Mycophenolic acid

Long-term toxicity to fish

Administrative data

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20. June, 2017 - 26. October, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Detailed Review Paper (DRP) No. 95 on Fish life-cycle tests

Version / remarks:

2008

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

July 2013

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD 229: "Fish Short Term Reproduction Assay"

Version / remarks:

October 2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Mycophenolic acid

EC Number:

246-119-3

EC Name:

Mycophenolic acid

Cas Number:

24280-93-1

Molecular formula:

C17H20O6

IUPAC Name:

6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-2-benzofuran-5-yl)-4-methylhex-4-enoic acid

Test material form:

solid: particulate/powder

Details on test material:

- Batch no. 116-172
- Assay (HPLC) 99.2%
- Analysis date August 12, 2016
- Retest date August 12, 2018
- Solubility in water: 45 mg/I, water (25 °C, pH 5), 710 mg/I, water (25 °C, pH 7) (MSDS)*
- Hydrolysis: 46% (5 d, pH 5, 50 °C); 29% (5 d, pH 7, 50 °C); 37% (5 d, pH 9, 50 °C); hydrolytically unstable, extrapolated half-life at 25 °C between 1 day and 1 year (MSDS)
- Appearance: white to practically white powder (according to Safety Data Sheet provided by the sponsor); light-brown powder (observed on current batch)

Specific details on test material used for the study:

- Batch No.: 116-172
- Storage: Keep container tightly closed in a cool, dry, dark and well-ventilated place.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method for chemical analysis (BIOLOGICAL PHASE):

- Immediately after sampling and until transfer to the analytical test site, the samples were stored protected from light in a freezer (e.g. at =< -18 °C) in glass bottles.
- Sample volume: 100 mL
- Note 1: Samples of each freshly prepared stock solutions (Si, S2 and S3) were taken immediately before use. Samples of each aged stock solutions were taken at the last day used to prepare the test solution.
- Note 2: If the number of fish are not sufficient to provide a complete set of samples for further analysis at Biobide, Gipuzkoa Scientific & Technological Park, Paseo Mikeletegi 56, 20009 San Sebastian, Spain, due to high mortality during the exposure phase, at first samples of single fish were taken (F0 samples). Thereafter, the remaining fish were used for the pooled fish sample (FF sample). The remaining fish were stored as pooled reserve samples, each sample containing at maximum of 5 fish per sampling vial.

- Sampling method for chemical analysis (ANALYTICAL PHASE)
The water samples were received at Battelle in good condition (frozen) on the 8th September and 25th October 2017 and placed in a freezer until analysis.
Prior to analysis, water samples were thawed and homogenised by briefly shaking and sonicating. A small volume was then transferred from the sample bottle to a suitable vial followed by centrifugation. An aliquot was taken for subsequent dilution to ensure concentrations were within the calibration range.
An aliquot was transferred to an auto-sampler vial prior to analysis by LC-MS/MS.

Test solutions

Vehicle:

yes

Remarks:

Reconstituted water

Details on test solutions:

PREPARATION OF STOCK SOLUTIONS
Due to difficult water solubility of the test item in modified reconstituted water, a stock solution of the test item at 10 mg test item/I was prepared by weighing the desired amount of the test item into alkalinised deionised water.

The preparation of the stock solution (S1) was arranged by following procedure:
• The test item was pestled before use.
• The required amount (30.0-51.0 mg) of test item was addeded to a defined volume of deionised water (3000 or 5000 ml).
• 3.5 ml 1-M NaOH per litre deionised water was added to the stock solution (S1)
• The stock solution (S1) was stirred over night at room temperature in the dark using a magnetic stirrer.
• After a short stirring period the pH of s1 was measured and adjusted to target range of 7.2-7.8, using 1-M HCl and 1-M NaOH.

After overnight stirring of S1 the stock solutions (S2, S3) were prepared by the following procedures:
The final stock solution Si was diluted with the test medium to prepare the stock solution S2 and S3. The nominal concentration of S2 was 0.25 mg test item/l and 0.05 mg test item/l for S3. The stock solution S1 was mixed into the test medium as homogeneously as possible by intense stirring (stainless steel propeller stirrer) for 2-3 minutes. Thereafter, the stock solution S2 and S3 were ready to use.

The stock solutions S2 and S3 were used to produce the test solutions by dilution with modified reconstituted water. Test solutions were produced continuously using a flowthrough device.

The stock solutions (S1—S3) were prepared at least once a week. After the stirring period, the stock solutions S2 and S3 were stored at room temperature in the dark.

