Phenol, 2-methoxy-, reaction products with 2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 Dec 2008 - 27 March 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Although the study presented here lacks detailed and precise information on the method used, this study was sponsored by the Ministry of Economy, Trade and Industry of Japan. The studies conducting for them are internationally recognized as of high quality and high relevance as they closely follow the guidelines.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test

Version / remarks:

CONCENTRATION TEST OF CHEMICAL SUBSTANCE IN FISH AND SHELLFISH FOR METHOD OF TESTING NEW CHEMICAL SUBSTANCE, ETC. > (NOVEMBER 21, 2003 Pharmaceuticals and Foods No. 1121002. Heisei 15.11.13 Manufacturing Bureau No. 2 Environmental Protection Company No. 031121002, Final revision: November 20, 2006)

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

Deviations:
- A dispersant was used
- Reported BCFs are not lipid- or growth-corrected

GLP compliance:

not specified

Remarks:

Only a summary report was available with no indication of whether or not the laboratory is accredited for GLP.

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms during the uptake phase: Sampling on days 7, 14, 28, 42 and 60
- Sampling intervals/frequency for test medium samples during the uptake phase: Sampling on days 0, 7, 14, 22, 28, 35, 42, 49 and 60

- Sampling intervals/frequency for test organisms during the depuration phase: Sampling on days 0, 0.7 and 3

- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): GC/MS

Vehicle:

yes

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: Control with dispersant only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): 2-methoxyethanol
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): 25 ppm (v/v)

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Lipid content at test initiation (mean and range, SD): 5.5 ( 5.2-5.7)
- Weight at day 7 (first measure) (mean and range, SD): range 3.68 - 6.76, mean 4.77 SD 1.05 (the summary does not indicate the weight at day 0, as an alternative, data for the fish analyzed at day 7 is reported here)
- Weight at termination (mean and range, SD): range 8.07 - 11.95, mean 10.27 SD 1.27

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: Recommended method

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

60 d

Total depuration duration:

> 3 - < 4 d

Details on test conditions:

TEST SYSTEM
- Renewal rate of test solution (frequency/flow rate): 800L/day
- No. of vessels per concentration (replicates): 2

Nominal and measured concentrations:

Nominal: 0.5 and 5 µg/L
Measured: 0.45-0.50 and 4.42-4.94 µg/L

Reference substance (positive control):

no

Lipid content:

>= 5.2 - <= 5.7 %

Time point:

start of exposure

Lipid content:

>= 7.1 - <= 8.8 %

Time point:

end of exposure

Conc. / dose:

5 µg/L

Type:

BCF

Value:

> 84 - < 312 L/kg

Basis:

normalised lipid fraction

Calculation basis:

steady state

Conc. / dose:

0.5 µg/L

Type:

BCF

Value:

> 167 - < 1 380 L/kg

Basis:

normalised lipid fraction

Calculation basis:

steady state

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.45 d

Remarks on result:

other: 5 µg/L

Elimination:

yes

Parameter:

DT50

Remarks on result:

other: < 0.7 days at 0.5 µg/L

Remarks:

Substance not detectable in fish after 0.7 days

Details on results:

No indication on mortality or other effects were available in the summary report.
As 2-methoxyethanol was used as a solvent, the authors ran a concomittent solvent control during the study. No detailed information is available on the outcome of this control. It can only be assumed that if no specific information was reported, it is because no significant effects were seen in those controls that would impact the results obtained with the test material.

Water concentration and uncorrected BCFss

		Component	7d	14d	28d	42d	60d
5 µg/L	Water concentration (µg/L)	A	4.58	4.48	4.37	4.35	4.31
		B	5.13	5.08	4.94	4.80	4.76
	Bioconcentration	A	186	174	139	122	112
			129	122	124	146	136
		B	326	287	322	257	291
			312	256	259	317	367
		C	401	416	558	446	311
			361	298	497	521	462
0.5 µg/L	Water concentration (µg/L)	A	0.462	0.454	0.445	0.439	0.434
		B	0.511	0.508	0.498	0.491	0.483
	Bioconcentration	A	< 173	335	253	257	226
			< 181	< 163	209	289	287
		B	499	916	535	421	307
			530	635	491	548	510
		C	< 1680	1880	1250	2180	1090
			< 1760	1740	1750	< 1120	1050

Site-specific analysis for Component C

Test concentration	Depuration period (days)	Replicate	Conc in final solution (mg/L)	Conc in fish body (µg/g)	Residual rate
5 µg/L	0			2.26	100%
	0.7	1	0.122	0.94	34.5%
		2	0.089	0.62
	3	1	< 0.050*	< 0.43*	N/A
		2	< 0.050*	< 0.40*
0.5 µg/L	0			2.26	100%
	0.7	1	< 0.050*	< 0.35*	N/A
		2	< 0.050*	< 0.37*

* Substance was not detected.

