Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 09 March 2021
Experimental completion date: 07 October 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

General Information

Chemical name: 1-(2,3 epoxypropoxy)-2,2-bis[(2,3-epoxypropoxy)methyl]butane

- a constituent of reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane.

CAS Number: 3454-29-3
Molecular formula: C15H26O6
Molecular weight: 302.4 (unlabeled)
Physical state: Clear, colourless liquid
Water solubility: 2.73 g/L at 20˚C
Log Kow: 0.467 at 20˚C

Radiolabeled Test Item:

Position of radiolabel: [hydroxymethyl-14C1]
Specific activity: 2.15 GBq/mmol (by mass spectrometry)
Batch number: TW55HH/DSL02/01
Radiochemical purity: 96.2% (determined prior to use)
Storage conditions: -20°C±10°C under nitrogen

Non-Radiolabeled Test Item:

Name: Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-
hydroxymethyl butane
EC Number: 701-135-4
Batch number: AAF1078500
Purity: 58% C15H26O6 and 25% C12H22O5
Supplier: Sponsor
Storage: Ambient in the dark
Expiry date: 31 August 2021

Radiolabelling:

yes

Details on sampling:

Fish Sampling

On Day 0, at initiation of the test, five individual fish were randomly sampled from the main stock tank for analysis. On Day 14, at the end of the exposure phase, ten individual fish were randomly sampled from the control and ten individual fish were sampled from the test group. During the depuration phase on Day 1, 4, 7, 14 and 21 five fish were taken from the control and treatment tanks for analysis. Also, on Day 27 all remaining fish (15 per tank) were taken from the control and treatment tanks and stored frozen for further analysis, if necessary.

Vehicle:

yes

Remarks:

Corn oil

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

Preparation and Administration of test substance

The fish were exposed to the test substance in a commercial diet treated at a nominal concentration of 1000 µg/g. The radio-labelled test substance [hydroxymethyl-14C1] was radio-diluted with non-radio-labelled substance prior to administration of the test item to the diet.

A portion of [hydroxymethyl-14C1] test substance stock solution (8.13 mL equivalent to 9.27 mg/65.9 MBq) was transferred to a volumetric flask (15 mL) together with a weighed aliquot of non-radio-labelled test substance solution (139.74 mg). Solvent was then removed under a steady stream of nitrogen. The test material was then diluted to volume with ethanol before being sonicated for ca 5 minutes. Triplicate aliquots (10 µL) were removed and diluted to volume (25 mL) with acetonitrile. Duplicate aliquots (250 µL) were removed from these dilutions to determine the radioactivity content by radio-assay (liquid scintillation counting, LSC) and calculate the specific activity.

A portion (13.09 mL) of the above radio-diluted treatment solution was added to corn oil (2.6 mL) and the ethanol removed under nitrogen. Replicate aliquots (10 µL) were removed from the corn oil and diluted to volume (25 mL) with acetonitrile. Duplicate aliquots (250 µL) were taken for radio-assay. The corn oil contained 115.86 mg of test substance; 2.47 mL of the oil was added to 110.07 g of feed, which was manually shaken followed by rotational agitation for five minutes. This process was repeated five times so that the feed had been shaken for a total of six times and rotated for approximately 30 minutes. Five weighed aliquots (approximately 0.01 g) were removed for assessment of homogeneity by combustion and radio-assay. The feed was stored frozen until use.

Control diet was prepared using a portion (110.07 g) of feed treated with 2.47 mL of corn oil, manually shaken followed by rotational agitation for five minutes. This was repeated five times so that the feed had been shaken for a total of 6 times and rotated for approximately 30 minutes. Five weighed aliquots (approximately 0.9 g) were removed for assessment of background radioactivity levels by combustion and radio-assay. The feed was stored frozen until use.

Test Item and Supporting Information

1) Preliminary Palatability Study (14C Diet):

Identification: Fish feed dosed with 14C-labelled test substance
Activity of Diet Received: 1000 µg/g (4.15 MBq)
10 µg/g (0.66 MBq)
Appearance: Brown solid granules
Diet Batch Expiry Date: 18 March 2023
Storage Conditions: Approximately -20 °C in the dark

2) Control Diet for Formal Exposure (coated with corn oil):

Identification: Control Diet
Diet Batch Expiry Date: 28 April 2023
Storage Conditions: Approximately -20 °C in the dark


3) Formal Exposure (14C Diet)

Identification: Fish feed dosed with 14C-labelled test substance:

Activity of Diet Received: 1000 µg/g (48.61 MBq)
Diet Batch Expiry Date: 28 April 2023
Storage Conditions: Approximately 20 °C in the dark

Diet for Depuration:
Identification: Inicio Plus 1.1 mm
Diet Batch: 3633086
Diet Batch Expiry Date: 06 November 2021
Storage Conditions: Approximately -20 °C in the dark

Feeding rate:

Groups of juvenile rainbow trout were exposed to treated feed at nominal concentrations of 10 and 1000 μg/g relative to feed for a period of up to 7 days at a feeding rate of approximately 2% bodyweight under dynamic (continuous flow) test conditions.