Each time when prepared, the volumes of the stock solutions (S1—S3) were large enough to prepare all test concentration levels and all analytical samples at once.

The test vessels containing the test solutions were placed into a temperature controlled water bath at test temperature for temperature adaptation.


PREPARATION OF TEST SOLUTIONS
At start of the exposure of the F0 generation the test solutions were manually prepared to ensure an exposure of the adult fish to the correct required test item concentrations. Therefore 10000 ml of test solution was prepared for each test replicate. Before addition of the final test solution into the test vessels the control medium of the pre-exposure period was partly removed.

Thereafter the test solutions were prepared by a flow-through device. The stock solutions S2 and S3 were delivered by the flow-through system to mixing vessels. The test solutions were delivered directly from the mixing vessels to the designated test vessels using teflon tubing and a multi-channel peristaltic pump assembled with Tygone® tubes, set to deliver the desired volume.

General pump settings:
- F0 generation:
• Time (in seconds) for pumps to deliver the required volumes of solutions during one run (TR): 455 s.
• Number of runs per 24 h: 96 (900 s per run); July 04 to July 19, 2017.

- F0+F1 generation:
• Time (in seconds) for pumps to deliver the required volumes of solutions during one run (TR): 445 s.
• Number of runs per 24 h: 134 (645 s per run); July 04 to July 27, 2017.

- F1 generation:
• Time (in seconds) for pumps to deliver the required volumes of solutions during one run (TR): 445 s.
• Number of runs per 24 h: 38 (2274 s per run); July 27 to August 26, 2017.

S2: stock solution, nominal concentration 0.25 mg test item/I
S3: stock solution, nominal concentration 0.05 mg test item/I

During dosing into the flow-through system (between renewals), the stock solutions S2 and S3 were stored at room temperature in the dark. During storage, the tanks were closed with a lid to reduce air exchange and evaporation.

The flow rates (volume delivered per stroke) were measured volumetrically at least three times per week. The volume delivered/measured was divided by the number of strokes. The variation of the nominal stroke-volume was calculated by dividing the measured flow [ml/stroke] by the nominal volume per stroke of the designated pump expressed in percentage of nominal values.

The actual measured flow rates for nearly all treatments were within ±10% of the mean flow rates, except at the treatment level C0 (control) during the exposure period of the F0 generation where the flow rates exceeded the required range on one occasions (C0: 5.92 water exchanges per test vessel and day).

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish.
- Strain/clone: Danio rerio
- Origin: Fertilised eggs were collected from adult zebrafish from in-house cultures, which have been maintained and bred at ECT since September 2016.

-Number of animals:
--F0 generation: 2 replicates of 14 mature fish per concentration level, 8 presumed male and 6 presumed female fish.
--Fl generation: 4 replicates of 20 fertilized eggs per concentration level.

- Method of holding and fertilisation:
Holding conditions and fertilisation of test organisms: In the culture tanks, the fish were kept in reconstituted water (OECD guideline 203, 1992) diluted with deionised water (1:1; v/v) and supplemented with 1% of artificial seawater (Tropic Mahn, Dr. Biener GmbH Aquarientechnik, Wartenberg, Germany; salinity 28±2%0). Fish were held in glass aquaria at 26±2 °C with a 14 h/10 h photoperiod. They were fed a combined diet of newly hatched nauplii of Artemia sp. (Sanders Brine Shrimp Co., Morgan, UT 84050, USA) and TetraMin (Tetra Werke, MeIle, Germany).

Study design

Test type:

flow-through

Water media type:

other: Reconstituted water (OECD guideline No. 203) mixed with deionised water (1:1; v/v), supplemented with 1% artificial seawater

Limit test:

no

Total exposure duration:

57 d

Remarks on exposure duration:

F0 generation exposure duration: 23 d / F1 generation exposure duration: 34 d

Test conditions

Hardness:

F0 pre-exposure [°dH]: 11.0
F0 generation [°dH]: 9.2-10.8
F1 generation [°dH]: 9.4-10.4

Test temperature:

F0 pre-exposure: 25.0-26.9
F0 generation: 25.0-26.6
F1 generation: 25.3-26-4

pH:

F0 pre-exposure: 7.3-7.8
F0 generation: 7.2-7.4
F1 generation: 7.4-7.7

Dissolved oxygen:

F0 pre-exposure: 4.5-8.0 [mg/L] // 56-100 %
F0 generation: 4.0-7.9 [mg/L] // 61-99 %
F1 generation: 6.7-8.0 [mg/L] // 84-100 %

Salinity:

nominal salinity originating from artificial seawater: 0.028%

Conductivity:

758 - 809 uS/cm

Nominal and measured concentrations:

Nominal: 0.0, 0.0005, 0.0029, 0.0171, 0.1 mg test item/L
Measured: 0.0, 0.000256, 0.00132, 0.0109 and 0.0730 mg test item/1 (calculated geometric mean measured concentration)

Details on test conditions:

REPARATION OF TEST MEDIUM
Reconstituted water according to OECD guideline No. 203 was used to dilute the test item and to keep the fish before and during the period of the test.

