N,N'-(3,3'-dimethylbiphenyl-4,4'-ylene)di(acetoacetamide)

Appearance / physical state / colour

Administrative data

Endpoint:

appearance / physical state / colour

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: observation made during recent algae testing

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-09 to 2017-02-06

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:

2006, corrected in 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Nominal concentrations: 0, 0.625, 1.25, 2.5, 5 and 10 mg/L, sampled at 0h and after 72h
- Sample storage conditions before analysis: stored deep frozen (< -18°C) until transfer to analytical laboratory.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 10 mg/L was prepared, homogenisation by shaking and 60 minutes of ultrasonification. Lower concentrations were prepared by dilution in the AAP medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: SAG 61.81
- Source (laboratory, culture collection): commercial supplier MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3-4 days before test start, the test medium is inoculated and held under test conditions to produce pre-culture in state of exponential growth
- Method of cultivation: semi-continuous in sterile cultures, old medium periodically refreshed
- Culture conditions:
Illumination: continuous 4440-8880 Lux
Temperature: 21-24°C
CO2 supply by shaking

ACCLIMATION
- Acclimation period: 3-4 days under test conditions (not exposed to test material)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.3-22.8C

pH:

7.36 to 7.93

Nominal and measured concentrations:

Nominal concentrations: 0, 0.625, 1.25, 2.5, 5 and 10 mg/L
Measured concentrations: 0, 0.503, 1.01, 2.17, 3.78 and 9.04 mg/L. Measured concentrations varied between 65 and 102 % of the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer, 100 mL
- Type: closed with aluminium caps
- Material, size, headspace, fill volume: 50 mL test solution
- Aeration: continuous agitation, pivoted bogie
- Initial cells density: 0.5 × 10^4 cells per mL
- Control end cells density: 31.46 x 10^ cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP medium as described in OECD guideline


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, pH 7.5 +/- 0.1 - ranged from 7.36 to 7.73
- Temperature: 22.3-22.8°C
- Photoperiod: continuous light
- Light intensity and quality: 79.7 µEm-2s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence detection using calibration curve fluorescence signal compared to counted cell number (Neubauer chamber)
- Fluorescence reader: microplate reader (infinite 200Pro), emission wavelength 670 nm, evaluation with Tecan i-control


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 0, 0.001, 0.01, 0.1, 1 and 10 mg/L
- Results used to determine the conditions for the definitive study: No inhibition up to and including 10 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 9.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 9.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 9.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 9.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50 (growth rate): 1.64 mg/L; EC50 (yield): 1.03 mg/L

Reported statistics and error estimates:

Not required

Determined concentrations:

Nominal concentration (mg/L)	Sampling	Measured concentration (mg/L)	% of nominal	Geometric mean conc (mg/L)
Control	0h fresh	n.d.	-	-
	72h aged	n.d.	-
0.625	0h fresh	0.508	81	0.503
	72h aged	0.501	80
1.25	0h fresh	1.06	85	1.01
	72h aged	0.967	77
2.5	0h fresh	2.54	102	2.17
	72h aged	1.86	74
5	0h fresh	4.41	88	3.78
	72h aged	3.24	65
10	0h fresh	9.5	95	9.04
	72h aged	8.6	86

The average cell numbers for each samping time and concentration:

Nominal concentration (mg/L)	Measured concentration (geom. mean) (mg/L)	average cell numbers (10^4/mL)
		0h	24h	48h	72h
0	0	0.57	1.48	5.89	31.46
0.625	0.503	0.57	1.86	8.05	42.11
1.25	1.01	0.57	2.45	9.69	48.67
2.5	2.17	0.57	1.93	10.54	43.96
5	3.78	0.57	2.13	10.40	43.62
10	9.04	0.57	2.32	10.42	40.76

Average growth rate inhibition at the different concentrations:

Nominal concentration (mg/L)	Measured concentration (geom. mean) (mg/L)	% inhibition of growth rate
		0h-24h	0h-48h	0h-72h
0	0	0.0	0.0	0.0
0.625	0.503	-25.0	-14.3	-7.4
1.25	1.01	-54.2	-22.2	-11.0
2.5	2.17	-28.8	-25.9	-8.4
5	3.78	-39.5	-25.2	-8.2
10	9.04	-48.7	-25.1	-6.3

Average yield inhibition at the different concentrations:

Nominal concentration (mg/L)	Measured concentration (geom. mean) (mg/L)	% inhibition of yield
		0h-24h	0h-48h	0h-72h
0	0	0.0	0.0	0.0
0.625	0.503	-41.8	-40.6	-34.5
1.25	1.01	-106.6	-71.4	-55.7
2.5	2.17	-49.5	-87.4	-40.5
5	3.78	-71.4	-84.8	-39.4
10	9.04	-92.3	-85.2	-30.1

Validity criteria fulfilled:

yes

Conclusions:

The test item was found not to have significant inhibitory effects on growth rate or yield at test concentrations 9.04 mg/L. The EC10, NOEC and EC50 values were found to be > 9.04 mg/L (measured concentration).

Executive summary:

The toxicity of the test item to algae, Pseudokirchneriella subcapitata, was assessed according to OECD 201, in a static test set-up. The test was performed under GLP and there were no deviations to the OECD guideline.

In a dose-range finder study, test item concentrations 0, 0.001, 0.01, 0.1, 1 and 10 mg/L. Higher concentrations were not tested as this would be above the solubility of the test item. No inhibitory effects on growth rate or yield were observed. Based on the outcome of the dose range finder study, following nominal test concentrations were selected: 0, 0.625, 1.25, 2.5, 5 and 10 mg/L with a spacing factor of 2. The test concentrations at 0h and 72h were checked using a HPLC-MS/MS. The measured concentrations ranged between 65 and 102 % of the nominal concentrations. Hence, measured concentrations (geometric mean) were used to express results. The measured concentrations are: 0, 0.503, 1.01, 2.17, 3.78 and 9.04 mg/L

The test item was found not to be inhibitory to growth rate nor yield of the algae up to and including the highest concentration 9.04 mg/L. Therefore, the EC10 and EC50 values are > 9.04 mg/L (measured concentration). The NOEC was found to be 9.04 mg/L.

A reference item, potassium dichromate is also tested at the test facilities twice per year . The EC50 value found for potassium dichromate are in line with the expectations.

The validity criteria were evaluated and were all met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

no guideline followed, the results are obtained from observations made during a recent study on Algae testing of the substance.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-(3,3'-dimethylbiphenyl-4,4'-ylene)di(acetoacetamide)

EC Number:

202-111-1

EC Name:

N,N'-(3,3'-dimethylbiphenyl-4,4'-ylene)di(acetoacetamide)

Cas Number:

91-96-3

Molecular formula:

C22H24N2O4

IUPAC Name:

N-{2-methyl-4-[3-methyl-4-(3-oxobutanamido)phenyl]phenyl}-3-oxobutanamide

Test material form:

solid

Details on test material:

- State of aggregation: solid
- Analytical purity: 97.1%
- Purity test date: 07/12/2017
- Lot/batch No.: 16031556
- Expiration date of the lot/batch: 21-10-2018

Results and discussion

Physical state at 20°C and 1013 hPa:

solid

Form / colour / odour

Key result

Form:

solid

Colour:

grey-beige

Substance type:

organic

Applicant's summary and conclusion

Conclusions:

The test substance is a grey-beige solid product.

Executive summary:

The results are obtained from observations made during a recent study on Algae testing of the substance

The substance is a grey-beige solid product.