Control and Treated Feed Sampling:

A subsample (approximately 3 – 5 g) of the control and treated feed was taken for measurement of radioactivity and test substance on Day 0, from the bulk prepared feeds, and following the uptake phase (Day 14) from the remaining treated feed.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The fish (Rainbow trout (Oncorhynchus mykiss)) were acclimatized in holding tanks for a period of at least 2 weeks and fed commercial trout pellets. No mortality was observed during the acclimatization period.

Prior to initiation of exposure (Day -1) in the main test, the weights and lengths of a subsample of ten fish, selected at random from the batch to be used in the test, were determined. The mean weight of these fish was calculated as 3.64 g (with a standard deviation of 0.61) and the mean length of these fish was calculated as 7.0 cm. From the subsample weights it was determined that the maximum fish weight to be assigned to the test would be 4.25 g (mean weight plus one standard deviation) and therefore the minimum fish weight would be 2.83 g (two-thirds of the maximum fish weight). Prior to initiation of the test the fish were weighed to ensure that each test tank had fish that fell within a weight range of 2.83 g to 4.25 g, ensuring that the smallest fish were no smaller than two-thirds of the weight of the largest fish.

At the start of the test 50 fish were placed in each of the 1000 µg/g and control tanks respectively. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 13 °C to 17 °C with a maximum deviation of ±2 °C and a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 14 days for the uptake phase.

After 14 days of dietary exposure, the fish were transferred to equivalent fresh clean tanks for the depuration period. The depuration phase was conducted for a total of 27 days.
The fish were not individually identified.

Fish were fed with Inicio Plus 1.1 mm trout pellets (56% protein and 18% fat content) either treated or untreated. Feeding occurred daily, except from day 14 of the exposure phase and day 27 of the depuration phase. The fish (treatment group) during the uptake phase were exposed to 14C-labelled test item as a feed at a nominal concentration of 1000 µg/g. The fish (control group) during the uptake phase were exposed to the same type of feed as that given to the treatment group, however, this feed was treated with corn oil only.

Route of exposure:

feed

Justification for method:

other: The aqueous route of exposure was considered not to be appropriate due to low accumulation and high intra-fish variability.

Test type:

flow-through

Water / sediment media type:

natural water

Remarks:

Dechlorinated tap water

Total exposure / uptake duration:

14 d

Total depuration duration:

27 d

Hardness:

Water hardness of approximately 140 mg/L as CaCO3.

Total water hardness was in the range 158 to 160 mg/L as CaCO3 at the beginning of test.

Test temperature:

For the duration of the test (exposure and depuration phases), the temperature of the water in each test tank was maintained within the range 14.0 ±1°C.

pH:

pH was maintained within the range 7.8 to 8.3.

Dissolved oxygen:

Oxygen saturation greater than 60% (9.3 to 10.5 mg O2/L equivalent to 90 to 102% Air Saturation Value).

TOC:

Total organic carbon determined in each test tank both prior to the exposure period was found to be less than the Limit of Quantification (LOQ) of the analytical method (determined to be 1.0 mg C/L).

Method detailed in "Any other information on materials and methods incl. tables" section.

Salinity:

Not applicable

Details on test conditions:

Flow Through System (Main Test)

Two glass aquaria (approximately 260 L in volume, one test and one control), fitted with overflows to maintain a volume of approximately 100 L, were set up with a continuous flow through of approximately 1152 L per day (nominal flow rate 800 mL/minute). Laboratory tap water (also used for the preliminary palatability study) was dechlorinated by passage through an activated carbon filter (Fleck 2750 Duplex Dechlorination Unit). A proportion of the incoming water was softened (Elga Nimbus 1248D Duplex water softener) and then remixed with the main supply to give a water hardness of approximately 140 mg/L as CaCO3, and a pH of 6.0 to 8.5. After dechlorination and softening the water was passed through a series of computer-controlled plate heat exchangers to achieve the required temperature. 

The temperatures in the test tanks were maintained at 13 to 14°C. The water was aerated to maintain oxygen levels equal to or greater than 60% oxygen saturation. A lighting regime of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods was maintained.