Local tap water was treated by reverse osmosis and ion exchange to prepare deionised water. Therefore, a contamination with heavy metals, pesticides and total organic carbon is excluded.

The reconstituted water consists of deionised water and salts in the following final concentrations:
A) CaCl2 x 2 H20 - 294.0 mg/I
B) MgS0.4 x 7 H20 - 123.0 mg/I
C) NaHCO3 - 64.8 mg/I
D) KCI - 5.75 mg/I
The required amount of reconstituted water was prepared within four weeks before use. During storage reconstituted water was aerated.

Prior to use, the reconstituted water was diluted with deionised water (1:1; v/v) and supplemented with 1% of artificial seawater. After aeration of several hours, the following physico-chemical characteristics of this medium were determined: pH, conductivity and total hardness.



TEST SYSTEM
• Number of animals & replicates:
F0 pre-exposure: 12 replicates of 14 mature fish in intreated test medium, 8 presumed male and 6 presumed female fish.
F0 generation: 2 replicates of 14 mature fish per concentration level, 8 presumed male and 6 presumed female fish.
F1 generation: 4 replicates of 20 fertilized eggs per concentration level.
• Temperature (target): 26±1.5 °C.
• pH of the test solutions (target): 6.5-8.5, not varying by more than ±0.5 units.
• Photoperiod: light/dark cycle 12 h/12 h.
• Light intensity: measured (target): 550-1000 lx, measured: 513-809 lx.
• Aeration: the test vessels were gently aerated during the test using teflon tubes.
• The test was performed under flow-through conditions.
• The test vessels were set up in a quasi-stochastic manner.
• Each test vessel was labelled with the study number, the concentration or concentration code and specific letters for each replicate; each test vessel was closed by a lid.
• The spent test solutions (effluents) were treated with activated charcoal and disposed of according to current valid local regulations.


EXPOSURE CONDITIONS

PRE-EXPOSURE PERIOD
The test was initiated with sexually dimorphic mature zebrafish (e.g. with clear secondary sexual characteristics visible) from ECT stock culture to establish breeding groups. These parental fish were pre-exposed in untreated test medium for 11 days. The determined fecundity (number of eggs), the fertilization success in percent of total number of eggs and the hatching success were determined once during the pre-exposure phase.

After temperature adaptation of the test medium (control water) in the vessels, the required number of mature fish were transferred from the stock into the test vessels by using a onestage randomisation procedure. This was done by sequentially adding one fish per test vessel until all test vessels contained 6 presumptive female and 8 presumptive male fish. During the pre-exposure period, the fish were incubated under the same conditions which prevail in the subsequent test. Fish were fed ad libitum with a combined diet of newly hatched nauplii of Artemia sp. (once per workday) and dry fish food (TetraMin; twice per workday and once at weekend) throughout the exposure phase. Egg production was assessed every day during the pre-exposure phase. Spawning was observed in all replicate tanks prior to inclusion in the exposure phase of the assay.


