2(1H)-Pyridinone, 4-amino-5-methyl-

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-25 to 2018-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline; EU Method; GLP

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:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-5-methyl-1,2-dihydropyridin-2-one

EC Number:

682-818-3

Cas Number:

95306-64-2

Molecular formula:

C6 H8 N2 O

IUPAC Name:

4-amino-5-methyl-1,2-dihydropyridin-2-one

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test medium of the only test concentration and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 48 hours).
All samples were diluted by a factor of 2 with acetonitrile.
Additional samples of the control and the dilution solvent were taken at test start and test end without any sample treatment.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentration: 100 mg test item/L and a control
Control: In the control, test water was used without addition of the test item.
Dosage of Test Item: The test medium of the only test item concentration of nominal 100 mg/L was prepared by dissolving 51.3 mg test item into 513 mL test water by intense stirring for 10 minutes.
The test media was prepared just before introduction of the daphnids (= start of the test).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 0.25 to 19.25 hours old Sex: Female
Origin: The daphnids introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate

Test temperature:

20 °C at test start;
21 °C at test end

pH:

7.9 at test start;
7.8 at test end; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.9 mg/L at test start;
8.8 mg/L at test end

Nominal and measured concentrations:

100 mg test item/L and a control

Details on test conditions:

Test Environment: Controlled environment room
Measurement of pH, Dissolved Oxygen and Water Temperature:
The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
Water Temperature: 20.3 C at test start;
20.9 C at test end
pH-Values: 7.9 at test start;
7.8 at test end; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration: 8.9 mg/L at test start;
8.8 mg/L at test end
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 720 to 1000 lux (measured once during the test).

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours of exposure no immobilisations of the test animals was observed in the control and in the only test concentration of nominal 100 mg test item/L.

Results with reference substance (positive control):

In the most recent test with the reference item potassium dichromate performed in January 2018 , the EC50 after 24 hours was determined to be 1.06 mg test item/L, indicating that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).
In the reference test performed in June 2017 with the reference item potassium dichromate, the EC50 after 24 hours was determined to be 1.58 mg test item/L and the EC50 after 48 hours was determined to be 1.07 mg test item/L.

Reported statistics and error estimates:

No statistical analysis was performed.
The EC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Control Immobilisation Rate was 0 % and furthermore no daphnid showed signs of disease or stress. Dissolved Oxygen Concentration was above or equal 8.8 mg O2/L in the control and test vessels at the end of the test.

Conclusions:

The toxic effect of the test item 4-Amino-5-methylpyridinon to Daphnia magna was assessed in a static limit test. The 48-hour NOEC was determined to be ≥100 mg test item/L. The 48-hour LOEC was determined to be > 100 mg test item/L and the 48-hour EC50 value was determined to be > 100 mg test item/L. All reported endpoints were determined directly from the raw data.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test.

Executive summary:

Title: 4-Amino-5-methylpyridinon: Acute Toxicity to Daphnia magna in a Static 48-hour Immobilisation Limit Test

Purpose: The purpose of this study was to determine the influence of the test item 4-Amino-5-methylpyridinon on the mobility of Daphnia magna.

For this purpose, young daphnids (< 24 hours old) were exposed in a static test for 48 hours to the only concentration of nominal 100 mg/L under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours.

The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species.

The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium.

Guidelines:

-Commission Regulation (EC) No 440/2008, Annex, Part C, C.2.: "Daphnia sp. Acute Immobilisation Test", Official Journal of the European Union (EN), dated May 30, 2008

-OECD Guideline for Testing of Chemicals No. 202: "Daphnia sp., Acute Immobilisation Test" adopted April 13, 2004

-SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

 

Materials and Methods

Test Item: 4-Amino-5-methylpyridinon; batch no.: BXR7SWA; purity: 99.4 % (analytical) according to certificate of analysis

Test Species: Female Daphnia magna, clone 5; 0.25 to 19.25 hours old

Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.

Test Design: This study encompassed two treatment groups (one test item concentration at nominal 100 mg/L, and one control) each containing 20 individuals. The mobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours.

Endpoints: Number of immobile organisms after 24 and 48 hours

Test Concentrations: 100 mg test item/L and a control

Test Conditions: Water temperature: 20.3 to 20.9 °C; pH value: 7.8 to 7.9; dissolved oxygen concentration: 8.8 to 8.9 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 720 to 1000 lux; and thus were within the ranges requested by guideline OECD 202

 

Results

Biological test results: After 48 hours of exposure no immobilisation of the test animals was observed in the control and at the concentration of 100 mg test item/L.

Analytical test results: The quantification of the test item 4-Amino-5-methylpyridinon in the test samples was performed using liquid chromatography with UV detection.

At the start of the test 104 % of the nominal test concentration of 100 mg/L was found. After 48 hours test duration, 103 % of the nominal value was determined. During the test the daphnids were exposed to a mean of 104 % of nominal.

 

Conclusion: The toxic effect of the test item 4-Amino-5-methylpyridinon to Daphnia magna was assessed in a static limit test. The 48-hour NOEC was determined to be ≥100 mg test item/L. The 48-hour LOEC was determined to be > 100 mg test item/L and the 48-hour EC50 value was determined to be > 100 mg test item/L. All reported endpoints were determined directly from the raw data.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.