Mycophenolic acid

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31. October 2016 - 31. January 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

October 2012

Deviations:

no

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: Not specified

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method for chemical analysis: Samples were taken in duplicates at week 1, 2 and 3 directly after preparation of the test solutions and before renewal.
- Samples were stored protected from light in glass bottles of appropriate volume (e.g. bottle volume at least twice the sample volume) at =–18 °C, transferred to the analytical laboratory for chemical analysis (Battelle UK Ltd.) 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 and an aliquot 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:

3.5 ml 1-M NaOH per litre deionised water

Details on test solutions:

PREPARATION OF STOCK AND TEST SOLUTIONS
A solution was prepared of 3.5 ml 1-M NaOH per litre deionised water. This solution was diluted with deionised water at a rate of 1:10 (v/v). The test item (60 mg nominal) was mixed with 3000 ml of this solution to result in a concentration of 20 mg test item/l (S0). S0 was stirred overnight at ambient temperature in the dark.

Thereafter the pH of S0 was measured and adjusted to 7.8 with 1-M HCl.

Stock solution S0 was diluted to a nominal concentration of 10 mg test item/l by addition of double-concentrated test medium (see section 13.4) and was termed S1; the pH was measured and adjusted to 8.0 with 1-M HCl.

The stock solutions S0 and S1 were prepared once per exposure week. The stock solution S1 was stored in the refrigerator in the dark for up to 5 days while not in use. Before each use S1 was brought to ambient temperature and visually inspected for particles in the solution; none were observed.

Each time when prepared, the volume of the stock solution was large enough to prepare all test concentration levels and all analytical samples at once.

The test solutions were prepared once per exposure week and used without further storage.

The test vessels containing the test solutions were placed into a temperature controlled room at test temperature for temperature adaptation. The test vessels were placed on three trays that were arranged in the testing area in a randomised manner at start of the test and after each renewal of test solutions.

PREPARATION OF MEDIA
Elendt medium M4 was used to dilute the test item and to keep the daphnids during the period of the test.
The medium consists of trace elements, vitamins and macro-nutrients in the following final concentrations (in mg/L):

Trace elements (in mg/L):
H3BO3 - 2.86
MnCl2 × 4 H2O - 0.361
LiCl - 0.306
RbCl - 0.071
SrCl2 × 6 H2O - 0.152
NaBr - 0.016
Na2MoO4 × 2 H2O - 0.063
CuCl2 × 2 H2O - 0.017
ZnCl2 - 0.013
CoCl2 × 6 H2O - 0.010
KI - 0.0033
Na2SeO3 - 0.0022
NH4VO3 - 0.00058
Na2EDTA × 2 H2O - 2.5
FeSO4 × 7 H2O - 1.0

Macronutrients (in mg/L):
CaCl2 × 2 H2O - 293.8
MgSO4 × 7 H2O - 123.3
KCl - 5.8
NaHCO3 - 64.8
Na2SiO3 × 9 H2O - 10.0
NaNO3 - 0.274
KH2PO4 - 0.143
K2HPO4 - 0.184

Vitamins (in mg/L):
Thiamine hydrochloride - 0.075
Cyanocobalamin - 0.001
Biotin - 0.00075

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna clone M10
- Common name: Water flea
- Cultured at ECT Oekotoxikologie GmbH since December 22, 2011. Origin: The organisms were originally supplied by KU Leuven, Belgium.
- Age at study initiation: Neonate daphnids (parent animals; F0 females) aged less than 24 hours.
- Method of breeding: They were produced from a parthenogenetically reproducing female population of an age of 12–14 days and were not the first brood of these adult parental daphnids.
- Method of holding:
*The daphnids were held in a specific medium (Elendt Medium M4 as specified in the test guideline). The vessels containing the stock culture are on a 16/8 hours light/dark cycle at a target temperature of 20±2 °C. The daphnids are fed with green algae (Desmodesmus subspicatus) and yeast.
*Material of stock vessel: glass
*Volume of medium per stock vessel: 1.8 l
*Depth of medium in the stock vessel: approximately 14.5 cm
*Number of daphnids kept as stock per culture vessel (batch size): approximately 20
*Separation of adult and young daphnids: 2–5 times per week
*Renewal of medium: batchwise, twice per week
*Light intensity: 50–1000 lx
*Aeration: none
*Feeding frequency: 4 times per week The health of the Daphnia culture and the overall quality of the test organisms is monitored regularly by using the reference toxicant potassium dichromate (K2Cr2O7) in an acute test design in order to determine the sensitivity of the organisms from the culture and to demonstrate that the test conditions are appropriate.
*The selected holding vessels designated for production of the neonates for the test were cleared of all mixed-age young daphnids on the day before test start. These vessels contained parental daphnids aged less than 28 days. On the day of test start (day 0) all newly produced neonates of these vessels were <24 hours old. These neonates were collected for the test.