EXPOSURE OF F0 GENERATION
The parental fish were exposed to the test item for 23 days.
• The flow-through system was not conditioned with the test solutions prior to exposure of F0 generation.
• The test solutions were prepared manually at start of the exposure period (day 0), to provide sufficient test solution volumes for each test replicate. The flow-through system was adjusted to maintain the required nominal test concentrations.
• At start of the test (day 0) the water in the pre-exposure vessels were siphoned off down to a level of 5-6 cm water column. To ensure a sufficient volume of test solution for later
introduction of the spawning glass bowls, manually prepared test solutions were added to the test vessels.
• Renewal of the test solution during the test period: flow-through, two vessel volumes per vessel and day.
• A subsample of 14 pre-exposed fish were weighed in order to determine the mean weight of the population. This was done using fish not designated for later exposure (Code: B2).
• The test vessels were set up in a quasi-stochastic manner to ensure balanced distribution of replicates.
• Temperature: 26±1.5 °C (target); measured min/max: 24.7/26.6 °C (online); preexposure and exposure of F0 generation, June 23 — July 28, 2017, measured 25.0/26.6 °C (manual).
• The flow rate was adjusted so that the loading (biomass per volume of test solution) did not exceed a loading of 5 g fish/I of solution and a loading rate of 0.5 g fish/I per 24 hours at any time of the test (see below).
• To calculate the maximum loading, the total fish wet weight per vessel was derived from the wet weight of a representative group of fish determined at start of the exposure period (558 mg per fish) and additionally from all fish at the end of the exposure period of the F0 generation (517 mg per fish), multiplied by the total number of introduced fish (14 per test vessel) and divided by the test medium volume (10 l). The maximum loading was 0.78 g fish/l, respectively 0.724 g fish/I.
• The loading rate was calculated by dividing the maximum total fish wet weight per test vessel (representative group of fish: 7812 mg per fourteen fish, respectively all fish at the end of the exposure period: 7238 mg per fourteen fish) by the target flow rate of two vessel volumes per day (20 I/vessel/day). The maximum loading rate was 0.391 g fish/I per 24 hours, respectively 0.362 g fish/I per 24 hours.
• Fish were fed ad libitum with a combined diet of newly hatched nauplii of Artemia sp. (once per day) and dry fish food (TetraMin; twice per workday and once per day on weekends) throughout the exposure phase.
• Dead fish were removed from the test vessels upon daily observation throughout the test.


EXPOSURE OF F1 GENERATION
The F1 generation was started after an exposure period of the F0 generation of 20 days for the treatment levels and the control after 19 days (a time-staggered start was necessary since no sufficient number of eggs was collected at the fixed starting date.

On the evening before test start of the F1 generation, glass bowls covered with a stainless steel mesh were introduced in the test tanks of the adult zebrafish of the F0 generation. Glass plants were placed on the mesh, allowing the fish to spawn.

The spawning bowls were removed from the test tanks on the day of test start shortly after onset of illumination (i.e. 60 minutes), which triggers fertilization. The content of the bowls was poured over a sieve, rinsed with temperature-adjusted reconstituted water and collected in a glass vessel filled with temperature-adjusted test medium of the corresponding test solution. Immediately afterwards, the collected eggs were transferred to glass dishes containing test solution (>100 ml) of each designated exposure vessel, one for each test concentration and control, acclimated at test temperature. The dishes containing the collected eggs (n = ca. 120-150) were placed into an incubator set to 26 °C (25±1.5 °C) for 1.25 hours. Thereafter, groups of fertilised eggs were transferred to an additional set of preexposure vessels (four replicates per test concentration) containing temperature-adjusted test solutions using a randomisation procedure. This was done by sequentially adding groups of fertilised eggs (1-5 eggs per test vessel) from the first pre-exposure vessel for each test concentration into the corresponding replicate pre-exposure vessels until each test vessel contained 20 eggs (embryonic stage: high plastula stages). Thereafter the fertilised eggs (20 per pre-exposure vessel) were transferred into the test units.

• The flow-through system was conditioned with the test solutions prior to exposure of F1 generation.
• Renewal of the test solutions during the test period: flow-through, five vessel volumes per vessel and day. The test vessels were set up in a quasi-stochastic manner to ensure balanced distribution of replicates.
• Temperature: 26±1.5 °C (target); measured min/max: 25.3/26.3 °C (online); exposure of the F1 generation, July 22 — August 26, 2017, see section 18, 25.3/26.4 °C (manual).
• The flow rate was adjusted so that the loading (biomass per volume of test solution) did not exceed a loading of 5 g fish/I of solution and a loading rate of 0.5 g fish/I per 24 hours at any time of the test (see below).
• To calculate the maximum loading, the maximum total fish wet weight per vessel in the controls at test end (806.2 mg in the controls, replicate a) was divided by the test medium volume (0.8 I) per test vessel. The maximum loading was 1.01 g fish/I.
• The loading rate was calculated by dividing the maximum total fish wet weight per vessel (806.2 mg in the controls, replicate a) by the target flow rate of five vessel volumes per day (4.0 I/vessel/day). The maximum loading rate was 0.202 g fish/I per 24 hours.
• Feeding: On day 4 of exposure, when the first larva per test vessel was recorded to swim up, feeding was started by adding food to the vessels. The test organisms were fed on dry food (NovoBaby 01 and NovoBaby 02, JBL), live Artemia nauplius larvae and paramaecia (Paramecium caudatum) ad libitum. The daily ration was fed in 3-4 equal portions on workdays. On weekends the daily ration was fed in 2-4 portions. Uneaten food was removed once daily. The food ration was adjusted to the number of living fish per test vessel. Food was withheld from the fish for 24 hours prior to test end.
• Dead eggs, embryos or fish were removed from the test vessels upon daily observation throughout the test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