Validity criteria fulfilled:

not specified

Conclusions:

The test material is not considered as bioaccumulative as the measured BCFss was 84 - < 1380.

Only a summary report is available and not all of the information is available to evaluate all the validity criteria. However, although the study presented here lacks detailed and precise information on the method used, this study was sponsored by the Ministry of Economy, Trade and Industry of Japan. The studies conducting for them are internationally recognized as of high quality and high relevance as they closely follow the guidelines.

Executive summary:

Japanese authorities conducted an OECD 305 -I equivalent study using Cyprinus carpio. In this test, three isomers of the substance were analyzed and followed during a 60-day uptake phase for all three isomers, and a depuration phase for one of the three (the only one showing accumulation of a factor > 1000). The concentrations tested were 0.5 and 5 µg/L.

BCF steady-state (5 µg/L - 0.5 µg/L)

Component A: 133 – 264 (Corrected to 5% lipid: 84-167)

Component B: 319 – 496 (Corrected to 5% lipid: 202-314)

Component C: 493 – < 2180 (Corrected to 5% lipid: 312-<1380)

A steady-state was reached over the 60-day period for components A and B and for 5 µg/L for component C. For Component C at 0.5 µg/L no steady state was reached, so it is the range of BCFs displayed and not the steady state BCF calculated.

The depuration phase for component C lasted 3 and 4 days at 0.5 and 5 µg/L, respectively. The biological half-life for 5 µg/L was 0.45 days and at 0.5 µg/L, the half-life could not be determined as the substance was already not detectable after 0.7 days. The half-life was therefore < 0.7 days.

Reference 2

Endpoint:

bioaccumulation in aquatic species, other

Remarks:

in vitro test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 319B

Version / remarks:

June 2018

Deviations:

not specified

GLP compliance:

not specified

Remarks:

No information on the GLP status of the laboratory was available in the publication.

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency: 0, 20, 40, 60, 90 and 120 minutes. Reactions were initiated by the addition of a test article, and terminated by the addition of ice-cold methanol and then extracted with 0.600 mL of dichloromethane.

- Sample storage conditions before analysis: Samples were either analyzed immediately or stored at approximately -80°C

- Details on sampling and analysis of samples (e.g. sample preparation, analytical methods):
Before analysis, all samples, if not analyzed immediately, were thawed when applicable and vortexed for three 1-minute intervals with approximately a 30-second pause between intervals. Samples were covered with Sepra® seals and centrifuged for 10 minutes at 900 x g at room temperature. Samples were submitted for GC-MS or LC-MS/MS analysis.

Vehicle:

no

Test organisms (species):

other: Rainbow trout liver S9 fraction

Details on test organisms:

Rainbow trout liver S9 fraction (20 mg/mL stock) (Strains: Kamloops-PNNL Battelle, Sequin, WA and Emerson; Crystal Lake Fisheries, MO) were obtained from Life Technologies (acquired by ThermoFisher). Fish were about 1 year old. The rainbow trout liver S9 fraction was characterized (Life Technologies) by measuring the activity of several important phase I (7-ethoxyresorufin O-deethylase and testosterone hydroxylation) and phase II (estradiol glucuronidation, 7-hydroxycoumarin sulfotransferase and uridine-diphosphoglucuronate-glucuronosyltransferase activities) enzymes.

Route of exposure:

other: In vitro

Value:

> 214 - < 840 L/kg

Basis:

whole body w.w.

