Reaction mass of 2,6-bis(1-phenylethyl)phenol and 2,4,6-tris(1-phenylethyl)phenol

Biotransformation and kinetics

Administrative data

Endpoint:

biotransformation and kinetics

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study according to a sound study design similar (but not identical) to the OECD protocol for biotransformation using rainbow trout liver S9 fraction and hepatocytes that is currently undergoing validation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline for biotransformation using rainbow trout liver S9 fraction and hepatocytes (Guideline currently under validation)

Deviations:

yes

Remarks:

A single tube (in replicates) was used for the incubations procedure as opposed to several test tubes representing each time point.

GLP compliance:

yes

Type of medium:

animal

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-tris(1-phenylethyl)phenol

EC Number:

242-128-1

EC Name:

2,4,6-tris(1-phenylethyl)phenol

Cas Number:

18254-13-2

Molecular formula:

C30H30O

IUPAC Name:

2,4,6-tris(1-phenylethyl)phenol

Test material form:

liquid: viscous

Details on test material:

The test material is one of the main constituents of the reaction mass of 2,4,6-tris(1-phenylethyl)phenol and Bis(1-phenylethyl) phenol.

Results and discussion

Transformation products:

no

Any other information on results incl. tables

The hepatocyte assay was conducted with the biological activity being stopped using a 1:1 ACN:THF dilution. Tables 1 - 3 detail the biotransformation of TSP after 180 min incubation with rainbow trout hepatocytes in the three separate experiments.

In Experiment One, loss of TSP was observed in the reaction mix, as well as in all of the controls (e.g. solvent deactivated and fluroxypyr positive control). It was later identified that the source of the GC-MS was not workin properly and therefore, any observed loss was due to the malfunction of the GC-MS instrument itself.

 

There was also a concern that the solvent deactivation procedure was not stringent enough and therefore caused false positive responses in the controls. It was then decided thatthe hepatocytes would be heat deactivated (boiling hepatocytes at 100 °C for at least 1 min) as opposed tobeing deactivated using solvent as it was performed in Experiment One. Based on these two factors, twoadditional experiments were conducted with TSP and hepatocytes.

In Experiments Two and Three, no loss of TSP was observed in the heat deactivated negative controlsindicating that enzymes were successfully denatured. In addition, loss of the positive control, fluroxypyr,was observed. over the course of both experiments indicating that the hepatocytes were metabolically active. No loss of TSP was observed in the reaction mixes from Experiment Two and Three.

In conclusion there was no significant loss of parent TSP over time demonstrating that there was noenzymatic metabolism

It should be noted that higher levels of TSP and fluroxypyr were detected in the S9 study, as opposed to this study, even though both studies utilized the same concentration. While a definitive reason cannot begiven, it is likely due to differences in binding within the cell suspension and ultimate bioavailability. Itis likely that these compounds had a greater affinity for the hepatocytes compared to the S9 suspension, thus lowering the overall bioavailable free measurable fraction. The fluoroxypyr concentrations detected in both experiments are within the range typically detected from our laboratory.

Further, the absolute Time 0 initial concentrations are different between concentrations. The Time 0samples were taken immediately after the addition of the last reagent. Recent discussions with thistechnique suggest that this does not represent a true Time 0 as biotransformation may begin immediately. Therefore, the reported Time 0 is a relative value. Given that the bioavailability of TSP appears to be different with S9 cells and cell suspensions, the bioavailability in the reaction mix likely attributed to differences in starting concentrations.

Table 1: Experiment One

		TSP Reaction Mix	Solvent Deactivated Control		Fluroxypyr Positive Control	Fluroxypyr Deactivated Control
Run	Time point	TSP Conc. (ppm)	TSP Conc. (ppm)	Time point	TSP Conc. (ppm)	TSP Conc. (ppm)
1	0 min	3.57	1	0 min	41.64	52.84
2	0 min	1.62	0.12	0 min	47.87	36.63
3	0 min	1.83	0.44	0 min	48.55	38.37
Avg.	0 min	2.34	0.52	0 min	46.02	42.61
1	30 min	2.16	0.77	5 min	38.84	8.25
2	30 min	1.32	0.69	5 min	35.81	36.63
3	30 min	1.5	0.67	5 min	39.05	38.37
Avg.	30 min	1.66	0.71	5 min	37.9	16.08
1	60 min	0.27	0.44	10 min	28.94	14.59
2	60 min	0.25	0.08	10 min	13.23	26.6
3	60 min	0.52	0.19	10 min	13.14	24.13
Avg.	60 min	0.35	0.24	10 min	18.44	21.77
1	120 min	0.61	0.08	20 min	2.49	7.94
2	120 min	0.16	0.27	20 min	10.89	4.68
3	120 min	nd	nd	20 min	11.63	16.49
Avg.	120 min	0.39	0.18	20 min	8.34	9.70
1	180 min	0.42	nd	30 min	4.06	15.76
2	180 min	0.79	0.18	30 min	1.41	13.74
3	180 min	nd	0.12	30 min	nd	9.04
Avg.	180 min	0.61	0.15	30 min	2.74	12.85