23 d

Dose descriptor:

EC10

Effect conc.:

0.019 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

adult mortality

Remarks:

/ survival of the parental fish

Effect concentrations 2

Key result

Duration:

23 d

Dose descriptor:

EC50

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

adult mortality

Remarks:

/ survival of the parental fish

Effect concentrations 3

Key result

Duration:

23 d

Dose descriptor:

NOEC

Effect conc.:

0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

adult mortality

Remarks:

/ survival of the parental fish

Effect concentrations 4

Key result

Duration:

23 d

Dose descriptor:

LOEC

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

adult mortality

Remarks:

/ survival of the parental fish

Effect concentrations 5

Key result

Duration:

23 d

Dose descriptor:

EC10

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

fertility

Remarks:

/ fecundity

Effect concentrations 6

Key result

Duration:

23 d

Dose descriptor:

EC50

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

fertility

Remarks:

/ fecundity

Effect concentrations 7

Key result

Duration:

23 d

Dose descriptor:

NOEC

Effect conc.:

0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

fertility

Remarks:

/ fecundity

Effect concentrations 8

Key result

Duration:

23 d

Dose descriptor:

LOEC

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

fertility

Remarks:

/ fecundity

Effect concentrations 9

Key result

Duration:

23 d

Dose descriptor:

EC10

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ fertilisation success

Effect concentrations 10

Key result

Duration:

23 d

Dose descriptor:

EC50

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ fertilisation success

Effect concentrations 11

Key result

Duration:

23 d

Dose descriptor:

NOEC

Effect conc.:

0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ fertilisation success

Effect concentrations 12

Key result

Duration:

23 d

Dose descriptor:

LOEC

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ fertilisation success

Effect concentrations 13

Key result

Duration:

34 d

Dose descriptor:

EC10

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks:

/ hatching success

Effect concentrations 14

Key result

Duration:

34 d

Dose descriptor:

EC50

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks:

/ hatching success

Effect concentrations 15

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks:

/ hatching success

Effect concentrations 16

Key result

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks:

/ hatching success

Effect concentrations 17

Key result

Duration:

34 d

Dose descriptor:

EC10

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch survival F1

Effect concentrations 18

Key result

Duration:

34 d

Dose descriptor:

EC50

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch survival F1

Effect concentrations 19

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

0.011 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch survival F1

Effect concentrations 20

Key result

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

post-hatch survival F1

Effect concentrations 21

Key result

Duration:

34 d

Dose descriptor:

EC10

Effect conc.:

0.032 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

/ length of the surviving fish F1

Effect concentrations 22

Key result

Duration:

34 d

Dose descriptor:

EC50

Effect conc.:

> 0.073 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

/ length of the surviving fish F1

Effect concentrations 23

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

Length of the surviving fish F1

Effect concentrations 24

Key result

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

0.011 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

Length of the surviving fish F1

Effect concentrations 25

Key result

Duration:

34 d

Dose descriptor:

EC10

Effect conc.:

0.011 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weigth of the surviving fish F1

Effect concentrations 26

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight of the surviving fish F1

Effect concentrations 27

Key result

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

0.011 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight of the surviving fish F1

Details on results:

F0 PRE-EXPOSURE:
- A quantitative guidance on desirable daily egg production cannot be provided at this stage, but average spawns of >10 eggs/female/day are commonly observed. All spawning groups showed an average spawn of >19 eggs/female/day, the number of female was supposed to be 6 per test vessel. Nevertheless, the number of female fish varied between 6 and 10, as determined/verified at the end of the exposure period.


F0 EXPOSURE:
An influence of the test item on the survival of parental fish, fecundity and fertilisation success could not be observed.
Statistically significant differences on the survival of parental fish, fecundity and fertilisation success were not observed between treated and control groups at p 0.05. No clear correlation was observed between the concentration of the test item and the survival of parental fish.

Behaviour & health:
- All fish appeared healthy during the exposure, i.e. behavioural or morphological abnormalities were not observed. All fish showed normal feeding behaviour throughout the test. Since no unhealthy fish and/or other abnormalities were observed throughout the exposure, no statistical evaluation was performed on the number of healthy fish. A concentration-relationship and/or increase of abnormal behaviour of fish was not observed at all test item concentrations.

Hatching Success
- The hatching success test conducted during the exposure of the F0 generation (days 3, 9, 15, 20 and 22) showed no influence of the test item on the development of the fish eggs in the parental fish.The hatching success in the treatments was always equal to or higher than the control values. Also the post hatch success seems not to have been influenced by any effect of the test item on the development of the fish eggs in the parental F0 fish.

