Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

Bioaccumulation: aquatic / sediment

Description of key information

The bioaccumulation potential was assessed for the constituents of this UVCB substance.

As worst case the experimental value of 1730 L/kg is used for the CSA. Therefore, according to Annex XIII of regulation 1907/2006/EC and this UVCB substance and its constituents do not fulfil the criterion “bioaccumulative (B)” or “very bioaccumulative (vB)”. do not meet the criteria for bioaccumulation according to REACh.

 

boundary composition 2:

Boundary composition 2 contains the impurity Diphenylamine. According to the EU Risk assessment report for Diphenylamine, BCF values in the range of 51-253 l/Kg are reported for fish (see Chap. 13.2).

 

Based on a ECHA decision on a substance evaluation (referrence SEV-D-2114398715-33-01/F), the substance is subject to a further tiered testing strategy. These tests comprise currently an evalaution in a simulation study according to OECD 309 and a possible subsequent bioaccumulation study in fish (OECD 305) as well as a possible toxicity study in fish (OECD 210).

 

Key value for chemical safety assessment

BCF (aquatic species):

1 730 dimensionless

Additional information

 

In Article 13 of Regulation (EC) No 1907/2006, it is laid down that information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI (of the same Regulation) are met. Furthermore according to Article 25 of the same Regulation testing on vertebrate animals shall be undertaken only as a last resort.

According to Annex XI of Regulation (EC) No 1907/2006 (Q)SAR results can be used if (1) the scientific validity of the (Q)SAR model has been established, (2) the substance falls within the applicability domain of the (Q)SAR model, (3) the results are adequate for the purpose of classification and labeling and/or risk assessment and (4) adequate and reliable documentation of the applied method is provided.

For the assessment of the UVCB substance CAS 68411-46-1 (Q)SAR results were used for aquatic bioaccumulation. The criteria listed in Annex XI of Regulation (EC) No 1907/2006 are considered to be adequately fulfilled and therefore the endpoint(s) sufficiently covered and suitable for risk assessment. The BCF of the individual representative constituents was determined. As alkylation in para position of the aromatic ring is preferred representative structures of the constituents of the target substance are used to estimate the bioaccumulation potential and adsorption potential of the constituents of CAS 68411-46-1. Additionally, experimental data with a similar substance (CAS 27177-41-9 corresponds to a constituent of CAS 68411-46-1) is available.

Therefore, and for reasons of animal welfare, further experimental studies on bioaccumulation are not provided.

The bioaccumulative potential of the substance was assessed in a weight of evidence approach including several QSAR estimations, experimental data and data on the molecular size and log Kow. The single QSAR models, studies and their results and further evidences are summarized in the table below.

 

 

	Substance A	Substance B	Substance C	Substance D	Substance E	Substance F Read across
	C8C8 dialkylated DPA	C4C8 dialkylated DPA	C8 monoalkylated DPA	C4C4 dialkylated DPA	C4 monoalkylated DPA	C9 monoalkylated DPA
SMILES	N(c2ccc(C(C) (C) CC(C) (C) C) cc2) c1ccc(C(C) (C) CC(C) (C) C) cc1	N(c2ccccc2) c1ccc(C(C) (C) CC(C) (C) C) cc1	N(c2ccc(C(C) (C) C) cc2) c1ccc(C(C) (C) C) cc1	c1cc(ccc1Nc2ccc(cc2) C(C) (C) C) C(C) (C) C	N(c2ccccc2) c1ccc(C(C) (C) C) cc1	CCCC(C)CC(C)(C)c1ccc(Nc2ccccc2)cc1
Molecular weight	393.66	337.55	281.44	281.44	225.34	295.47
logKow experimental				6.7 23°C, pH 6.7
LogKow (KowWin v1.68)	10.82	8.96	7.05	7.11	5.2	7.58
Water solubility experimental (mg/L)				0.004 20°C, pH 6.6
Water solubility (WSKowv1.42) (mg/L)	1.939e-006	0.0001629	0.01487	0.01331	1.167	0.004363
Experimental BCF						1730
OASIS Catalogic v5.11.17/5.11.19
DiamMax Average (Å)	18.724	17.761	15.564	16.572	14.293	16.6
BCFcorrected / (BCFmax) L/kg / Mitigating factors	8 / (80) / Metabolism (0.8) and size (0.7)	23 / (1714) / Metabolism (0.6) and size (0.5)	1047 / (27797) / Metabolism (0.6) and size (0.4)	617 / (26607) / Metabolism (0.6) and size (0.4)	603 / (7211) / Metabolism (0.6) and size (0.4)	832 / (15596) / Metabolism (0.5) and size (0.5)
BCFBAFv3.01
Meylan (BCF l/kg wet wt)	179	1450	12500	11700	1250	6890
BCFBAF upper limit incl biotransformation (BCF l/kg wet wt) / (biotransformation rate of zero)	10 / (14)	331 / (771)	1719 / (11180)	4176 (10460)	1347 (10800)	722 (5810)

