N,N''-(methyl-m-phenylene)bis[N',N'-dimethylurea]

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:

The target substance consists of the same constituents as the source substance.
These constituents are 3-[3-(dimethylcarbamoylamino)-4-methylphenyl]-1,1-dimethylurea (CAS-No. 17526-94-2, EC-No. 241-523-6) and 3-[3-(dimethylcarbamoylamino)-2-methylphenyl]-1,1-dimethylurea (CAS-No. 17607-23-7, no EC-No. assigned). Due to the different typical concentrations of the main constituent and the 2nd constituent it became necessary to submit two registrations in order to distinguish between the mono-constituent substance (source) and the multi-constituent substance (target).
As the constituents of source and target are the same, the approach “read-across from supporting substance (structural analogue or surrogate)” is applicable because it is obvious that the physical-chemical, ecotoxicological and toxicological profiles are identical.

Reason / purpose for cross-reference:

read-across source

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)

Key result

Duration:

96 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:

A) Range-finding test:
No mortality or clinical effects were observed at any of the test concentrations during the test period. Based on these results samples taken from nominal 100 mg/l were analysed. Analyses showed that the measured concentration was stable and in agreement with nominal during the test period (101-102%,). Therefore, the expected LC50 was above 100 mg/l.
All test conditions were maintained within the limits prescribed by the protocol.

B) Limit test:
1) Measured concentrations
Analysis of the samples taken from 100 mg/l at the start and the end of the test showed that the measured concentration was stable and in agreement with nominal (100%).
2) Mortality and other effects
The mortality data recorded during the limit test confirmed that TDI-Urone was not toxic for carp up to 100 mg/l. No effects were observed.

Results with reference substance (positive control):

96-hour acute toxicity study in the carp with pentachlorophenol (PCP); NOTOX Project 498549 (Batch K11-06):
The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, the procedures were designed to meet the test methods of the ISO International Standard 7346-1: Static method, 1996. (Start: 05 January 2012, End: 09 January 2012).

This reference test was carried out to check the sensitivity of the test system as used by NOTOX. The reference substance was pentachlorophenol (PCP, SIGMA, Art. P2604, Batch 06324ED-405).

Concentrations: 0.10, 0.22 and 0.46 mg/l in ISO-medium.

During the test the pH, oxygen concentration and the temperature of the medium were within the optimal ranges for fish.

Under the conditions of the present test with carp exposed to PCP, the 96h-LC50 was 0.25 mg/l based on nominal concentrations, with a 95% confidence interval between 0.20 and 0.41 mg/l. This effect was already reached within 24 hours of exposure.

The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/l based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.

Sublethal observations / clinical signs:

Acceptability of the test

1. No mortality was observed in the control at the end of the test.

2. Test conditions were maintained constant throughout the test.

3. The dissolved oxygen concentration has been at least 60% of the air saturation value throughout the test (>5 mg/l at 22°C).

4. The concentrations of the substance being tested have been at least 80% of the nominal concentration throughout the test.

Mortality and other effects

Table 1 shows the mortality data recorded during the limit test. The responses confirmed that TDI-Urone was not toxic for carp up to 100 mg/l. No effects were observed.

Table 1: Incidence of mortality and total mortality during the limit test

Concentration TDI-Urone (mg/l)	Initial number of fish	Cumulative mortality					Total mortality (%)
		4h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
100	7	0	0	0	0	0	0

Calibration curves

Calibration curves were constructed using five concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration² weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.

Procedural recovery samples

The mean recoveries of the procedural recovery samples fell within the criterion of 70-110%. It demonstrated that the analytical method was adequate for the determination of the test substance in the test samples.

Test samples

The results for the test samples are given in Table 2 and Table 3.