Preparation and Administration

Fish were fed with Inicio Plus 1.1 mm trout pellets (56% protein and 18% fat content) either treated or untreated, feces were removed from each tank prior to feeding. Flow through of the dilution water and aeration were paused temporarily during administration of treated or control feed. The tanks were cleaned of any remaining food debris and feces, approximately 30 minutes after the feed was given.

The fish (treatment group) during the uptake phase were exposed to 14C-labelled test item as a feed at a nominal concentration of 1000 µg/g. The fish (control group) during the uptake phase were exposed to the same type of feed as that given to the treatment group, however, this feed was treated with corn oil only. The treated diets (including control) were prepared then shipped on dry ice and stored in the freezer upon receipt until required. 

The fish in both the treatment and control vessels were fed at a rate of 2% of body weight during the exposure and depuration phases.

The feeding rate during the depuration phase was adjusted on sample days (with the exception of the first week of depuration when Day 0 depuration weights were used for the first week feeding rates) and were based on the weight of the fish removed for analysis. Both groups of fish were fed untreated pellets during this phase of the study.

Treated Feed Sampling

Samples of the treated feed were taken for analysis on Day 0 and 14. These were transferred for quantitative and qualitative analysis.

Preliminary Palatability Test

A preliminary test was conducted in order to assess palatability and to ensure accumulation of radioactivity in fish tissue was sufficient to monitor a 95% decline in body burden during the depuration phase of the main exposure. Groups of juvenile rainbow trout were exposed to treated feed at nominal concentrations of 10 and 1000 µg/g relative to feed for a period of up to 7 days at a feeding rate of approximately 2% bodyweight under dynamic (continuous flow) test conditions.

In this preliminary test, five rainbow trout were placed in each test vessel (approximately 65 L in volume) and maintained at approximately 14°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 7 days under flow through test conditions. Two glass aquaria, fitted with overflows to maintain a volume of approximately 45 L, were set up with a continuous flow through of approximately 460 L per day (nominal flow rate 320 mL/minute). Each test vessel was covered to reduce evaporation. Any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish on a daily basis. Prior to initiation of exposure in the palatability test, the weights and lengths of a subsample of ten fish, selected at random from the batch to be used in the test, were determined. The mean weight of these fish was calculated as 2.89 g (with a standard deviation of 0.31) and the mean length of these fish was calculated as 6.73 cm. From the subsample weights it was determined that the maximum fish weight to be assigned to the test would be 3.20 g (mean weight plus one standard deviation) and therefore the minimum fish weight would be 2.13 g (two-thirds of the maximum fish weight). Prior to initiation of the test the fish were weighed to ensure that each test tank had fish that fell within a weight range of 2.13 g to 3.20 g, ensuring that the smallest fish were no smaller than two-thirds of the weight of the largest fish.

Observations were conducted on the feeding rate of the fish to ensure that the diet was palatable.

Five fish were taken from each test group for analysis of accumulated radioactivity on Day 7 during the exposure phase. The length and weight of each fish were also determined on Day 7.

TEST SYSTEM
- Test vessel: aquaria
- Type: open tank covered to reduce evaporation
- Material, size: Glass, 2609 L, filled to 100 L
- Aeration: yes
- Type of flow-through: continuous
- Renewal rate of test solution (flow rate): 800 mL/minute, 1152 L /day
- No. of organisms per vessel: 50
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water/dechlorinated
- Particulate matter: not reported
- Metals: Cu 0.0764 mg/L, Pb <1.5 microgram/L, Ni 4.6 microgram/L
- Pesticides: <0.004 microgram/L
- Hardness: moderate
- Sodium: 46.4 mg/L
- Conductance: 513 micro S /cm at 20

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hrs light, 8 hrs dark, 20 min dawn/dusk transition period
- Light intensity: 795 – 845 LUX
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall daily feeding rate used in the study: 2% of bodyweight
- For OECD 305 part III (dietary exposure fish bioaccumulation), number of feeds per day: one
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food before test start taking into account the contribution from the corn or fish oil vehicle, if used: not reported
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food after end of exposure taking into account the contribution from the corn or fish oil vehicle, if used: not reported

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: 1000 µg/g and 10 µg/g
- Results used to determine the conditions for the definitive study: yes, 1000 µg/g used in definitive study as there was no mortality or sub-lethal effects and feeding rates were normal at this concentration.