Weight & length:
- Statistically significant differences on length and weight of parental fish were not observed between treated and control groups at p >=0.05. No correlation was observed between the concentration of the test item and the length and weight of parental fish.



F1 EXPOSURE:
Behaviour & health:
- Concentration-relationship and/or increase of abnormal behaviour of fish larvae was not observed at all test item concentrations

Hatching Success:
- The fish started hatching on day 2 after start of exposure. Hatching was finished on day 9 for all treatments. A concentration-response relationship could not be observed.
In controls the mean hatching success was 97.5%. In all treatments the mean hatching success ranged between 90.0 and 100%. In all treatments the mean hatching success ranged between 90.0 and 100%.
An influence of the test item on the hatching success could not be observed. Statistically significant differences on the hatching success were not observed between treated and control groups at p 50.05. No correlation was observed between the concentration of the test item and the hatching success.

Post-hatching success (survival):
- In the control the post-hatch success (survival) was 86.5%; the validity criterion of >=75%, was met for the control. In the test item treatment groups the mean post-hatch success ranged between 2.7 and 88.7%.
An influence of the test item on the post-hatch success (survival) of fish could be observed. Statistically significant differences of post-hatch success of fish were observed between treated and control groups. Statistically significant differences of post-hatch success of fish were observed between
treated and control groups at p50.05. A clear correlation was observed between the concentration of the test item and the post-hatch success of fish.

Weight & Length:
-Weight: In the control the mean weight was 47.56 mg. In the test item treatment groups the mean dry weight ranged between 42.08 and 48.36 mg, except at the highest treatment level with a mean fish dry weight of 68.85 mg, An influence of the test item on the weight of the surviving fish could be observed.
Statistically significant differences of wet weight of fish were observed between treated and control groups at p 50.05. A correlation was observed between the concentration of the test item and the wet weight of fish.

-Lenght:In the control the mean length was 14.70 mm. In the test item treatment groups the mean fish length ranged between 13.76 and 14.69 mm, except at the highest treatment level with a mean fish length of 16.75 mm.
An influence of the test item on the length of the surviving fish could be observed.
Statistically significant differences of length were observed between treated and control groups at p 50.05. A correlation was observed between the concentration of the test item and the length of fish.

Any other information on results incl. tables

Summary of measured concentrations of the test item in solutions throughout the test:

Nominal concentration [mg test item/L]	Test period [d]	Test period F0 generation [d]	Test period F1 generation [d]	Measured concentration test item [mg a.s./L]	Recovery [%]
0.05	45	-	27	0.0422	84.4
0.25	45	-	27	0.213	85.0
10.0	45	-	27	9.08	90.8
0.05	25	-	34	0.000304	0.6
0.25	25	-	34	0.0546	21.8

The calculated geometric mean measured concentration for each concentration levels are 0.000256, 0.00132, 0.0109 and 0.0730 mg test item/L. The biological endpoints were expressed based on nominal and on geometric mean measured concentrations.

The validity criteria required by the study plan and guideline were fulfilled as follows, except

were indicated otherwise:

F0 generation:

Required	Found
>= 90% survival of fish in the controls	96.4%
Dissolved oxygen level during routine water quality analyses not below 60% of air saturation.	Minimum: 56%*
The water temperature should be within a range of ±2.0 °C of the desired test temperature.	Min.: 24.7 °C, Max.: 26.6 °C (online measurement), Range: 1.9 °C Min.: 25.0 °C, Max.: 26.9 °C (manual measurement), Range: 1.9 °C
Test concentrations were within a range of ±20% of mean measured values per treatment level.	63-98%**

Fl generation:

Required	Found
>=75% post-hatch success in the controls	86.5%
>=70% hatching success in the controls	92.5%
Dissolved oxygen level during routine water quality analyses not below 60% of air saturation.	Minimum: 84%
Water temperature must not differ by more than ±1.5 °C between test vessels or between successive days.	<= 1.1 °C between test vessels on the same day <= 0.6 °C per vessel between successive measuring days
Water temperature must not differ by more than ±1.5 °C between test vessels or between successive days.	Min.: 25.3 °C, Max.: 26.3 °C (online measurement), Range: 1.0 °C Min.: 25.3 °C, Max.: 26.4 °C (manual measurement), Range: 1.1 °C
Test concentrations were measured.	Measured in all treatment levels
No significant effect of any solubilising agent as revealed by a solvent-only control.	None used

* This deviation occurred during the pre-exposure period of the F0 generation and has no impact on the integrity or overall validity of the study results.