 

 

 

Representative trialkylated DPAs

 

	C4C4C4 trialkylated DPA	C4C8C8 trialkylated DPA	C8C8C8 trialkylated DPA
SMILES	c1cc(ccc1Nc2ccc(cc2C(C)(C)C)C(C)(C)C)C(C)(C)C	CC(C)(C)CC(C)(C)c1ccc(Nc2ccc(C(C)(C)CC(C)(C)C)cc2C(C)(C)C)cc1	CC(C)(C)CC(C)(C)c1ccc(Nc2ccc(C(C)(C)CC(C)(C)C)cc2)c(C(C)(C)CC(C)(C)C)c1
Molecular weight	337.55	449.77	508.88
LogKow (KowWin v1.68)	9.02	12.73	14.58
Water solubility (WSKowv1.42) (mg/L)	0.0001459	2.025e-08	2.234e-010
Experimental BCF
OASIS Catalogic v5.11.17
DiamMax Average (Å)	16.502	18.898	18.904
BCFcorrected / (BCFmax) L/kg / Mitigating factors	26.30 (1555.97) / Metabolism (0.7), size (0.4)	7.41 (11.83) / Metabolism (0.5), size (0.5), water solubility (0.3)	7.41 (9.27) / Water solubility (0.9)
BCFBAFv3.01
Meylan (BCF l/kg wet wt)	1348	20.3	3.16
BCFBAF upper limit incl biotransformation (BCF l/kg wet wt) / (biotransformation rate of zero)	282.7 (691.4)	1.04 (1.053)	0.8952 (0.8952)

 

EXPERIMENTAL DATA

A bioconcentration study has been performed according to test methods designated for New Chemical Substances in Japan under MITI with mono-nonyl diphenylamine (Substance F, CAS 27177-41-9) (Mitsubishi 2000).

The study was performed with Cyprinus carpio as test species. The exposure period was 42 days, the depuration period 42 days. The variations in the BCF on days 28, 35, and 42 were within 20%, indicating that the bioconcentration reached a steady state. The following BCF values were determined:

 

High exposure level:          0.10 mg/L            BCF  411             whole body w.w.

Low exposure level:           0.01 mg/L            BCF 1730           whole body w.w.

 

The low exposure level is in the solubility range of the substance. The BCF on whole body was 110-476 for the experiment at the high exposure level and 395-1870 for the experiment at the low exposure level. At depuration time 10 days the eliminated amount was 27%, at 20 days 66% and at 42 days 82% of the steady state concentration in the fish.

 

QSAR

In addition to these experimentally determined BCF values a number of QSAR calculations were performed Per QSAR model the results of these calculations are tabulated and evaluated.

 

The Meylan and the Arnot-Gobas predictions are considered to be reliable for Substance C, D, E, and F. For Substance A is in the applicability domain of the Meylan model. Due to the high logKow the results of the other models maybe uncertain. Substance B is within applicability domain of the Meylan model and Arnot-Gobas BAF/BCF model. Due to the high logKow the results of the other submodel (biotransformation rate in Fish) maybe uncertain.