Nominal and measured concentrations:

Achieved concentration from direct analysis of bulk treated food was 953 µg/g (nominal 1000 µg/g)

% sample radioactivity by HPLC:

Day 0 96.0%
Day 14 96.6%

Not corrected for extractability (94.1 and 94.9% of applied radioactivity).

Reference substance (positive control):

no

Lipid content:

3.88 %

Time point:

end of exposure

Remarks on result:

other: Day 14

Lipid content:

>= 3.34 - <= 4.18 %

Time point:

other: depuration phase

Remarks on result:

other: Day 1-21

Key result

Conc. / dose:

1 000 µg/g food

Type:

BCF

Value:

752.9 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic, corrected for growth

Remarks:

Lipid normalized

Remarks on result:

other: test substance

Key result

Conc. / dose:

1 000 µg/g food

Type:

BCF

Value:

668.7 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic, corrected for growth

Remarks:

Lipid normalised

Remarks on result:

other: Metabolite M8

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

5.5 d

Remarks on result:

other: For test substance, calculated using the growth corrected-lipid normalized biomagnification factor (BMFKgl) of 0.023

Key result

Parameter:

DT50

Depuration time (DT):

1.05 d

Remarks on result:

other: For metabolite M8, calculated using the growth corrected-lipid normalized biomagnification factor (BMFKgl) of 0.027

Key result

Rate constant:

growth rate constant (d-1)

Value:

0.026

Remarks on result:

other: For test substance

Key result

Rate constant:

growth rate constant (d-1)

Value:

0.026

Remarks on result:

other: For metabolite M8

Details on kinetic parameters:

Test substance: Kinetic Parameters Derived from Mean Concentrations of test substance after 14 days treatment:
k2 (first order depuration rate constant) 0.173 1/day
K2g (growth corrected depuration rate) 0.146 1/day
kf (uptake rate constant from food) 0.00116 1/day

Metabolite M8: Kinetic Parameters Derived from Mean Concentrations of test substance after 14 days treatment:
k2 0.84634 1/day
K2g 0.82034 1/day
kf 0.00483 1/day

Metabolites:

Chromatographic analysis of fish extracts indicated that parent substance was present in only the Day 1 depuration sample accounting for 1% (0.046 μg equiv/g) of sample radioactivity.

Radioactivity in fish extracts was generally associated with up to eleven unknown metabolites M1 – M11.

M8 (tentatively identified as an oxidized metabolite of the parent substance was the most prominent metabolite accounting for 49.7% (5.65 μg equiv/g) at Day 14 of exposure and 59.0% (2.83 μg equiv/g) and 62.5% (1.19 μg equiv/g) at Day 1 and 4 depuration, respectively.

Two unknown components, M3 and M7, were also observed at notable levels accounting for 15.7% (1.79 μg equiv/g) and 9.8% (1.11 μg equiv/g) sample radioactivity, respectively. A further
unknown component, M2, was also of note accounting for 3.4% (0.382 μg equiv/g) sample
radioactivity. All remaining components accounted for ≤2.3% of sample radioactivity.

The proportion of test substance related metabolites were derived from direct evaluation of the radio-chromatograms using Laura software.

Results with reference substance (positive control):

Not applicable

Details on results:

No mortalities or sub lethal effects of exposure were observed throughout the test.

Preliminary Experiments

The homogeneity of [hydroxymethyl-14C1] test substance was determined for feed treated at nominal treatment rates of 10 and 1000 µg/g. Test item concentrations were consistant with nominal targets (within < 20%) with no indication of high variance between replicate aliquots taken for radioassay. The method of preparation was therefore deemed appropriate for the main experiment.

The palatability of [hydroxymethyl-14C1] test substance was determined for each treatment concentration up to 7 days. The 10 and 1000 µg/g dose levels were well tolerated by the fish with the daily feed generally rapidly consumed. The concentration of radioactivity in fish, fed at a nominal treatment rate of 1000 µg/g, was in the range 7.70 – 13.6 µg equivalents/g after 7 days. The lowest amount of radioactivity in any one fish was equivalent to 213324 dpm/g. This amount of radioactivity was sufficient for a 95% reduction in body burden to be determined with a limit of detection of twice the background. Therefore the 1000 µg/g exposure level was chosen for the main experiment.

The results of the palatibility experiment are presented in tables below.