** This deviation from the required test concentration range during the F0 generation has no impact on the integrity or overall validity of the study results.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions of this study, no concentration-response relationship was observed for all measured parameters determined for the F0 generation, especially for survival of parental fish, fecundity and fertility. Also, the hatchability test performed with eggs collected from the F0 generation and exposed under control conditions showed no effects on the eggs. Similarly, for the hatching success (hatchability) determined for the F1 generation no concentration-response relationship was observed.

In contrast, a clear concentration-response relationship was observed for post-hatch success and a lesser concentration-response relationship was observed for length and wet weight of fish larvae during the F1 generation. Therefore, it may be concluded that the test item has not negatively influenced the parental fish nor the reproduction of the parental fish during the 23 days of exposure of the F0 generation.

In contrast, juvenile fish exposed to the test item over 32 days were clearly influenced showing effects on post-hatch success as well as weight and length of the F1 fish larvae.

Executive summary:

EXECUTIVE SUMMARY BIOLOGICAL PHASE

Report: Daniel Gilberg & James Chambers: "Mycophenolic acid: A Shortened Life-cycle test with Zebrafish - Investigation of Effects on F0-Reproduction, Fl-Early-Life Stages and Fl-Survival"

Source: ECT Oekotoxikologie GmbH, unpublished report No.: 17AZ1FZ October 26, 2018

Guideline:

• OECD (2008). Detailed Review Paper (DRP) No. 95 on Fish Life-Cycle Tests. OECD Environment, Health and Safety Publications. Series on Testing and Assessment No. 95. ENV/JM/MON0(2008)22. Organisation for Economic Co-Operation and Development, Paris.
• OECD (2012). OECD Guideline for Testing of Chemicals, No. 229 "Fish Short Term Reproduction Assay". Adopted on 02 October 2012. Organisation for Economic Co-Operation and Development, Paris.
• OECD (2013). OECD Guideline for Testing of Chemicals, No. 210 "Fish, Early-life Stage Toxicity Test". Adopted on 26 July 2013. Organisation f or Economic Co-Operation and Development, Paris.

GLP: Yes (certified laboratory)

Dates of experimental work: June 23, 2017 — August 26, 2017 (biological phase)

Test item:

- Name: Mycophenolic acid

- Chemical name: 6-(1,3-Di hyd ro-4-hyd roxy-6-methoxy-7-methy1-3-oxo-5-isobenzofurany1)-4-methy1-4-hexenoic acid

Material and methods:

Test organism: Zebrafish (Danio rerio, Hamilton-Buchanan 1822)

Test medium: Reconstituted water (OECD guideline No. 203) mixed with deionised water (1:1; v/v), supplemented with 1% artificial seawater

Endpoints: EC/LC, NOEC, LOEC

Biological parameters:

*F0 generation:

- Survival of the mature fish;

- Fecundity;

- Fertilisation success;

- Numbers of atretic eggs;

- Any changes in appearance and any abnormal behaviour;

- Sex of the parental fish.

*Fl generation:

- Hatching success;

- Number of larvae hatching each day;

- Time to start of hatching and end of hatching;

- Cumulative mortality;

- Number of deformed or abnormally appearing larvae;

- Number of organisms exhibiting abnormal behaviour;

- Length and wet weight of the surviving fish.

Test duration:

F0 generation: 23 days

F1 generation: 34 days

Temperature (target): 26±1.5°C

Photoperiod: 12/12 hours light/dark cycle

Light intensity (target): 550-1000 lx

Test units:

F0 generation: Stainless steel, 18 I volume

F1 generation: Glass vessels, 0.9 I volume

Test concentrations: 0.0005, 0.0029, 0.0171 and 0.100 mg test item/I plus a control

No. of replicates per treatment: F0 generation: 2; F1 generation: 4

Renewal of test solution during exposure: Permanent (flow-through)

Chemical analysis of test concentrations: Control, 0.0005, 0.0029, 0.0171 and 0.100 mg test item/L.

Data evaluation: Probit analyses, Weibull analyses, 2- and 3-param. normal CDF, Shapiro-Wilk's test on normal distribution, Levene's test on variance homogeneity, Fisher's Exact Binomial test, Chi' 2x2 Table Test, Welsh t-test, Step-down Cochran-Armitage test Procedure, Williams Multiple Sequential t-test Procedure and Dunnett's Multiple t-test Procedure.

The biological phase of the study was conducted at ECT Oekotoxikologie GmbH, Flörsheim am Main, Germany. Samples of test solutions were analysed at Battelle UK Ltd under the responsibility of the principal investigator for analysis.