- Feeding during test: three times per week, fresh algae suspension of Desmodesmus subspicatus


ACCLIMATION
- Acclimation conditions: After temperature adaptation of the test solutions the daphnids were transferred into the test solutions in a randomised manner by using upside-down glass pipettes.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

14.0-14.2 [°dH], 250-154 mg/LCaCO3

Test temperature:

19.3–20.5 °C (manual measurement; n = 12)
17.6–22.5 °C (automatic measurement in test medium; n = 442)

pH:

7.7-9.0

Dissolved oxygen:

93-128 [%ASV], 8.4-11.5 mg/L

Conductivity:

640 [µS/cm]

Nominal and measured concentrations:

Nominal concentrations[mg/L], high to low :
10.0 / 3.16 / 1.00 / 0.316 / 0.100

Measured concentrations [mg/L] corresponding to the nominal concentration:
Week 1, fresh: 10.3 / 1.34 / 0.701 / 0.19 / 0.0756
Week 1, old: 6.92 / 1.83 / 0.681 / 0.198 / 0.0832

Week 2, fresh: 8.24 / 2.44 / 0.767 / 0.22 / 0.0766
Week 2, old: 5.76 / 1.71 / 0.496 / 0.174 / 0.0444

Week 3, fresh: 8.28 / 1.85 / 0.626 / 0.162 / 0.0598
Week 3, old: 6.68 / 1.48 / 0.541 / 0.14 / 0.036

% of nominal:
Week 1, fresh: 102.8 / 42.4 / 70.1 / 60.1 / 75.6
Week 1, old: 69.2 / 58.0 / 68.1 / 62.6 / 83.2

Week 2, fresh: 82.4 / 77.3 / 76.6 / 69.7 / 76.6
Week 2, old: 57.6 / 54.2 / 49.6 / 55.0 / 44.4

Week 3, fresh: 82.8 / 58.6 / 62.6 / 51.1 / 59.8
Week 3, old: 66.8 / 46.9 / 54.1 / 44.4 / 36.6

Details on test conditions:

TEST SYSTEM
- Test vessel: 100-ml glass beakers (height: 7 cm; diameter: 5.3 cm), covered by glass lids, containing 50-60mL of test solution / test vessel.
- No. of organisms per Vessel: 1 daphnid (F0 female) aged less than 24 hours at the beginning of the test.
- Replicates for test solutions: at least 10 replicates.
- Replicates for Controls: at least 10 replicates.

TEST MEDIUM / WATER PARAMETERS:
- Test solutions were renewed three times per week.
- Temperature: 19.3–20.5 °C (manual measurement; n = 12); 17.6–22.5 °C (automatic measurement in test medium; n = 442); the maximum daily range exceeded 2 °C by 0.5 °C on 3 occasions

OTHER TEST CONDITIONS
- Light regime: 16 h light/8 h dark
- Light intensity: 8.9–10.5 µE m–2 s–1 (n = 3)

DEVIATIONS FROM STUDY PLAN OR GUIDELINES
Deviating from the study plan the automatically recorded temperature was between 17.6 and 22.5 °C, i.e. the temperature range over the total exposure period exceeded the required limit of ±1 °C by ±1.5 °C. The maximum daily temperature range exceeded 2 °C by 0.5 °C on three occasions. As the reproduction in the controls exceeded the minimum required number of offspring per parent, an iNfluence of the temperature range on the integrity of the study is not expected.

Reference substance (positive control):

yes

Remarks:

potassium dichromate, separate study performed from May 31 to June 01, 2016 (Study No. IDA1605)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.929 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ fecundity

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

4.285 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ fecundity

Effect concentrations 3

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ fecundity

Effect concentrations 4

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ fecundity

Effect concentrations 5

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.53 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

/ immobility of parental daphnids

Effect concentrations 6

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 7.53 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

/ immobility of parental daphnids

Effect concentrations 7

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks:

length of parental daphnids

Effect concentrations 8

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks:

length of parental daphnids

Effect concentrations 9

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.53 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

intrinsic rate of population increase

Effect concentrations 10

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 7.53 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

intrinsic rate of population increase

Details on results:

A clear concentration-response relationship of daphnid reproduction was observed for reproduction.

Sublethal effects as observed in the living parental daphnids were only recorded at the highest concentration level. For example, immobile or floating neonates as well as individual and temporarily floating parental daphnids were observed. In none of the treatments below 10 mg/L (nominal) were any sublethal effects recorded.

The mortality of the parent animals (female Daphnia) in the controls (required: =< 20%): 0.0%
The mean number of live offspring produced per parent animal in the controls surviving at the end of the test (required: >= 60%): 78.7%

Results with reference substance (positive control):

The reference test with potassium dichromate performed from May 31 to June 01, 2016 (Study No. IDA1605) resulted in an EC50(24 h) of 1.68 mg/l.

According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h) values for potassium dichromate obtained from different laboratories should range between 0.6 mg/l and 2.1 mg/l.
The toxicity of the reference item is within this range. Therefore the results of this reference test are acceptable and the test conditions are reliable and the sensitivity of the test system could be demonstrated.

Any other information on results incl. tables

The time-weighted mean measured concentrations are shown in the following table:

treatment	nominal concentration	time-weighted mean concentration	recovery
	mg/L	mg/L	(% of nominal)
C1	0.100	0.0615	61.5
C2	0.316	0.183	57.8
C3	1.00	0.630	63.0
C4	3.16	1.80	57.1
C5	10.0	7.53	75.3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A clear concentration-response relationship was observed for reproduction.