Achieved concentration of [hydroxymethyl-¹⁴C₁] Test Substance in Feed Treated at a Nominal Concentrations of 10 and 1000  µg/g - Palatability

Nominal Concentration (µg/g)	Aliquot	dpm/g	Mean dpm/g	Specific activity(µg/g)	Mean µg equivalents/g
10	1	4284389	4430329	427000	10.4 (104%)
	2	4402740
	3	4328870
	4	4598290
	5	4537355
1000	1	21777127	23595661	27700	852 (85.2%)
	2	24146082
	3	20751724
	4	20870370
	5	30433001

Value in parenthesis indicates % of nominal target

Concentration and Accumulation in Fish Treated with [hydroxymethyl-14C1] test substance - Palatability

The specific activity of the 10 µg/g feed was 427000 dpm/µg and specific activity of the 1000 µg/g feed was determined to be 27700 dpm/µg.

Exposure Days	Nominal Feed Concentration (µg/g)	Fish Number	dpm/g	µg equivalents/g	Mean µg equivalents/g
7	10	1	51684	0.121	0.130 ± 0.019
		2	46178	0.108
		3	64270	0.151
		4	51454	0.121
		5	63928	0.150
	1000	1	377865	13.6	12.2 ± 2.5
		2	213324	7.70
		3	361759	13.1
		4	364826	13.2
		5	365034	13.2

 

Radiochemical Purities

The radiochemical purity of [hydroxymethyl-14C1] test substance prior to preparation of the treated diet was determined by HPLC to be 96.2%.

The radiochemical purity of radiodiluted [hydroxymethyl-14C1] test substance in extracts of treated feed (1000 µg/g) was determined (HPLC) to be 96.9% at time zero and 96.6% after 14 days exposure. These results confirmed the stability of the test item in the treated feed for the duration of the exposure phase.

Homogeneity and Achieved Concentrations

The variance between replicate aliquots of feed treated with [hydroxymethyl-14C1] test substance, at a nominal concentration of 1000 µg/g, was 7.3%, indicating that the bulk sample was homogeneous. The specific activity of the feed was calculated to be 26500 dpm/µg. The achieved concentration on feed was 953 µg/g (95.3% of nominal). The achieved concentration on feed was considered acceptable for meeting the objectives of the study.

Recoveries of Radioactivity from Treated Feed

At the start (time zero) and end of the exposure phase (Day 14), the extractability of radioactivity from the treated feed was 94.1 and 94.9% of applied radioactivity, respectively. The remaining applied radioactivity in the post extraction solid unextracted amount was 5.9 – 5.1%. As detailed previously, HPLC analysis of the extracts confirmed the stability of the test item in the treated feed for the duration of the exposure phase.

Total Radioactive Residues in Fish

Concentrations are reported as µg equivalents of test substance (µg equiv/g).

Exposure Phase

After 14 days of exposure to [hydroxymethyl-14C1] test substance, at a nominal concentration of 1000 µg/g, the mean concentration of total radioactivity in fish tissue was 11.4 µg equivalents/g.

Depuration Phase

At Day 1 of the depuration phase, the mean concentration of radioactivity in whole fish had declined to 4.79 µg equiv/g, a reduction in total body burden of 58.0%. At Day 14, the concentration in fish tissue had declined further to 0.455 µg equiv/g, a reduction in total body burden of 96.0%. The depuration phase was terminated at Day 21; the concentration in fish tissue at this time was 0.266 µg equiv/g representing a reduction in total body burden of 97.7%.

Extraction of Radioactivity

Due to the limited amount of radioactivity in fish samples, only extracts of pooled fish from Day 14 of exposure and Day 1 and Day 4 of depuration were extracted and analysed by HPLC.
The amounted extracted from fish at Day 14 exposure was 95.9% (post extraction solid accounted for 4.1%). At Day 1 and Day 4 of depuration, the amount extracted was 95.2% and 93.6%, respectively (post extraction solids accounted for 4.8% and 6.4%)

Proportions and Concentrations of Test Substance in Fish Extracts

Up to 11 unknown components were detected in fish extracts (M1 – M11), however not all components were detected in each sample. Test substance was detected only in the Day 1 depuration sample where it accounted for 1% (0.046 µg equiv/g) sample radioactivity.

At Day 14 of exposure, the majority of the radioactivity was associated with unknown metabolite M8 accounting for 49.7% (5.65 µg equiv/g) sample radioactivity. Two unknown components, M3 and M7, were also observed at notable levels accounting for 15.7% (1.79 µg equiv/g) and 9.8% (1.11 µg equiv/g) sample radioactivity, respectively. A further unknown component, M2, was also of note accounting for 3.4% (0.382 µg equiv/g) sample radioactivity. All remaining components accounted for ≤2.3% of sample radioactivity.

