2-[3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazoniol-5-yl]ethyl dihydrogen diphosphate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to the analogue approach justification document provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Source: CAS 10023-48-0, Gado & Broschard, 2017

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Source: CAS 10023-48-0, Gado & Broschard, 2017

Conclusions:

The key values retained for the assessment of the toxicity of the target substance to aquatic algae are an ErC10 (72 h) > 100 mg/L and an ErC50 (72 h) > 100 mg/L (geometric mean measured, OECD 201, P. subcapitata, CAS 10023-48-0); read-across

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Apr - 28 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted 23 Mar 2006, corrected 28 Jul 2011

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany (18 Nov 2014)

Analytical monitoring:

yes

Remarks:

HPLC-UV

Details on sampling:

- Concentrations: Control, 6.25, 12.5, 25.0, 50.0, and 100 mg/L (nominal)
- Sampling method: Two replicates of each sample were taken directly after preparation on each day of exposure (0, 24, 48, and 72 h). One replicate was used for analysis and the other was kept as backup. For the chemcial analyses, a separate replicate without algae and two separate replicates with algae were prepared for every treatment level.
- Sample storage conditions before analysis: Samples without algae were directly stored at ≤ - 20 °C until analysis. Samples with algae were first centrifuged and the supernatant was stored at ≤ - 20 °C. The stability of the test item during freezing was demonstrated in a separate non-GLP study.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 100 mg/L test item in reconstituted water was prepared by mixing for 30 min with a magnetic stirrer. The stock solution was diluted to the different test item concentrations. The pH values was not adjusted.
- Controls: Reconstituted water (untreated test medium)
- Evidence of undissolved material: The test media were clear preparations and stayed unchanged throughout the study.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green alga
- Strain: SAG 61.81
- Source: Pflanzenphysiologisches Institut der Universität Göttingen, Germany
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: The permanent algae cultures, the pre-culture and the algae cultures of the study were cultivated in an air-conditioned room with a water temperature of 21.0 - 24.0 °C ± 2 °C. For cultivation, 300 mL Erlenmeyer flasks filled with 100 mL algae suspension were covered with air permeable stoppers (Heinz Herenz Medizinalbedarf GmbH). The cultures were shaken continuously at about 120 rpm (Universalschüttler SM25) under 4440 - 8880 lux (fluorescent tubes, Lumilux T5 new FLH1 HO 80W/840, Osram GmbH).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L CaCO3 (0.24 mmol/L) (reconstituted water)

Test temperature:

23.5 - 24.2 °C

pH:

6.93 - 8.24

Nominal and measured concentrations:

Control, 6.25, 12.5, 25.0, 50.0, and 100 mg/L (nominal)
< LOD, 5.19, 10.72, 21.60, 44.14, and 91.10 mg/L (measured after 72 h, without algae)
< LOD, 21.5 - 97.5% of nominal (measured after 72 h, with algae)

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks filled with 100 mL test medium covered with air permeable stoppers.
- Type: Open
- Initial cells density: 10000 cells/mL
- Control end cells density: 222418 cells/mL
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The components of the reconstituted water were dissolved in fully demineralized water (Milli-Q Water Purification System, Merck).
- Culture medium different from test medium: Culuture medium same as test medium. The pre-culture used as inoculum was prepared in dilution water.
- Intervals of water quality measurement: The pH was measured in the control group and the test item groups with algae and in one additional falsk of each group without algae at the start and end of the test. The water temperature was continuously measured in a separate Erlenmeyer flask containing fully demineralized water only kept under the same experimental conditions.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test item solution was not adjusted.
- Light intensity and quality: 7548 lux (mean at test start) and 7650 lux (mean at test end)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Electronic particle counter (Coulter-Counter Z2, Beckman Coulter GmbH)
- Cell count: After 24, 48, and 72 h (± 1 h)
- Morphological effects: Assessed at the end of the test by microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes
- Results used to determine the conditions for the definitive study: Yes, growth inhibition was seen between 1.0 and 100.0 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: No morphological abnormalities occurred.
- Any observations that might cause a difference between measured and nominal values: The test media were clear preparations and stayed unchanged throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the EC50 values are in the range of ± two standard deviations of the current historical means.
- ErC50 (72 h): 0.86 mg/L, 95% confidence interval 0.84 - 0.88 mg/L
- Other: The last reference test dates from 16 - 19 Mar 2017.

Reported statistics and error estimates:

The EC-values were calculated by Probit analysis using the software ToxRat Professional Version 2.10.

ANALYTICAL RESULTS

Overall, the analytical results showed decreased concentrations of test item after 72 h in samples with algae, indicating that the test item might be bioavailable and metabolized by the algae (see Tables 1 and 2).

Therefore, the EC values were based on the geometric mean measured concentrations.

ANALYTICAL RESULTS WITH ALGAE

The recoveries of all test medium samples with algae ranged from 21.5 - 97.6% of the nominal concentrations over 72 h (Table 1). Due to the low recoveries after 72 h especially of the lower concentrations, all 72 -h sampleswere re-analysed. Re-analysis confirmed the low recoveries.