Table 2: Experiment Two

		TSP Reaction Mix	Solvent Deactivated Control		Fluroxypyr Positive Control	Fluroxypyr Deactivated Control
Run	Time point	TSP Conc. (ppm)	TSP Conc. (ppm)	Time point	TSP Conc. (ppm)	TSP Conc. (ppm)
1	0 min	3.84	1.07	0 min	3.85	3.58
2	0 min	2.68	1.38	0 min	2.98	2.98
3	0 min	2.98	1.24	0 min	3.68	2.79
Avg.	0 min	3.17	1.23	0 min	3.50	3.45
1	30 min	3.86	1.22	5 min	3.12	3.48
2	30 min	3.11	nd	5 min	2.45	4.62
3	30 min	2.89	1.02	5 min	2.98	3.37
Avg.	30 min	3.29	1.12	5 min	2.85	3.82
1	60 min	3.44	1.02	10 min	1.45	5.01
2	60 min	3.48	nd	10 min	1.99	3.45
3	60 min	3.74	1.17	10 min	1.32	4.12
Avg.	60 min	3.55	1.09	10 min	1.59	4.19
1	120 min	3.95	1.97	20 min	0.95	3.97
2	120 min	3.61	nd	20 min	0.78	4.82
3	120 min	3.51	1.99	20 min	nd	3.12
Avg.	120 min	3.69	1.98	20 min	0.87	3.97
1	180 min	3.46	1.46	30 min	0.01	4.63
2	180 min	3.69	nd	30 min	0.15	4.12
3	180 min	2.87	1.64	30 min	nd	3.46
Avg.	180 min	3.34	1.55	30 min	0.08	4.07

Table 3: Experiment Three

		TSP Reaction Mix	Solvent Deactivated Control		Fluroxypyr Positive Control	Fluroxypyr Deactivated Control
Run	Time point	TSP Conc. (ppm)	TSP Conc. (ppm)	Time point	TSP Conc. (ppm)	TSP Conc. (ppm)
1	0 min	2.23	5.21	0 min	2.13	4.12
2	0 min	3.21	4.28	0 min	2.58	4.78
3	0 min	2.79	5.01	0 min	2.93	3.87
Avg.	0 min	2.74	4.83	0 min	2.55	4.26
1	30 min	3.05	4.749	5 min	1.78	3.98
2	30 min	2.87	5.12	5 min	2.19	4.12
3	30 min	2.01	4.31	5 min	2.01	4.45
Avg.	30 min	2.64	4.74	5 min	1.99	4.18
1	60 min	2.21	6.23	10 min	0.98	5.12
2	60 min	2.12	4.29	10 min	1.12	3.78
3	60 min	2.03	4.82	10 min	0.84	4.29
Avg.	60 min	2.12	5.11	10 min	0.98	4.40
1	120 min	2.35	5.19	20 min	0.12	4.59
2	120 min	3.01	4.76	20 min	0.24	5.81
3	120 min	2.08	4.92	20 min	0.08	4.19
Avg.	120 min	2.48	4.96	20 min	0.15	4.86
1	180 min	2.13	4.18	30 min	0.01	5.08
2	180 min	3.02	5.64	30 min	nd	4.76
3	180 min	2.52	4.58	30 min	nd	4.38
Avg.	180 min	2.56	4.8	30 min	0.01	4.74

Applicant's summary and conclusion

Conclusions:

There was no significant loss of parent material over time due to the active biotransformation of TSP in hepatocytes from rainbow trout.

Executive summary:

Three separate experiments were conducted where 5µM of TSP was incubated with rainbow trout hepatocytes for 180 min. In the first experiment, a loss of TSP was observed in the reaction mix and allnegative controls. It was found that the GC-MS was compromised andthe results were not reliable. Twoadditional experimentswereconductedutilizing a different GC-MS instrument. One-way ANOVA followed by Dunnett’s post hoc testing showed no significant loss of TSP relative to time 0 minutes for two independent hepatocyte assays (p ≥ 0.05). TSP showed no loss in the Heat Deactivated or No NADPH controls in the hepatocyte assay.

In conclusion, there was no significant loss of parent material over time due to the active biotransformation of TSP in hepatocytes from rainbow trout.