At Day 1 of depuration, the majority of the radioactivity was associated with unknown metabolite M8 accounting for 59.0% (2.83 µg equiv/g) sample radioactivity. Unknown components M2 was a notable metabolite at this time accounting for 12.1% (0.579 µg equiv/g) sample radioactivity. M3 and M6 were also of note accounted for 5.8% (0.278 µg equiv/g) and 3.8% (0.182 µg equiv/g) sample radioactivity, respectively. All remaining components accounted for ≤1.6% of sample radioactivity.

At Day 4 of depuration, the majority of the sample radioactivity was associated with unknown metabolite M8 accounting for 62.5% (1.19 µg equiv/g) sample radioactivity. Unknown components M3 was a notable metabolite at this time accounting for 11.2% (0.214 µg equiv/g) sample radioactivity. M2 and M7 were also of note accounted for 4.1% (0.078 µg equiv/g) and 5.0% (0.094 µg equiv/g) sample radioactivity, respectively. All remaining components accounted for ≤1.1% of sample radioactivity.

Lipid Analysis

Measured fish lipid content ranged from 3.34 to 4.18% tissue weight. After 14 days of exposure to [hydroxymethyl-14C1] test substance (1000 µg/g), the mean concentration of total radioactivity in fish tissue corrected to 5% lipid content was 14.7 µg equiv/g. The corrected concentration of metabolite M8 was 7.31 µg equiv/g.

At Day 1 of the depuration phase, the mean concentration of radioactivity in whole fish had declined to 7.17 µg equiv/g and by Day 14, the concentration in fish tissue had declined further to 0.615 µg equiv/g. The depuration phase was ended at Day 21 where the concentration was 0.318 µg equiv/g.

Concentrations of metabolite M8 declined to 4.24 µg equiv/g at Day 1 of the depuration phase, then to 1.48 µg equiv/g at Day 4.

Preliminary Palatability Test

No mortalities or sub lethal effects of exposure were observed throughout the test when fish were exposed to treated feed at nominal concentrations of 10 and 1000 µg/g.
Observations of feeding rates showed all food was consumed within two minutes of being administered on each occasion and was considered to be normal throughout the duration of the test.

Conditions of Definitive Exposure

One day prior to initiation of the exposure phase, a sample of ten fish was selected at random from the main stock tank. A fish length (cm) range of 6.5-7.5, with a mean and standard deviation of 7.0 and 0.24 respectively, was recorded. A fish weight (g) range of 3.07-5.18, with a mean and standard deviation of 3.64 and 0.61 respectively, was recorded. Based on this sample the fish for the test were selected.

Water Quality Characteristics

The temperature measurements recorded in the control tank by a Testo temperature logger were shown to have been maintained at between 13.0 and 13.6 °C.

For the duration of the test (exposure and depuration phases), the temperature of the water in each test tank was maintained within the range 14.0 ±1°C, oxygen saturation at greater than 60% (9.3 to 10.5 mg O2/L equivalent to 90 to 102% Air Saturation Value) and pH was maintained within the range 7.8 to 8.3. Total water hardness was in the range 158 to 160 mg/L as CaCO₃ at the beginning of test. The light intensity was observed to be in the range 795 to 845 Lux.

Total organic carbon determined in each test tank both prior to the exposure period was found to be less than the Limit of Quantification (LOQ) of the analytical method (determined to be 1.0 mg C/L (Method detailed in "Any other information on materials and methods incl. tables" section.).

The control and test concentration were observed to be clear, colorless solutions by visual inspection throughout the exposure and depuration phases of the study.

Mortality and Observations of the Test Fish

No mortalities or sub lethal effects of exposure were observed throughout the test. 

Validity Criteria

During the in-life phase of the definitive study the following validity criteria were met (validity criteria were not applied during the preliminary test):

	Required	Achieved
Temperature variation	Less than 2 °C	Test temperatures recorded in the control tank by a Testo data logger (hourly) were 13.0 to 13.6 °C The temperature recorded by a digital thermometer daily in each tank showed that the temperature was maintained within the range 14 ±1 °C
Dissolved oxygen concentration	Does not fall below 60% Air Saturation Value (ASV)	All dissolved oxygen concentrations recorded as equal to or greater than 9.3. mg O2/L (equivalent to being equal to or greater than 90% ASV).
Mortality or adverse effects in control and treated fish	Less than 10%	There was no mortality or adverse effects in the control or test groups.

