate and CO2. The CO2 originates from the carboxyl adjacent to the carbonyl group; it was collected and the C13 content deterinined on the mass spectrometer.

GLP compliance:

no

Oxygen conditions:

aerobic

Initial conc.:

0.01 mol/L

Based on:

act. ingr.

Parameter:

other: CO2 fixation by α-ketoglutarate

Remarks:

mM uptake with malonate

Value:

239

Remarks on result:

other: sampling time was not reeported

Parameter:

other: CO2 fixation by α-ketoglutarate

Remarks:

mM uptake without malonate

Value:

135

Remarks on result:

other: sampling time was not reported

Validity criteria fulfilled:

not applicable

Interpretation of results:

readily biodegradable

Conclusions:

The present study was performed in order to provide evidence that α-ketoglutarate is able to take up CO2 if the oxidative decarboxylation of α-ketoglutarate is reversed. Thus, it was demonstrated that α-ketoglutarate is an essential part of the intermediary metabolism in E. coli.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Principles of method if other than guideline:

- Principle of test:
Investigation of the effects of α-Ketoglutarate phosphoanalogues on the activity of α-ketoglutarate dehydrogenase complexes (α-KGDC) from Escherichia coli (E.coli) and pigeon breast muscle.
- Short description of test conditions:
Enzyme preparations: An extract of E.coli was prepared as previously described. Isolation of α-KGDC from E. coli and its activity determination by NAD+ reduction were carried out using a modified technique.
Effect of phosphoanalogues on dehydrogenyse activity in E.coli extract and in a model system: 50 µl of fresh E. coli extract was added to 50 µl of incubation mixture that contained 5 mM of one of the phosphoanalogues. The incubation was carried out at 37°C for 6 h. An aliquot (I0 µl) was taken at definite intervals of time to determine α-KGDC activity using the above method. In the model system purified enzyme preparations of aspartate aminotransferase from pig hearts and α-KGDC from E. coli were used. The phosphoanalogues were incubated with aspartate aminotransferase under conditions similar to those described, then aliquots were taken and added to the incubation mixture for determination of α-KGDC activity.

GLP compliance:

no

Oxygen conditions:

aerobic

Duration of test (contact time):

6 h

Based on:

not specified

Parameter followed for biodegradation estimation:

other: determination of α-Ketoglutarate concentration during inhibition of α-KGDC activity.

Key result

Parameter:

other: Reduction of α-KGDC activity to 5%

Remarks:

measured in E.coli

Value:

5

Sampling time:

6 h

Key result

Parameter:

other: Redution of α-KGDC activity to 38%

Remarks:

measured in pigeon breast muscle

Value:

38

Sampling time:

6 h

Validity criteria fulfilled:

not applicable

Interpretation of results:

readily biodegradable

Conclusions:

In the present study the inhibition of α-KGDC activity by Glu-phohoanalogues was measured in E.coli and pigeon breast muscle extracts. α-KGDC activity was determined by the α-ketoglutarate consumption in these cells extracts. However, the results demonstrate that α-ketoglutarate is an integral part of the intermediary metabolism and substantiate the assumption that it is readily biodegradable.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Principles of method if other than guideline:

- Principle of test:
TFDA, the enzyme for the first step in 2,4-dichlorophenoxyacetic acid degradation, from Alcaligenes eutrophus was overexpressed in E.coli cells and its activity measured by the 2,4 Dichlorophenol (2,4-DCP) generation. Additionally the generation of radiolabeled CO2 from this reaction was determined by using α-ketoglutarate as a substrate.
- Short description of test conditions:
Escherichia coli JM109 cells were transformed with pUS311 (containing the tfdA gene), grown overnight at 30°C on modified LB medium and harvested by centrifugation. Cultures (2 liters) of E. coli JM109(pUS311) were grown overnight at 30 or 37°C with moderate aeration. Cells were harvested by centrifugation. TFDA was purified 19-fold from cell extracts (obtained from cultures that were grown at 30°C) by chromatography on DEAE-Sepharose at 4°C. The 2,4-Dichlorophenoxyacetic acid (2,4-D)degrading activities of cell extracts and partially purified TFDA were determined by measuring 2,4-Dichlorophenol (2,4DCP) formation after 10 min of incubation at 30°C. The A510 was determined after 20 min with a Gilford Response spectrophotometer. The succinate concentration was determined by using an Aminex HPX-78H column (Bio-Rad) with an eluent of 8 mM sulfuric acid at a flow rate of 0.5 mL/min with concomitant monitoring with a differential refractive index detector. The identities of succinate and glyoxylate in reaction mixtures were confirmed by well-established gas chromatography-mass spectrometry (GC-MS) methods. Partially purified TFDA was incubated with 2.1 nmol of [1-14C]α-ketoglutarate (1.8 GBq/mmol; New England Nuclear) in the presence of unlabeled α-ketoglutarate (99 µM), 2,4-D (100 µM), ascorbate (50 µM), and (NH4)2Fe(SO4)2 (100 µM) in 2 ml of 10 mM MOPS buffer (pH 6.5) at 30°C.