Calculation basis:

steady state

Remarks on result:

other: Determined from clearance rate in an invitro assay

Rate constant:

other: S9 intrinsic clearance rate

Value:

0.36

Remarks on result:

other: mL/mg protein/h

Details on kinetic parameters:

S9 CLint (mL/mg protein/h): 0.36
Kmet determined using the calculated fu: 0.2281
Resulting BCF (calc fu): 840 L/kg
Kmet determined using fu =1: 2.3773
Result BCF (fu=1): 214 L/kg

fu: ratio of free chemical concentration in blood plasma to that in the in vitro assay
Kmet: whole body biotransformation rate

Results with reference substance (positive control):

The test material was also tested in a control. In that assay the S9 fraction was heat-treated to deactivate the enzymes. No disappearance of the compound was noted in this control, therefore confirming that the clearance observed in the assay was due to metabolic activity.

Details on results:

The clearance rate of the test material was characterized as "fast" by the authors.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met, with the exception of the R² value which is unknown and so this criteria cannot be evaluated (see details above).

Conclusions:

In an in vitro assay similar to the OECD TG 319B, the authors of the publication have demonstrated that the intrinsic clearance rate of the test material using rainbow trout liver S9 was fast (>= 0.3 ml/mg protein/h). The resulting calculations show the BCFss estimated to be between 214 and 840 L/kg.

Executive summary:

In a recent publication, Weeks et al (2020) have tested several substances, including Sandela, in an in vitro assay to evaluate the metabolic stability of the test materials in rainbow trout (Oncorhynchus mykiss). In order to do so, they followed the methods of Johanning et al. (2012) and determined the BCF utilizing the in vitro to in vivo extrapolation model by Nichols, Hugget et al (2013). These methods references are the same as those constituting the recently adopted OECD TG 319B "Determination of in vitro intrinsic clearance using rainbow trout liver S9 sub-cellular fraction (RT-S9)".

For the test material considered here, one isomer was followed to determine the intrinsic clearance rate (CLint) using a concentration of 1 µM and a concentration of active liver S9 concentration of 2 mg/ml protein. Rainbow trout liver S9 fraction was obtained from an outside source and characterized by measured the activity of the enzymes. Preliminary incubations at 1 and 10 µM were performed to determine incubation times and protein concentration before the final assay was performed. Negative controls included trout liver S9 samples inactivated by heat treatment. All incubations were performed in triplicate at 12°C with active or inactive (heat-treated) S9. Reactions were initiated by the addition of the test substance and terminated by the addition of ice-cold methanol. Samples were terminated at six time points: 0, 20, 40, 60, 90 and 120 minutes. Negative controls were terminated at 0 and 120 minutes.

The samples were analyzed using GC-MS analysis. Sample concentrations were calculated against two calibration curves. The mean concentrations determined at various time points were compared with that of t0 minutes to determine the percentage remaining parent. The metabolic rate was calculated based on the rate of disappearance or loss of the parent compound. The slope of the regression of the ln of the concentration vs time was expressed as the in vitro intrinsic clearance (CLint). This rate was then used in an in vitro to in vivo extrapolation model (Nichols, Hugget et al 2013) to determine the BCF.

This extrapolation requires the determination of the ratio of free chemical concentration in blood plasma to that in the in the in vitro assay (fu). It can be assumed to be 1 or estimated using calculations (Han et al 2009). The authors derived whole fish steady-state BCFs using both methods.

The CLint measured for the test material indicated a fast clearance rate according to the author's scale. Heat-treated controls showed that the material was stable throughout the incubation perodio was there was no or little degradation. The CLint determined was 0.36 mL/mg protein/h, resulting in a whole body steady-state BCF determination of 214 and 840 when calculated fu or default fu =1 was used respectively.

These estimated BCF values indicate that the test material does not have a potential for bioaccumulation.

Description of key information

The potential for bioaccumulation of Phenol, 2-methoxy-, reaction products with 2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated was investigated in multiple studies using different approaches including a full in vivo fish bioaccumulation test, a screening in vivo fish bioaccumulation test and an in vitro assay of its metabolic degradation using fish S9. These studies were also complemented with QSAR estimations of the BCF.

 

Based on all the information available and in accordance with the recommendations of Chapter R.7c of the Guidance on IR&CSA on applying a Weight-of-Evidence approach, it is concluded that IBCH is not bioaccumulable or very bioaccumulable in aquatic organisms, considering in particular the fact that the BCF results from both in vivo studies and the in vitro assay are appreciably below the threshold of 2000.

 

The highest value reported for the in vivo data was used for the CSA, i.e. BCF 1380 L/kg ww.