Metabolites

The proportion of Metabolites in Extracts of Whole Fish Following Exposure to Diet Treated with test substance (1000 μg/g) is shown in the table below:

Components	Sample radioactivity (%)
	Exposure	Depuration
	Day 14	Day 1	Day 4
M1	2.0	1.6	1.2
M2	3.4	12.1	4.1
M3	15.7	5.8	11.2
M4	2.3	1.4	1.7
M5	1.2	ND	ND
M6	1.2	3.8	ND
M7	9.8	ND	5.0
M8	49.7	59.0	62.5
M9	1.2	1.0	ND
M10	1.1	ND	ND
M11	1.4	ND	ND
Parent substance	ND	1.0	ND
Remainder	6.9	9.5	7.9
Total Extract	95.9	95.2	93.6
Post extraction solid	4.1	4.8	6.4
Total	100	100	100

          ND = Not detected (less than 1% radioactivity)

         Remainder - Accounts for areas on the radiochromatogram which sum above zero but which                   contain no discrete area or component of radioactivity that accounted for greater than 1% of dose

         Post extraction solid - Proportion of radioactivity remaining in the post extraction solid following               extraction of the pooled fish

 

Concentrations of Metabolites in Extracts of Whole Fish During Exposure to Diet Treated with the test substance (1000 μg/g) are shown in the table below:

Components	µg equivalents of [hydroxymethyl-14C1] test substance/g
	Exposure	Depuration
	Day 14	Day 1	Day 4
M1	0.229	0.078	0.023
M2	0.382	0.579	0.078
M3	1.79	0.278	0.214
M4	0.262	0.068	0.032
M5	0.131	ND	ND
M6	0.142	0.182	ND
M7	1.11	ND	0.094
M8	5.65	2.83	1.19
M9	0.142	0.046	ND
M10	0.120	ND	ND
M11	0.164	ND	ND
Parent substance	ND	0.046	ND
Remainder	0.785	0.456	0.150
Total Extract	10.9	4.56	1.78
Post extraction solid	0.500	0.230	0.120
Total	11.4	4.79	1.90

        ND = Not detected (less than 1% radioactivity)

        Remainder -  Accounts for areas on the radiochromatogram which sum above zero but which                  contain no discrete area or component of radioactivity that accounted for greater than 1% of dose

        Post extraction solid - Proportion of radioactivity remaining in the post extraction solid following              extraction of the pooled fish

Metabolite identification

A diluted stock solution of [14C] test substance and an extract of pooled fish treated with [14C] test substance (1000 μg/g) for 14 days were submitted for LC-MS analysis coupled with off-line radio-detection to generate radiochromatograms and generate full scan and MS/MS data.

Radioactivity (cpm) was measured in LC fractions that were collected at 6s intervals for 26 minutes into DeepWell LumaPlates and air dried completely. A Microbeta2 scintillation counter was used to count each plate, with wells simultaneously counted for 2 minutes.

Compound related molecular ions were identified by comparison of retention times with the 14C profiles. The mass spectrometer was operated in electrospray ionization positive ion/negative ion switching mode, and positive electrospray ionization mode (ESI+) data are presented in this report. Whilst 14C ion was the major species in the molecular ion cluster in the dose solution, 12C ion was the major species observed in the analysed sample. For accurate mass confirmation of a suspected metabolite, the measured accurate mass had to be within 5 ppm of the theoretical value in order to confirm the molecular formula (unless otherwise stated), and the fragmentation given is based on the 12C ion unless stated otherwise.

The structure, parent mass, and characteristic fragment ions of [14C] test substnace are presented in the table below:

Summary of Protonated Molecular Ion and Characteristic Fragment Ions for [14C] test substance:

Retention Time (minutes)	[^14C-M+H]^+	Structure and Proposed Fragmentation Pathway	Characteristic Fragment Ions, m/z
18.74	305	Refer to attachment	101, 87(A)^*, 87(B)^*, 83^*, 71, 75^*, 59^*, 57^*

Note: Retention time obtained from the reference standard

*Mass accuracy > 5 ppm but < 15 ppm

Summary of Protonated Molecular Ions and Characteristic Fragment Ions for Metabolites M3, M5 and M8:

Identifier	Retention Time (minutes)	[M+H]^+	Proposed Structure and Fragmentation Pathway	Characteristic Fragment Ions (m/z)
M3^A	NA	ND	NA	NA
M5^A	NA	ND	NA	NA
M8	8.97	357	refer to attachment	265, 173, 103, 99^*, 70, 75^*, 57^*

^A Peaks observed in radiochromatogram were not identified by LC-MS (data not shown)

^* Mass accuracy > 5 ppm but < 18 ppm

 

 

 

 

 

Validity criteria fulfilled:

yes

Remarks:

Details are given in "Any other information on results incl. tables".

Conclusions:

Test substance - the growth corrected-lipid normalized biomagnification factor (BMFKgl) was calculated to be 0.023 with a half-life of approximately 5.5 days.

