(S)-7-[[2-O-6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]oxy]-2,3-dihydro-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 105.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Read-across from analogue substance

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

105.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Read-across from analogue substance

Details on results:

The results of the read-across approach from the experimental data obtained for the supporting substance are expressed as the estimated toxicity based on molecular weights. No other adaptation is necessary.

Validity criteria fulfilled:

yes

Conclusions:

Based on the available information for the read-across approach, the target substance has a 96h-EC50 > 105.1 mg/L and a NOEC ≥ 105.1 mg/L.

Executive summary:

An acute toxicity test on zebrafish (Brachydanio rerio) was performed on the analogue substance naringin, in accordance with OECD Guideline 203 / EU method C.1, under GLP conditions. Based on the results obtained in a preliminary range-finding test where no toxic response was observed, a Limit Test was carried out using only one test concentration (100 mg/L) and one control group in a semi-static system. The renewal period of the water was 48h, due to the stability of the test item. Test concentrations were analytically determined (by means of a validated method) at the beginning and at the end of each renewal period and did not deviate more than 20%. Seven fish per group were exposed to the test item or the control for 96 h. All validity criteria were met.The 96h-EC50 of the test item in zebrafish was determined to be higher tan 100 mg/L and the NOEC was 100 mg/L, both based on mortality. Based on the available information for the read-across approach, the target substance has a 96h-EC50 > 105.1 mg/L and a NOEC ≥ 105.1 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 17th to July 21st, 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 016G050
- Expiration date of the lot/batch: 21/07/2016
- Purity test date: 21/07/2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%), protected from light and humidity.
- Stability under test conditions: stable during test period.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 100 mg/L.
- Sampling method: The test concentration was analytically determined at the start and at the end of the renewal periods (0, 48, 96h) by HPLC-UV method.
- Sample storage conditions before analysis: No storage was necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a concentration of 100 mg/L (nominal) was prepared with direct addition of the test item, mixed into the test medium (aquarium water) using ultrasonic bath (approximately 45-50 minutes). As a limit test was carried out, further dilution of stock solution was not performed.
- Eluate: aquarium water.
- Controls: The dilution water (circulated and filtered) was used without of addition of the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.
- As the sample was only stable for 2 days, fresh test item solution was prepared by the method described above at each renewal period.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Brachydanio rerio
- Source: “Black Molly” Fish Farm, Veszprém, Hungary (preliminary test); István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary (definitive test)
- Age at study initiation (mean and range, SD): not specified.
- Length at study initiation (length definition, mean, range and SD): 2.6 – 2.9 cm
- Weight at study initiation (mean and range, SD): mean weight 1.45 – 1.54 g

ACCLIMATION
- Acclimation period: more than 12 days
- Acclimation conditions (same as test or not): same as test.
- Health during acclimation (any mortality observed): Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. There was no mortality of the population for seven days before the test.

FEEDING DURING TEST
- The fish were not fed during the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

194 mg/L (as CaCO3)

Test temperature:

20.3 – 24.6 ºC

pH:

7.61 – 8.20

Dissolved oxygen:

81 – 99 % of the air saturation value at the temperature used.

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 100 mg/L.
- Measured concentrations: 0 (control), 102 and 101 mg/L (at the start of the 48h-renewal periods); 91.7 and 96.2 mg/L (at the end of the renewal periods).

Details on test conditions:

TEST SYSTEM
- Test vessel: one aquarium for the test group and another one for the control group.
- Material, size, headspace, fill volume: fill volume 5L test solution.
- Renewal rate of test solution (frequency/flow rate): every 48h (as seen in study 16/333-316AN, the sample was only stable for 2 days in aquarium water).
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: aquarium water (circulated and filtered)
- Culture medium different from test medium: no.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The observations of mortality and sub-lethal effects of the fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. The test conditions (pH, temperature, oxygen saturation) were measured daily (twice after 48 hours - at the end of the first as well as at the beginning of the second renewal period) during the test. The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (1.0 g fish/litre testing liquid).

TEST CONCENTRATIONS
- Range finding study: Four fish were exposed to each concentration of the test item plus a control, for 96 hours under semi-static conditions (with 24-hour renewal).
- Test concentrations: In the range-finding study, the concentrations tested were 0 (control), 0.1, 1, 10, 100 mg/L test item. As the measured concentrations deviated not more than 20 per cent from the nominal, the biological results are based on the nominal concentration.
- Results used to determine the conditions for the definitive study: Because no toxic response was observed during the preliminary range-finding test, only one test concentration (100 mg/L)) and one control group was tested in the definitive study in a semi-static system (limit test).