Table 1. Analytical results of samples with algae.

Exposure time	0 h^1)		24 h		48 h		72 h
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	[%]^3)	Meas. conc. [mg/L]	[%]^3)	Meas. conc. [mg/L]	[%]^3)	Meas. conc. [mg/L]	[%]^3)
0	< LOD	-	< LOD	-	< LOD	-	< LOD	-
6.25	6.100	97.6	5.509	88.1	5.182	82.9	3.861	61.8
							4.163^2)	66.6
12.5	11.684	93.5	11.359	90.9	10.837	86.7	2.686	21.5
							3.129^2)	25.0
25.0	23.145	92.6	22.568	90.3	21.772	87.1	13.546	54.2
							14.788^2)	59.2
50.0	46.642	93.3	45.749	91.5	44.401	88.8	41.106	82.2
							42.695^2)	85.4
100.0	94.729	94.7	91.867	91.9	90.668	90.7	87.993	88.0
							91.393^2)	91.4

1) measured without algae

2) repeat analysis to confirm 1st result (not used for EC50 value calculation)

3) Rounded to the first decimal place

LOD = Limit of detection

ANALYTICAL RESULTS WITHOUT ALGAE

The recoveries in all test media without algae ranged from 83.1 - 97.6% of the nominal concentrations over 72 h (Table 2). Thus, the substance was stable in the test medium for the duration of the test.

Table 2. Analytical results of samples without algae.

Exposure time	0 h		24 h		48 h		72 h
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	[%]^1)	Meas. conc. [mg/L]	[%]^1)	Meas. conc. [mg/L]	[%]^3)	Meas. conc. [mg/L]	[%]^1)
0	< LOD	-	< LOD	-	< LOD	-	< LOD	-
6.25	6.100	97.6	5.593	89.5	5.331	85.3	5.191	83.1
12.5	11.684	93.5	11 .215	89.7	10.890	87.1	10.717	85.7
25.0	23.145	92.6	22.547	90.2	21.664	86.7	21.599	86.4
50.0	46.642	93.3	45.672	91 .3	43.863	87.7	44.137	88.3
100.0	94.729	94.7	91.978	92.0	91.359	91.4	91.102	91.1

1) Rounded to the first decimal place

LOD = Limit of detection       

BIOLOGICAL RESULTS

No significant inhibition of algae growth was determined.

VALIDITY CRITERIA

The study fulfilled the validity criteria laid down by the OECD guideline 201 (Table 3).

Table 3: Validity criteria OECD guideline 201.

Criterion from the guideline	Study results	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	Biomass in the control increased by a factor of 222.0 over 72 h.	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficient of variation of the daily growth rates in the control during 72 h was 26.6%.	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	The coefficient of variation of the average specific growth rates in the replicates of the control group after 72 h was 2.3%	Yes

 

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

ErC10 (72 h) > 100 mg/L (geometric mean measured, OECD 201, P. subcapitata); read-across

ErC50 (72 h) > 100 mg/L (geometric mean measured, OECD 201, P. subcapitata); read-across

Additional information

There is no study available, in which the toxicity of the target substance 2-[3-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazoniol-5-yl]ethyl dihydrogen diphosphate (CAS 136-09-4) to aquatic algae was investigated. Therefore, read-across to the structurally and physico-chemically related source substance 2-{3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl}ethyl hydrogen phosphate (CAS 10023-48-0) was conducted in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5. Based on the physico-chemical similarity, the source substance is considered a suitable proxy for the assessment of the toxicity of the target substance to aquatic algae. A detailed analogue approach justification is provided in IUCLID section 13 of the technical dossier.

 

In the available study, the toxicity of the source substance (CAS 10023-48-0) to aquatic algae was investigated according to OECD guideline 201 and GLP. In a static test, exponential phase Pseudokirchneriella subcapitata at an initial cell density of 10000 cells/mL was exposed to five nominal concentrations of 6.25, 12.5, 25.0, 50.0, and 100 mg/L test item in an open system for 72 h. Test item concentrations were analytically verified by HPLC-UV at the beginning of the test (0 h) and then every 24 h in replicates with and without algae.

Substance recovery over 72 h ranged from 83.1 – 97.6% of the nominal concentrations in all samples without algae and from 21.5 – 97.6% of the nominal concentrations in all samples with algae. Particularly low recoveries were obtained after 72 h in samples with algae, indicating that the substance may be bioavailable and metabolized by algae. Therefore, the EC values were based on the geometric mean measured concentrations.

After 72 h no significant inhibition of algae growth was observed and the reported effect concentrations are an ErC10 (72 h) of > 100 mg/L and ErC50 (72 h) of > 100 mg/L.

Based on the high degree of structural and chemical similarity of the target and source substance, the target substance is expected to exhibit a similar ecotoxicological profile as the source substance. Therefore, it can be concluded that the target substance 2-[3-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazoniol-5-yl]ethyl dihydrogen diphosphate (CAS 136-09-4) does not cause toxicity to aquatic algae up to a nominal concentration of 100 mg/L.