Findings:

Biological findings:

Under the experimental conditions of this study, no concentration-response relationship was observed for the parameter survival of the parental fish, fecundity and fertilisation success evaluated for the F0 generation.

At the end of the F1 generation no concentration-response relationship was observed for the parameter hatching success, whereas a clear concentration-response relationship was observed for the parameter post-hatch success (survival), wet weight and length of the surviving fish.

To determine the actual test item concentrations, samples were taken from the test solutions for analytical measurement. Samples were measured in test solutions at regular intervals throughout the exposure period and the mean measured concentrations were calculated.

Analytical findings:

The concentrations measured during the exposure of the F0 generation were not within ±20% of nominal concentration. The recoveries range between 63% and 99% of the nominal concentration. Nevertheless, the measured concentrations, showed no timedependent trend, and may therefore be stated to be stable within this range.

The concentrations measured during the exposure of the Fl generation range between <LOD and 104% of the nominal concentration. Only at the end of the Fl generation was a considerable decrease of the measured concentrations over the exposure period observed.

The geometric mean measured concentration from the samples taken throughout the whole test period (F0 and F1 generation) were used. The calculated geometric mean measured concentrations for each concentration level are 0.000256, 0.00132, 0.0109 and 0.0730mg test item/I, respectively.

The biological endpoints (ECAC. and NOEC/LOEC) were corrected and reported based on the geometric mean measured concentration in the test solutions.

Validity of the test:

The validity criteria required by the study plan and guideline were fulfilled, except two deviations that had no impact on the integrity or overall validity of the study results.

EXECUTIVE SUMMARY ANALYTICAL PHASE

Experimental Start date (sample receipt) September 08, 2017

Experimental Termination date (final analysis) October 26, 2017

Materials and Methods

The purpose of the analytical phase of the study was to determine the concentration of Mycophenolic Acid in water samples from the toxicity test, in order to meet the requirements of the test guideline. The method for the determination of Mycophenolic Acid involved quantification by LC-MS/MS. The method for water involved direct injection of samples following dilution and required no prior validation.

The limit of detection (LOD) for MPA, based on 3 x noise, is 0.000008 mg/I.

The limit of quantification (LOQ) for MPA, based on 10 x noise, is 0.00003 mg/I.

Findings:

• The concentrations of Mycophenolic Acid found in the FO generation solutions of the Cl dose group, sampled at 1, 3, 6, 13 and 23 days, were 0.000404, 0.000428, 0.000354, 0.000356 and 0.000398 mg/I, respectively. The concentrations found in the F1 generation solutions of the Cl dose group, sampled at 18, 24, 31, 38, 45 and 52 days, were 0.000470, 0.000400, 0.000344, 0.000306, 0.000230 and <LOD mg/I, respectively.
• The concentrations of Mycophenolic Acid found in the FO generation solutions of the 02 dose group, sampled at 1, 3, 6, 13 and 23 days, were 0.00244, 0.00224, 0.00204, 0.00224 and 0.00248 mg/I, respectively. The concentrations found in the F1 generation solutions of the C2 dose group, sampled at 18, 24, 31, 38, 45 and 52 days, were 0.00270, 0.00272, 0 .00234, 0.00188, 0.00135 and <LOD mg/I, respectively.
• The concentrations of Mycophenolic Acid found in the FO generation solutions of the C3 dose group, sampled at 1, 3, 6, 13 and 23 days, were 0.0129, 0.0130, 0.0116, 0.0107 and 0 .0142 mg/I, respectively. The concentrations found in the F1 generation solutions of the 03 dose group, sampled at 18, 24, 31, 38, 45 and 52 days, were 0.0164, 0.0152, 0.0137, 0 .0133, 0.0113 and 0.00174 mg/I, respectively.
• The concentrations of Mycophenolic Acid found in the F0 generation solutions of the C4 dose group, sampled at 1, 3, 6, 13 and 23 days, were 0.0866, 0.0860, 0.0782, 0.0636 and 0 .0978 mg/I, respectively. The concentrations found in the F1 generation solutions of the C4 dose group, sampled at 18, 24, 31, 38, 45 and 52 days, were 0.104, 0.0938, 0.0896, 0 .0942, 0.0682 and 0.0155 mg/I, respectively.
• During sample analysis, the lowest nominal fortification level in test medium for recovery efficiency testing was 0.0005 mg/I.
• The mean recovery efficiency from 6 tests conducted on the day of analysis was 99% with an RSD of 4.6%.
• There were no residues of Mycophenolic Acid detected in control samples analysed during sample analysis.