Sublethal effects as observed in the living parental daphnids were only recorded at the highest concentration level. For example, immobile or floating neonates as well as individual and temporarily floating parental daphnids were observed. In none of the treatments below 10 mg/L (nominal) were any sublethal effects recorded.

Executive summary:

Report: PHILIPP EGELER & JAMES G. CHAMBERS: "Mycophenolic acid: A Study on the Chronic Toxicity to Daphnia magna"

Source: ECT Oekotoxikologie GmbH, unpublished report No.: 16AZ9DB, 31 January 2018

Guideline: OECD (2008). OECD Guideline for the Testing of Chemicals, No. 211, "Daphnia magna, Reproduction Test" adopted October, 2012

GLP: Yes (certified laboratory)

Dates of experimental work: 01 November–23 November 2016 (biological phase)

Test item:

Name: Mycophenolic acid

- Chemical name: 6-(1,3-Dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5- isobenzofuranyl)-4-methyl-4-hexenoic acid

- CAS number: 24280-93-1

Material and methods:

Test system: Daphnia magna (Straus)

Medium: Elendt Medium M4

Endpoints: ECx, NOEC/LOEC

Biological parameters:

- reproduction as assessed by fecundity (total number of living offspring (F1) per parental animal alive at the end of the test);

- mortality/survival as assessed by immobility of parental (F0) daphnids;

- length of surviving parental daphnids

- intrinsic rate of population increase

Test duration: 21 days

Temperature: 19.3–20.5 °C (manual measurement; n = 12) 17.6–22.5 °C (automatic measurement in test medium; n = 442)

Adjustment of pH in the test solutions: none

Photoperiod: 16 h light/8 h dark

Aeration: no aeration during exposure

Test units: 100 ml glass beakers, covered by glass lids

Volume of test solution per test vessel: 50–60 ml

Number of daphnids per test vessel: one at start of exposure

Feeding during exposure: three times per week, fresh algae suspension of Desmodesmus subspicatus; 0.15–0.20 mg C/daphnia × d

Test item concentrations (nominal): 10, 3.16, 1.00, 0.316 and 0.100 mg test item/l plus a control

Number of replicates per test concentration and control: 10

Renewal of test solution during exposure: semi-static conditions; test solutions renewal three times per week

Chemical analysis of test concentrations: once each week of exposure (fresh and aged solutions)

Data evaluation: Williams test, Fisher's Exact test, Multiple sequentiallyrejective U-test after Bonferroni-Holm, Probit analysis

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, Chelmsford, UK, under the responsibility of the principal investigator for analysis.

Validity of the test:

All validity criteria were fulfilled as required by the study plan:

Required		Found
The mortality of the parent animals (female Daphnia) in the controls: (required: 20%)		0.0%
The mean number of live offspring produced per parent animal in the controls surviving at the end of the test: (required: 60%)		78.8%

Findings:

A clear concentration-response relationship was observed for reproduction.

Thus, the following endpoints were established based on nominal concentrations: Endpoints (ECx, NOEC, LOEC) for reproduction as assessed by fecundity (total number of living offspring per parent animal alive at the end of the test), mortality as assessed by immobility of parental daphnids, length of parental daphnids and intrinsic rate r based on statistical evaluation of biological results and nominal concentrations in mg/l.

Endpoints [mg/L nominal concentration]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Reproduction/fecundity	1.746	2.711	6.290	1.00	3.16
Mortality/immobility of parental daphnids	na	na	na	10.0	>10.0
Length of parental daphnids	na	na	na	1.00	3.16
Intrinsic rate of population increase	na	na	na	10.0	>10.0

na: not applicable since ECx values could not be determined due to the lack of a meaningful concentration/response relationship

The time-weighted mean measured concentrations are shown in the following table:

treatment	nominal concentration	time-weighted mean concentration	recovery
	mg/L	mg/L	(% of nominal)
C1	0.100	0.0615	61.5
C2	0.316	0.183	57.8
C3	1.00	0.630	63.0
C4	3.16	1.80	57.1
C5	10.0	7.53	75.3

The biological endpoints were additionally expressed based on time-weighted mean measured concentrations (see table below).

Endpoints (NOEC, LOEC) for reproduction as assessed by fecundity (total number of living offspring per parent animal alive at the end of the test), mortality as assessed by immobility of parental daphnids, length of parental daphnids and intrinsic rate r based on statistical evaluation of biological results and on time-weighted mean measured concentrations (mg/l) as determined over the complete exposure period (day 0 to day 21).

Endpoints [mg/L measured concentration]
Parameter	EC10	EC20	EC50	NOEC	LOEC
Reproduction/fecundity	0.929	1.570	4.285	0.630	1.80
Mortality/immobility of parental daphnids	na	na	na	7.53	>7.53
Length of parental daphnids	na	na	na	0.630	1.80
Intrinsic rate of population increase	na	na	na	7.53	>7.53

na: not applicable since ECx values could not be determined due to the lack of a meaningful concentration/response-relationship