Key value for chemical safety assessment

BCF (aquatic species):

1 380 L/kg ww

Additional information

The potential for bioaccumulation of Phenol, 2-methoxy-, reaction products with 2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated was investigated in multiple studies using different approaches including a full in vivo fish bioaccumulation test, a screening in vivo fish bioaccumulation test and an in vitro assay of its metabolic degradation using fish S9. These studies were complemented with QSAR estimations of the BCF. Further details are provided below.

Based on all the information available and in accordance with the recommendations of Chapter R.7c of the Guidance on IR&CSA on applying a Weight-of-Evidence approach, it is concluded that IBCH is not bioaccumulative or very bioaccumulative in aquatic organisms, considering in particular the fact that the BCF results from both in vivo studies and the in vitro assay are appreciably below the threshold of 2000.

Source	Type of study	Result BCF in L/kg ww (exposure concentration in mg/L)	Klimish score	Remark
Full in vivo study  2009	In vivo, Cyprinus carpio, OECD 305-I	BCFss 84 (0.005) – < 1380 (0.0005) Half-life 0.45 - < 0.7 days	2	BCFss, corrected for standard lipid content Used 25 ppm of dispersant
Screening in vivo study  2015	In vivo, Oncorhynchus mykiss, similar to minimized OECD 305 (OECD 305-II)	BCFk 96 (0.010) Half-life 2.3 days	3	BCFk No correction for lipid or growth due to limited information Half-life probably overestimated due to limited sampling time points
In vitro study  2020	In vitro assay using trout liver S9	BCFss 214 – 840	2	From metabolic rates
ISIDA predictions 2020	QSAR	BCF 340 – 403	2	4/4 components In domain of applicability
Oasis predictions 2020	QSAR	BCF 741-776	2	3/4 components In domain of applicability
T.E.S.T. predictions 2020	QSAR	BCF 29.79 - 825.26	2	4/4 components In domains of applicability
EPIWIN BCFBAF predictions 2020	QSAR	BCF 1985 – 2096 Half-life 10-17d	3	4/4 components In domain of applicability
VEGA predictions	QSAR	Not reported as the components are out of the domain of applicability of the different models

In vivo fish bioaccumulation tests

1/ Full in vivo study

In a 2009 study, Japanese authorities conducted an OECD 305-equivalent study using Cyprinus carpio. In this test, three isomers of the substance were analyzed and followed during a 60-day uptake phase for all three isomers, and a depuration phase for one of the three (the only one showing accumulation of a factor > 1000). The concentrations tested were 0.5 and 5 µg/L.

BCF steady-state (5 µg/L - 0.5 µg/L)

Component A: 133 – 264 (Corrected to 5% lipid: 84-167)

Component B: 319 – 496 (Corrected to 5% lipid: 202-314)

Component C: 493 – < 2180 (Corrected to 5% lipid: 312-<1380)

A steady-state was reached over the 60-day period for components A and B and for 5 µg/L for component C. For Component C at 0.5 µg/L no steady state was reached, so it is the range of BCFs displayed and not the steady state BCF calculated.

The depuration phase for component C lasted 3 and 4 days at 0.5 and 5 µg/L, respectively. The biological half-life for 5 µg/L was 0.45 days and at 0.5 µg/L, the half-life could not be determined as the substance was already not detectable after 0.7 days. The half-life was therefore < 0.7 days.

2/ Screening in vivo study

In a 2015 study, a modified version of the OECD 305 guideline using Oncorhynchus mykiss was used to screen the substance for its bioaccumulation potential. The design of the study was intended to output a kinetic BCF based on whole fish analysis (4 combined samples from 6 fish at each sampling point). Only one concentration was used (0.010 mg/L), one tank, and no controls were conducted in parallel. The fish were only sampled after 28 days of uptake and 14 days of depuration, no intermediary analysis was performed. k1, k2 and the kinetic BCF were calculated based on the equations from the EPA guidance on bioaccumulation.

kinetic BCF: 96

Half-life: 2.3 days.

Although this study shows important deficiencies in its design compared to the recommended guideline, the results indicate that the substance does not have a significant potential for bioaccumulation. Also, the half-life is most probably overestimated as it is calculated from the concentration in whole fish at the end of the uptake phase and that at the end of the depuration phase, 14 days later, a time point at which the substance was below the limit of quantification of the method. As a worst-case scenario, the authors used a value of ½ LOQ (0.188 mg/kg) to generate the half-life. However, it is possible that the concentration in fish was even lower than that and/or that the concentration fell below the LOQ much sooner than 14 days (see information from the other in vivo study above).