 

Based on and the Arnot-Gobas sub-model the BCF values for substance A, B are < 2000 L/kg (upper trophic level including biotransformation and without biotransformation) For Substance E the Meylan sub-model gives BCF values < 2000. The BCF value calculated with the Arnot Goabs model for Substance C, D, E and F is > 5000 but a significant reduction of BCF is observed when biotransformation rates are taken into account for Substance C, E and F resulting in BCF values < 2000 L/kg (upper trophic level). Furthermore, a lipid content of 10.7% was assumed as default lipid content for upper trophic level in that model. Normalising to 5% lipid content would result in a BCF value of 1951 L7kg for substance D.

Concerning the trialkylated DPAs only the C4C4C4 trialkylated DPA is in the applicability domain of the Meylan model. With increasing substitution the structures do not meet the applicability domain of the BCFBAF model (logKow and number of instances) applicability domain. However, it can be seen that the predicted bioaccumulation potential decreases. According to the model none of the trialkylated DPAs has BCF values > 2000.

 

It can be concluded that based on the BCFBAF model Substance A, B and E and the trialkylated DPAs have BCF values < 2000 L/kg. Substances C, E and F have BCF values > 2000 only based on the Meylan model. As this model does not consider metabolism, it can be anticipated that this model overestimates the bioaccumulation potential.

 

BCF (OECD 305) estimation based on OASIS Catalogic v.5.10.17, BCF baseline model v. 02.09

The base-line concept for modelling the bioconcentration (OECD 305) of substances is based on a re­fer­ence curve delineating the empirically observed maximum bioconcentration dri­ven by hydrophobicity of substances. In fact, this is the highest log BCF (log BCFmax) which can be reached for a given log K_owvalue assuming that the mole­cu­les (small sized, not ionized) exhibit maximal bioavailability and are not metaboli­zed.

 

Mitigating phenomena and substance properties that can reduce bioconcentration potential, such as molecular size and flexibility, ionisation, biotransformation, etc., are used as reducing factors of the maximum bioconcentration determined via the base-line. The metabolism simulator is using the machinery utilizing a heuristic algorithm to generate plausible metabolic maps using the set of principal trans­forma­tions.

Without mitigating factors Substances C, D and F have the highest bioaccumulation potential (BCF > 2000, > 5000).Based on these results the C4 mono alkylated diphenylamine has a lower bioaccumulation potential compared to the di-alkylated diphenylamines and mono alkylated diphenylamines with higher molecular weight. With increasing substituents the calculated BCF is increasing. C8 mono-alkylated diphenylamine (Substance C) is identified to have the highest bioaccumulation potential. With increasing substitution the and increasing substituents the bioaccumulation potential is decreasing. None of the trialkylated DPAs has BCF values > 2000, even without mitigation factors.

However, all BCF values for the above mentioned structures are below 2000 when mitigating factors are considered.

 

 

INDICATIONS OF REDUCED BIOACCUMMULATION POTENTIAL

 Log Kow

At very high Log Kow (> ca.7), a decreasing relationship with BCF is observed for organic substances. Based on current knowledge, a calculated log Kow >10 is taken as an indicator of reduced bioconcentration for PBT assessments. This cut-off does not apply to Substance A (CAS15721-78-5 logPow 8.96).

The calculated n-octanol/water partition coefficient (log value) is > 5.1. Calculation of the logPow with KOWWIN v.1.68 for the five representative structures for the identified main constituents and the read across substance to one main constituent resulted in values between 5.2 (Substance E, C4 mono-alkylated diphenylamine) and 14.58 (C8C8C8 tri-alkylated diphenylamine). The corresponding water solubility values calculated with WSKOW v1.42 range from were 1.17 mg/L for substance E to 2.2E-10 mg/L for C8C8C8 tri-alkylated diphenylamine, respectively. Depending on their molecular weight the structures the logPow values decrease with decreasing molecular weight, see table above

There are two counteracting trends which both determine the bioaccumulation potential: Whereas lipophilicity (relevant for storage in fatty tissues) increases, water solubility (relevant for bioavailability) decreases. A correlation analysis of logPow versus BCF values performed with > 140 substances from different classes shows that BCF values increase at higher logPow in the range < 9.5. At logPow values >9.5, the BCF values decrease with increasing logPow. Regarding the components of this UVCB substance, lipophilicity increases in parallel to the mol mass.