For the main metabolite (M8) - the growth corrected-lipid normalized biomagnification factor (BMFKgl) was calculated to be 0.027 with a half-life of approximately 1.05 days

The estimated BCFs are below the bioaccumulation triggers used in EU member countries for a bioaccumulative (“B”: 2000 L/kg) or very bioaccumulative (“vB”: 5000 L/kg) substance.

Executive summary:

The potential for 1 (2,3 epoxypropoxy) 2,2 bis [(2,3 epoxypropoxy) methyl]butane (a constituent of reaction mass of 1 (2,3 epoxypropoxy)-2,2-bis ((2,3 epoxypropoxy)methyl) butane and 1-(2,3- epoxypropoxy)-2-((2,3 epoxypropoxy)methyl)-2-hydroxymethyl butane) to bioaccumulate via the diet in rainbow trout (Oncorhynchus mykiss) was studied according to the OECD Guidelines for Testing of Chemicals (2012) No. 305 "Bioaccumulation in Fish: Aqueous and Dietary Exposure" in a continuous flow through system.

Fish were exposed to the test item dosed on to commercial diet at a nominal concentration of 1000 µg/g, over a 14-Day exposure period. Depuration of the test item was determined over a further 27 Day period. The dietary exposure level of 1000 µg/g was selected on the basis of results generated during a preliminary 7 Day palatability study at concentrations of 10 and 1000 µg/g.

The achieved concentration of [hydroxymethyl-14C1] test substance from direct analysis of the bulk treated feed was 953 µg/g. The extractability of radioactivity from the treated feed was 94.1 and 94.9% of applied radioactivity. Chromatographic analysis of the feed extracts showed [hydroxymethyl-14C1] test substance accounted for 96.9% and 96.6% sample radioactivity, confirming the stability of the test item on feed for the duration of the 14 day exposure phase.

After 14 days of exposure to [hydroxymethyl-14C1] test substance, the mean concentration of total radioactivity in fish tissue was 11.4 µg equivalents/g. During the depuration phase, approximately 83% of the accumulated radioactivity was eliminated by Day 4 of depuration, and by Day 21 of depuration, approximately 98% radioactivity had been eliminated.

For the duration of the depuration phase, the overall range of lipid content (% body weight) measured was 3.34 to 4.18%. The mean concentrations of total radioactivity normalized to an average 5% fish lipid content declined from 14.7 µg equivalents/g after 14 days of exposure to 0.318 µg equiv/g after 21 days of depuration.

Chromatographic analysis of fish extracts indicated that test substance was present in only the Day 1 depuration sample accounting for 1% (0.046 µg equiv/g) of sample radioactivity. Radioactivity in fish extracts was generally associated with up to eleven unknown metabolites M1 – M11. M8 was the most prominent metabolite accounting for 49.7% (5.65 µg equiv/g) at Day 14 of exposure and 59.0% (2.83 µg equiv/g) and 62.5% (1.19 µg equiv/g) at Day 1 and 4 depuration, respectively. Two early eluting metabolites, M2 and M3 were also notable accounting for 3.4% (0.382 µg equiv/g) and 15.7%
(1.79 µg equiv/g) of sample radioactivity at end of exposure, respectively, and 4.1% - 12.1% (0.078 – 0.579 µg equiv/g) and 5.8% - 11.2% (0.214 – 0.278 µg equiv/g) of sample radioactivity during Day 1 and 4 of depuration.

Description of key information

The growth corrected-lipid normalized biomagnification factor (BMFKgl) was calculated to
be 0.032 with a half-life of approximately 5.5 days. Estimations of BCF ranged from 0.003
to 752.9 based on various methods used in study 8465605.

The estimated BCFs are below the bioaccumulation triggers used in EU member
countries for a bioaccumulative (“B”: 2000 L/kg) or very bioaccumulative (“vB”: 5000 L/kg)
substance.

Key value for chemical safety assessment

BCF (aquatic species):

752.9

BMF in fish (dimensionless):

0.027

Additional information

Similar results were found for M8, the main metabolite of the substance. For M8, the growth corrected-lipid normalized biomagnification factor (BMFKgl) was calculated to be 0.027 with a half-life of approximately 1.05 days. For M8, estimations of BCF ranged from 0.001 to 668.7 based on various methods and the full results for BCF estimation are presented in Table 5. The estimated BCFs are below the bioaccumulation triggers used in EU member countries for a bioaccumulative (“B”: 2000 L/kg) or very bioaccumulative (“vB”: 5000 L/kg) substance.