In vitro fish bioaccumulation test

In a published study from 2020, Weeks et al. have investigated the potential for bioaccumulation of various fragrances. Within their target compounds, they have also investigated the potential for bioaccumulation of IBCH (Sandela) using one of its isomers as a proxy. 

They used an in vitro metabolism method using rainbow trout S9 cell fractions. From the results of these in vitro assays, the authors derived an in vivo BCF estimate using the method of Nichols, Huggett et al. (2013).

The isomer of IBCH underwent fast enzymatic degradation with a clearance rate (metabolic degradation due to enzymes) of 0.36 mL/mg protein/h. The resulting calculated BCF ranged between 214 (based on a ratio of free chemical concentration in blood plasma to that in the in vitro assay of 1) and 840 (based on the calculated free chemical concentration in blood plasma to that in the in vitro assay).

QSAR estimations of BCF

For all four programs used, the four major components of IBCH were assessed. 

As presented below, the isomers all have similar results within each prediction. This indicates that the structural differences between the isomers do not significantly impact the results of the predictions. Therefore, the combined predictions of these 4 isomers should correctly represent the UVCB.

1/  ISIDA predictions

Detailed information on the model for BCF can be found in F. Lunghini, G. Marcou, P. Azam, R. Patoux, M.H. Enrici, F. Bonachera, D. Horvath and A. Varnek, QSPR models for bioconcentration factor (BCF): are they able to predict data of industrial interest?, SAR QSAR Environ. Res. 30 (2019), pp. 507–524. The predictor tool is publicly available at http://infochim.u-strasbg.fr/cgi-bin/predictor_reach.cgi 

This model uses fragments as molecular descriptors and multiple algorithms to derive an estimation.

Component	Log BCF	BCF	In applicability domain?
1	2.605	402	Yes - 17/17 models - Optimal reliability
2	2.583	382	Yes - 17/17 models - Optimal reliability
3	2.531	340	Yes - 16/17 models - Optimal reliability
4	2.605	402	Yes - 17/17 models - Optimal reliability

2/ OASIS predictions

For its predictions of BCF, Oasis takes into account a mitigating factor which accounts for the role of metabolism in BCF determination. Also, the training set of OASIS actually contains experimental data for the BCF of two of the four isomers (experimental BCF reported: 916). The predictions are based on the Log Kow which is itself predicted.

Component	Log BCF	BCF	In applicability domain?
1	2.56	776	Yes - Optimal reliability In the training set with an experimental value of BCF 916 and log BCF 2.96
2	2.87	741	Yes
3	2.93	851	NO
4	2.89	776	Yes - Optimal reliability In the training set with an experimental value of BCF 916 and log BCF 2.96

3/ T.E.S.T. predictions

The T.ES.T software includes five different methods to estimate BCF. The sixth method is the consensus method which simply averages the results of the predictions from the other five methods. The latter is typically the one providing the highest accuracy for the prediction since extreme predictions are dampened by the predictions of the other methods.

Component	Log BCF	BCF	In applicability domain?
1	2.29 - 2.89	196.36 - 778.01 Consensus: 360.76	Yes - for all 6 models
2	1.47 - 2.89	29.79 - 778.01 Consensus: 247.81	Yes - for all 6 models
3	2.32 - 2.92	208.73 - 825.26 Consensus: 435.22	Yes - for all 6 models
4	2.29 - 2.89	196.36 - 778.01 Consensus: 360.76	Yes - for all 6 models

4/ EPIWIN BCFBAF predictions

EPIWIN’s submodel from Meylan et al. (1997) (regression-based) was used to estimate the BCF of the different components. The predictions are based on the Log Kow which is itself predicted using KOWWIN. However, unlike OASIS, there is no correction factor applied. The values predicted by EPIWIN are unrealistically high compared to the valid experimental results. therefore, although the substance is within the applicability domain of the model, the results are regarded as unreliable and not taken into account for the assessment.

Component	Log BCF	BCF	Half-life (days)	In applicability domain?
1	3.298	1985	17	Yes
2	3.298	1985	17	Yes
3	3.298	1985	17	Yes
4	3.321	2096	10	Yes