 

In conclusion, it is considered that some components of the UVCB substance show a high and others a small potential for bioaccumulation. Due to lipophilicity (logPow), the lower mol mass components like mono-alkylated diphenylamine and C4 dialkylated DPA (Substance C, D, E, F) have a high, the high mass components like di-alkylated diphenylamine (Substance A and B) a small potential for bioaccumulation.

 

Molecular size

The decreasing log Kow/BCF relationship is considered to be due also, at least in part, by reduced uptake due to increasing molecular size. The molecular size of a substance is reflected by, among others, the average maximum diameter (Dmax-aver). Very bulky molecules will less easily pass through cell membranes which results in a reduced BCF of the substance.

The software OASIS Catalogic v.5.10.17 and 5.11.19 has been used to calculate the maximal molecular diameter of a molecule. Please note that the following structures are idealised, branched as well as linear substituents can occur.

The substance does not fulfill the criteria for indication of reduced bioaccumulation based on molecular weight and average maximum diameter according to ECHA guidance on information requirements part R.11.According to the Guidance on information requirements and chemical safety assessment Chapter R.11 (PBT assessment, 2014)) an average molecular diameter of >17 Å clearly mitigates the bioaccumulation potential. The DiamMax average range from 14.293 Å (Substance A) to 16.6 Å (Substance F) for the monoalkylated substances. For the dialkylated substances the DiamMax average range from 16.6 Å (Substance D) to 18.8 Å (Substance A). and for the trialkylated DPAs from 16.5 to 18.9 Å. The DiamMax average of Substance A, B and the two bigger trialkylated DPAs is above > 17 Å. However, as the molecular weight of the components is below 700g/mol they are still considered to be bioavailable and expected to pass cell membranes.

However, for most of the structures, the OASIS Catalog model which considers size and flexibility of the structure identifies molecular size as an important mitigating factor resulting in a reduced bioaccumulation potential.

 

Metabolism

The Arnot-Gobas model predicts significant biotransformation potential. In theOASIS BCF baseline v2.09 model metabolism is identified as main mitigating factor for reduced bioaccumulation (exemption: C8C8C8 tri-alkylated DPA, water solubility)

 

Toxicological studies

The bioaccumulation potential for the UVCB substance is determined as low based on toxicological data (see IUCLID section 7.1 for further information).

 

Conclusion:

In summary, in a weight-of-evidence approach balancing different QSAR estimations and experimental data significant accumulation of the certain constituents of the UVCB substance in organisms are expected. However, all data on representing the main components of the UVCB substance and a substance similar to one constituent showed a BCF <2000. Substances C, D and F have BCF values > 2000 only based on the Meylan model. As this model does not consider metabolism, it can be anticipated that this model overestimates the bioaccumulation potential. This is supported by experimental data on Substance F. Constituents corresponding to substance C and D (C8 mono-alkylated diphenylamine, and C4, C4 di-alkylated diphenylamine) have the highest bioaccumulation potential from all representative constituent of the uvcb substance.

Considering the available experimental data and the QSAR data of the different models, the BCF based on experimental data is used as worst case to assess the bioaccumulation potential and used for the CSA: 1730 L/kg

 

It is concluded that the BCF is < 2000. Therefore, according to Annex XIII of regulation 1907/2006/EC and according to the Guidance on information requirements and chemical safety assessment Chapter R.11 (PBT assessment, May 2008) this UVCB substance and its constituents do not fulfil the criterion “bioaccumulative (B)” or “very bioaccumulative (vB)”.