2,4-bis[N'-(4-methylphenyl)ureido]toluene

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 April 2003 - 4 April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,4-bis[N'-(4-methylphenyl)ureido]toluene

EC Number:

411-790-5

EC Name:

2,4-bis[N'-(4-methylphenyl)ureido]toluene

Cas Number:

54390-87-3

Molecular formula:

C23H24N4O2, C24H34N4O2

IUPAC Name:

3-(2-methyl-3-{[(4-methylphenyl)carbamoyl]amino}phenyl)-1-(4-methylphenyl)urea; 3-(2-methyl-5-{[(4-methylphenyl)carbamoyl]amino}phenyl)-1-(4-methylphenyl)urea; 3-{2-methyl-3-[(octylcarbamoyl)amino]phenyl}-1-(4-methylphenyl)urea

Details on test material:

- Name of test material (as cited in study report): KY-MA
- Substance type: white to light pink powder
- Physical state: solid
- Stability in water: at least 96 h
- Storage condition of test material: at RT in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: at t=0 and t=72 duplicate samples from test concentration and control
- Sample storage conditions before analysis: Not applicable (analyzed immediately)

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: supersaturated solution (10 mg/L) achieved via 4 days magnetic stirring and filtration (5 µm)
- Controls: treated in the same manner as test substance solutions
- Evidence of undissolved material (e.g. precipitate, surface film, etc): prior to filtering larger undissolved test substance particles

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: fresh water algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): in-house laboratory culture
- Method of cultivation: growth medium M1 innoculated with algal cells from pure culture on agar; continuous light (6000-10000 lux) and aeration at 23 +/- 2 degrees C

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (Ca+Mg)/ 24 mg CaCO3/L

Test temperature:

22.7-24.2 degrees C

pH:

8.0 - 8.5

Nominal and measured concentrations:

A supersaturated solution of 10mg/L was filtered through paper (5um) and this solution was used for the limit test. Subsequent analysis showed an initial concentration (@ t=0) of 0.015 mg/L and a final concentration (@ t=72h) of 0.004 mg/L, giving an average concentration of 0.008 mg/L. The limit of solubility of KY-MA in water is 0.006 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, glass
- Initial cells density: 10^4 cells/mL
- Control end cells density: 44.8 x 10^4
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO (reverse osmosis purified tap water)
- Culture medium different from test medium: M1 for culture, M2 for test

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24/7
- Light intensity and quality: 6330-8555 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber at start of test, followed by spectrophometric measurement at 720 nm at 24, 48 and 72h

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.008 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.008 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 (for algal test): yes
There were no effects seen in the limit test. A 10 mg/L supersaturated solution was filtered through paper (5um). This solution was then used (6 replicates) in the limit test. Analysis showed that the actual starting concentration of KY-MA in the solutions was 0.015 and the actual final concentration was 0.004, giving an average concentration of ~0.008 mg/L. The limit of solubility for KY-MAin water is 0.006 mg/L, thusit can be concluded that the 72h-EC50 value and the 73h-NOEC value are above the maximum solubility in water of KY-MA.

Results with reference substance (positive control):

Results with reference substance are valid.
Under the conditions of the reference test, potassium dichromate inhibited cell growth at nominal concentrations of 1.0 mg/L and higher and reduced growth rate at 1.8 mg/L and higher. The EC50 for cell growth inhibition was 1.1 mg/L with a 95% confidence interval of 0.73 -1.8 mg/L, which was within the historical ranges for this test facility. The EC50 for growth rate reduction was 1.9 mg/L with a 95% confidence interval of 1.5 -2.4 mg/L, which was within the historical ranges for this facility.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See the attached confidential document "Comments to Draft Decision EC 411-790-5 KY-MA.pdf"

Conclusions:

In this limit test, no effects were observed at the tested concentration. Based on the overall average measured test concentration, the 72h-ErC50 value was determined to be higher than 0.008 mg/L and the 72h-NOEC value was determined to be 0.008 mg/L. Since these values exceeds the maximum solubility of KY-MA in water (0.002 mg/L), it can be concluded that the 72h-EC50 value and the 72h-NOEC value are above the maximum solubility in water of KY-MA.

Executive summary:

A limit test was performed for KY-MA according to OECD 201 "Algae, Growth Inhibition Test". There were no deviations that would affect the results or data interpretation. A 10 mg/L supersaturated solution was filtered through paper (5um). This solution was then used (6 replicates) in the limit test. Analysis showed that the actual starting concentration of KY-MA in the solutions was 0.015 and the actual final concentration was 0.004, giving an average concentration of ~0.008 mg/L. Since no inhibition was observed, the 72h-EC50 value is higher than 0.008 mg/L and the 72h-NOEC is 0.008 mg/L. The limit of solubility for KY-MA in water is 0.006 mg/L, thus it can be concluded that the 72h-EC50 and the 72h-NOEC value are above the maximum solubility in water of KY-MA.