Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Bioaccumulation: aquatic / sediment

Administrative data

Endpoint:

bioaccumulation in aquatic species: fish

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:

BCF Base-line model developed by Laboratory of Mathematical Chemistry, OASIS-LMC Ltd., Burgas, Bulgaria

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Guideline:

other: REACH Guidance on QSARs R.6, May 2008

Principles of method if other than guideline:

Calculated with BCF base-line model developed by Laboratory of Mathematical Chemistry

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

EC Number:

277-146-9

EC Name:

Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Cas Number:

72968-71-9

Molecular formula:

C23H29N7O4S

IUPAC Name:

methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Test material form:

solid

Specific details on test material used for the study:

- Related to pure substance
- Smiles code: CC1=C(C(=O)OC)SC(N=Nc2c(C)c(C#N)c(NCCCOC)nc2NCCCOC)=C1C#N
- Molecular mass: 499.59 g/mole

Radiolabelling:

no

Test organisms

Test organisms (species):

other: none, estimated by calculation

Results and discussion

Bioaccumulation factor

Type:

BCF

Value:

6.5 L/kg

Basis:

other: calculation

Any other information on results incl. tables

Bioaccumulation prediction:

The predicted by CATALOGIC BCF base-line model bioconcentration factor for the target chemical) Maclolex Rot B is as follow:

•       log BCF = 0.81 in L/kg wet.

It is based on experimental log Kow = 6.1 and experimental water solubility 0.003 mg/L.

If calculated log Kow = 4.7 and calculated water solubility 99.7 mg/L are used the predicted bioconcentration is:

•       log BCF = 0.63 in L/kg wet.

Applicability domain of prediction:

The target substance belongs 100 % to the parametric and mechanistic sub-domains of the BCF base-line model and only 36.7 % to the structural domain of the model. The chemical is out of the structural domain because 63.3 % of its ACFs are not present among the training chemicals with observed BCF values. The new sub-domain named metabolic domain was added to the BCF model. This domain describes how well the metabolism of the target is simulated based on the available observed metabolism in the database of the model. The target belongs 36.7 % to the metabolic sub-domain too. The chemical is out of the metabolic sub-domain because 60 % of its ACFs are not present among the chemicals in BCF base-line model database with observed metabolism (i.e. these are unknown fragments for the model) and other 3.3% of its ACFs belongs to chemicals with incorrectly predicted metabolism.

The prediction of bioconcentration of Macrolex Rot B is based on its lipophilicity accounting for the effect of metabolism and the molecular size. The substance is not ionic (i.e. no effect of ionisation is expected) and the effect of its water solubility is not affecting the model prediction. The predicted first level metabolic reaction which is reducing significantly the maximum bioconcentration potential (i.e. BCFMAX) is reaction of N reduction with very high probability.

No analogue chemicals of the target containing the all functionalities with observed BCF and observed metabolism were found in the training sets of the BCF model.  In this respect, the search was focused to find analogues containing some of the moieties presented in the target molecule.

In the training set of the BCF base-line model azo dyes are presented with 15 chemicals. The details: CAS, name, SMILES, structure, observed and predicted BCFs for these chemicals are summarized in Appendix 2. Azo chemicals with experimental BCFs in BCF base-line model training set. All azo dyes found in the training set are not bioaccumulative having observed log BCFs in the range 0.2÷1.8 in L/kg wet. All azo days are correctly predicted by the model as not bioaccumulative with predicted log BCFs in the range 0.3÷0.9 L/kg wet.

In the available databases in OECD QSAR Toolbox 4.2 nine azo days with experimental BCFs were found. The details: CAS, SMILES, structure, observed BCFs, test species and the source database for these chemicals are given in Appendix 3. Azo chemicals with experimental BCFs in OECD QSAR Toolbox 4.2. All azo dyes found in OECD QSAR Toolbox 4.2 are not bioaccumulative with observed log BCFs in the range -0.22÷1.7 in L/kg wet.

In the database of BCF base-line model with observed metabolism 18 azo days were found. For 14 chemicals the predicted metabolism is with sensitivity 100% (all observed metabolites are predicted by the model), for 2 chemicals predicted metabolism is with sensitivity 75÷80% and only for 2 chemical the observed metabolism is not predicted (sensitivity 0%). Except for these 2 azo days the observed metabolism is starting with N-reduction of azo bond and formation of the corresponding amines. In Appendix 4. Azo days with observed metabolism in BCF base-line model database, the name, structure, sensitivity and reference from where the observed metabolism is taken are shown.