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: No abnormal behaviour, appearance or any other effect on test animals were detected.
- Observations on body length and weight: There was no considerable difference observed concerning body weights between the groups. Body lengths were within the acceptable ranges.
- Mortality of control: 0/7.
- Other adverse effects control: no.
- Abnormal responses: no.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: no.

Reported statistics and error estimates:

No statistical analysis was performed because of the lack of toxic effects. The LC50, NOEC, LOEC and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Table 2. Cumulative mortality data in the Limit Test

Test Group	Cumulative Mortality (number of dead fish) [initial population = 7 fish]
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
100 mg/L (nominal)	0	0	0	0	0	0

No sublethal effect was observed.

 

Table 3. Measured and calculated data of body weight

Test Group	Measured weight of 7 fish (g)	Calculated mean weight of 1 fish (g)	Loading of testing aquarium (g fish/L testing liquid)
Control	1.54	0.22	0.31
100 mg/L (nominal)	1.45	0.21	0.29

There was no considerable difference observed concerning body weights between the groups.

 

Table 4. Temperature

Test Group	Temperature (°C)
	Measuring
	0h^1)	24h	48^2)	48^1)	72	96^2)
Control	21.8	20.6	20.4	21.9	20.3	20.3
100 mg/L (nominal)	24.6	20.6	20.4	24.1	20.4	20.3

  

Table 5. Dissolved oxygen concentration

Test Group	Dissolved oxygen concentration (%)
	Measuring
	0h^1)	24h	48^2)	48^1)	72	96^2)
Control	99	94	91	97	92	89
100 mg/L (nominal)	91	87	85	89	84	81

 

Table 6. pH

Test Group	pH
	Measuring
	0h^1)	24h	48^2)	48^1)	72	96^2)
Control	8.11	7.98	8.02	8.17	8.03	7.93
100 mg/L (nominal)	8.18	8.12	7.96	8.20	7.94	7.61

¹⁾measurement at the start of the renewal period in the fresh test medium

²⁾measurement at the end of the renewal period in the old test medium

 

Table 7. Body length of test fish at the end of the test

Test Group	Body length [cm]
	1	2	3	4	5	6	7
Control	2.8	2.7	2.7	2.9	2.8	2.6	2.7
100 mg/L (nominal)	2.7	2.9	2.8	2.6	2.7	2.7	2.8

Remark: At the end of the test, body length of test fish were measured in order to check their compliance with the size range recommended for the species by the test guideline [Commission Regulation (EC) No 440/2008; Annex Part C, C.1]

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were fulfilled; see 'Overall remarks'.

Conclusions:

The 96h-EC50 of the test item in zebrafish was found to be higher tan 100 mg/L and the NOEC was 100 mg/L, both based on mortality.

Executive summary:

An acute toxicity test on zebrafish (Brachydanio rerio) was performed on the test item, in accordance with OECD Guideline 203 / EU method C.1, under GLP conditions. Based on the results obtained in a preliminary range-finding test where no toxic response was observed, a Limit Test was carried out using only one test concentration (100 mg/L) and one control group in a semi-static system. The renewal period of the water was 48h, due to the stability of the test item. Test concentrations were analytically determined (by means of a validated method) at the beginning and at the end of each renewal period and did not deviate more than 20%. Seven fish per group were exposed to the test item or the control for 96 h. All validity criteria were met. The 96h-EC50 of the test item in zebrafish was determined to be higher tan 100 mg/L and the NOEC was 100 mg/L, both based on mortality.

Description of key information

Key study. Study on the analogue substance naringin, according to OECD TG 302 / EU Method C.1 (GLP study). The 96h-EC50 of the test item in zebrafish was determined to be higher than 100 mg/L and the NOEC was 100 mg/L, both based on mortality. Based on the available information for the read-across approach, the target substance has a 96h-EC50 > 105.1 mg/L and a NOEC ≥ 105.1 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

105 mg/L

Additional information

An acute toxicity test on zebrafish (Brachydanio rerio) was performed for the analogue substance naringin in accordance with OECD Guideline 203 / EU method C.1, under GLP conditions. Based on the results obtained in a preliminary range-finding test where no toxic response was observed, a Limit Test was carried out with one dose (100 mg/L) and one control group in a semi-static system. The renewal period of the water was 48h, due to the stability of the test item. Seven fish per group were exposed to the test item or the control for 96 h. All validity criteria were met. Based on the available information for the read-across approach, the 96h-EC50 of the target substance in zebrafish was determined to be higher than 105.1 mg/L and the NOEC was 105.1 mg/L, both based on mortality.