GLP compliance:

no

Oxygen conditions:

aerobic

Duration of test (contact time):

> 10 - <= 30 min

Initial conc.:

0.99 µmol/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Key result

Parameter:

% degradation (CO2 evolution)

Value:

63.5

Sampling time:

30 min

Remarks on result:

other: 10µg TFDA used in the assay

Key result

Parameter:

% degradation (CO2 evolution)

Value:

72.2

Sampling time:

10 min

Remarks on result:

other: 25µg TFDA used in the assay

Validity criteria fulfilled:

not applicable

Interpretation of results:

readily biodegradable

Conclusions:

In the present study the activity of the TFDA enzyme from Alcaligenes eutrophus (α-ketoglutarate dependent dioxygenase) was determined in the absence and presence of α-ketoglutarate. CO2 evolution was also measured using radiolabeled α-ketoglutarate. The trapped radioactivity was 63.5 and 72.2 %, respectively. Thus, these results demonstrate that α-ketoglutarate is an indispensible part of the intermediary metabolism of Alcaligenes eutrophus and is readily degradable.

Description of key information

- QSAR estimation of biodegradability using EPISUITE v4.11, BIOWIN v4.10 prediction method, common functional groups within the substance indicating that the substance falls within the applicabiliy model, RL2, result: readily biodegradable.

- non-GLP, non-guideline study, RL3, detection of alpha-ketoglutarate metabolism in E.coli, supporting the assumption that 2 -oxoglutaric acid is readily biodegradable.

- non-GLP, non-guideline study, RL3, detection of alpha-ketoglutarate metabolism in Alcaligenes eutrophus, supporting the assumption that 2 -oxoglutaric acid is readily biodegradable.

- non-GLP, non-guideline study, RL3, detection of alpha-ketoglutarate metabolism in E.coli, supporting the assumption that 2 -oxoglutaric acid is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

A QSAR estimation of biodegradability using EPISUITE v4.11, BIOWIN v4.10 prediction method was performed. Common functional groups within the substance indicating that the substance falls within the applicabiliy model, thus, 2-oxoglutaric acid was considered to be readily biodegradable.

Additionally, several studies were available providing evidence that 2-oxoglutaric acid is metabolised to form CO2 and energy from various bacteria like Alcaligenes eutrophus or Escherichia coli. Although these informations are not regarded reliable for classification, they do support the QSAR estimation that 2 -oxoglutaric acid is readily biodegradable. Furthermore, it is well known that 2 -oxoglutaric acid is an integral part of the mammalian intermediary metabolism and to have some therapeutic properties (Halpern et al., 1961; Riedel et al., 1996; Bhattacharya et al., 2002; Zdzisińska et al., 2017). Thus, based on the presented relevant and adequate data 2 -oxoglutaric acid is considered to be readily biodegradable and further testing can be omitted.

References:

Halpern, Y. S., and H. E. Umbarger. "Utilization of L-glutamic and 2-oxoglutaric acid as sole sources of carbon by Escherichia coli."Microbiology26.2 (1961): 175-183.

Riedel, Eberhard, Michael Nündel, and Hannelore Hampl. "α-Ketoglutarate application in hemodialysis patients improves amino acid metabolism."Nephron74.2 (1996): 261-265.

Bhattacharya, R., and R. Vijayaraghavan. "Promising role of a-ketoglutarate in protecting against the lethal effects of cyanide."Human & experimental toxicology21.6 (2002): 297-303.

Zdzisińska, Barbara, Aleksandra Żurek, and Martyna Kandefer-Szerszeń. "Alpha-Ketoglutarate as a molecule with pleiotropic activity: well-known and novel possibilities of therapeutic use."Archivum immunologiae et therapiae experimentalis 65.1 (2017): 21-36.