Analogues found in the literature:

A literature search for BCF and metabolism for structurally related compounds was performed to support the BCF model prediction.  No BCF and metabolism data available for structurally similar compounds containing all functional groups represented in the molecule of the Macrolex Rot B were found.  Metabolism information for compounds containing part of the functional groups presented in the molecule of the Macrolex Rot B was found.

CAS No:  94-78-0, Phenazopyridine

IUPAC name: 3-[(E)-Phenyldiazenyl]-2,6-pyridinediamine

Canonical SMILES:

c1ccc(cc1)/N=N/c2ccc(nc2N)N

Phenazopyridine contains pyridineamine fragment as well as the target chemical. Phenazopyridine contains two primary amino groups at position 2 and 6, but substituents in ring at position 4 and 5 are missing in contrast with pyridine fragment of the target compound.

In vivo phenazopyiridine was absorbed after oral administration in mammals and metabolized by reductive cleavage of the azo bond and ring hydroxylation at the 4' and 5 positions as the principal metabolic pathways. Phase II metabolites were formed by N-acetylation of the primary amino group and conjugation of the phenol group with glucuronide and sulphate [7].

Proposed metabolic pathway of phenazopyridine in rat is shown in Figure 7. 2,3,6-Triaminopyridine (2), aniline (3) and 4-aminophenol (4) were undetected, but their occurrence as intermediates was imputed based on the occurrence of N-acetyl-4-aminophenol as a major metabolite , unchanged phenazopyridine accounted for <1% dose. [7,8].

It was found in metabolism studies of azo dyes that intestinal microorganisms and/or reductive enzymes in the liver catalyzed the reductive cleavage of the azo linkage to produce aromatic amines [9]. There is ample evidence that water-insoluble azo dyes are metabolized by reductases in the liver [10]. The azo reductase responsible for such metabolism was found in the fish and mammal liver microsomal fractions and its activity to the metabolic reduction of the azo bond was proven [10, 11].

Since the target compound Macrolex® Rot B is virtually insoluble in water (water solubility < 0.003 mg/L), it is assumed that its metabolism by cleavage of azo bond will proceed primarily in the liver. Because the ability of enzymes in the liver microsomal fractions of fish and mammals to reduce azo compounds was proven, the expected metabolites of the target compound which will be produced by the cleavage of the azo bond.

Summary of the properties of azo dyes:

•       All azo dyes found in the training set and in the available databases in OECD QSAR Toolbox 4.2 are not bioaccumulative.

•       For all azo dyes found in the training set the BCFs values are correctly predicted and simulated metabolism is identical with the observed.

•       Based on the observed metabolism of azo dyes and simulated multi-level metabolism of Macrolex Rot B it could be concluded that the simulated metabolism is correct although the target is out of the structural and metabolic sub-domain layers of the BCF base-line model.

Applicant's summary and conclusion

Conclusions:

The bioaccumulation factor (BCF) of Macrolex Rot B was etsimated to be 6.5 L/kg wet-wt since the BCF BAse-line model leads to the conclusion that the substance has a low potentila to bioccumulate in biota.

Executive summary:

i. Model predictions

The predicted by BCF base line model bioconcentration factor for Macrolex Rot B based on experimental log KOW = 6.1 and experimental water solubility 0.003 mg/L is as follows:

log BCF = 0.81 in L/kg wet.

Prediction based on calculated log KOW = 4.7 and water solubility 99.7 mg/L is as follows:

log BCF = 0.63 in L/kg wet.

The predicted log BCF is in agreement with the experimentally observed bioconcentration (log BCF in the range of -0.22 to 1.8) of 18 unique azo dyes (50 BCF values) found in the training set of the model and available databases in OECD QSAT Toolbox 4.2.

ii. Applicability domain. 

Macrolex Rot B belongs to the parametric and mechanistic domain of the model.  The chemical is out of the structural because 63.3% of its ACFs are not present among the training chemicals with observed BCF values. The chemical is out of the metabolic sub-domain because 60 % of its ACFs are not present among the chemicals in BCF base-line model database with observed metabolism (i.e. these are unknown fragments for the model) and other 3.3% of its ACFs belongs to chemicals with incorrectly predicted metabolism.

iii. Role of metabolism.  

Model prediction is based on lipophilicity and simulated first level metabolism for the target. Detailed analysis of documented metabolism of 16 azo dyes revealed that azo dyes where aromatic moieties linked together by azo (-N=N-) bond were metabolized in mammals by the cleavage of this bond. The simulated metabolism for Macrolex Rot B is in agreement with documented metabolism in fish and mammals which is a premise for correct prediction of the target BCF.