Table 2 Concentrations of the test substance in test medium - range-finding test

Time of sampling [hours]	Date of sampling [dd-mm-yy]	Date of analysis 1 [dd-mm-yy]	Concentration		Relative to nominal [%]	Relative to initial [%]
			Nominal [mg/l]	Analysed [mg/l]
0	09-01-12	16-01-12	100	101	101
24	10-01-12	16-01-12	100	101	101	99
96	13 -01 -2012	16 -01 -2012	100	102	102	101

1 Samples were stored in the freezer (≤ -15°C) until the day of analysis.

Table 3 Concentrations of the test substance in test medium - final test

Time of sampling [hours]	Date of sampling [dd-mm-yy]	Date of analysis 1 [dd-mm-yy]	Concentration		Relative to nominal [%]	Relative to initial [%]
			Nominal [mg/l]	Analysed [mg/l]
0	06-02-12	10-02-12	0 / 100	n.d. / 100	n.a. / 100
96	10-02-12	10-02-12	0 / 100	n.d. / 100	n.a. / 100	n.a. / 100

1 Samples were stored in the freezer (≤ -15°C) until the day of analysis.

n.d. Not detected.

n.a. Not applicable.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present test TDI-Urone induced no visible or lethal effects in carp at or below 100 mg/l (NOEC).
The 96h-LC 50 exceeded 100 mg/l based on analytically confirmed nominal concentrations.

Executive summary:

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.1, 2008 and the ISO International Standard 7346-1: Static method, 1996.

The batch of TDI-urone tested was a clear colourless powder with a purity of 97.5% active content (being a mixture of isomers) and completely soluble in test medium at the concentrations tested.

A limit test was performed based on the results obtained in a preceding range-finding test. Seven carp per test group were exposed to a control and a TDI-Urone concentration of 100 mg/l. The total test period was 96 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test period.

Analysis of the samples taken from 100 mg/l at the start and the end of the test showed that the measured concentration was stable and in agreement with nominal (100%).

The study met the acceptability criteria prescribed by the protocol and was considered valid.

TDI-Urone induced no visible or lethal effects in carp at or below 100 mg/l (NOEC).

The 96h-LC50 exceeded 100 mg/l based on analytically confirmed nominal concentrations.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 November 2011 - 20 March 2012

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:

yes

Remarks:

The study integrity was not adversely affected by the deviations (see section "Any other information on results incl. tables" for details).

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): TDI-Urone
- Substance type: organic
- Physical state: clear colourless powder
- Analytical purity: 97.5 % active content (being a mixture of the 4-methyl isomer and the 2-methyl isomer)
- Isomers composition: ratio of 4-methyl isomer and the 2-methyl isomer: approximately 80:20
- Lot/batch No.: 1268
- Expiration date of the lot/batch: 27 October 2012 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Sampling for analysis of test concentrations:
During the limit test singular samples for possible analysis were taken from both test groups according to the schedule below:
- Frequency at t=0 h and t=96 h
- Volume: 1.8 ml
- Storage: Samples were stored in a freezer until analysis.
Additionally, singular reserve samples of 1.8 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

Preparation of test solutions
The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface).

The batch of TDI-Urone tested was a clear colourless powder with a purity of 97.5% active content (being a mixture of isomers) and completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with the highest test concentration of 100 mg/l applying a short period of magnetic stirring (~10 minutes) to ensure complete dissolution in test medium. The lower test concentrations for the range-finding test were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
- Age at study initiation (mean and range, SD):
- Length at study initiation (mean and SD of 10 fish): Range-finding test: 2.9 ± 0.2 cm
Final test: 2.5 ± 0.2 cm
- Weight at study initiation (mean and SD of 10 fish): Range-finding test: 0.66 ± 0.14 g
Final test: 0.37 ± 0.07 g
- Characteristics: F1 from a single parent-pair bred in UV-treated water.
- Reason for selection: This system has been selected as an internationally accepted species.
- Total fish used: 26
- Feeding during test: No feeding from 24 hours prior to the test and during the total test period.
- Introduction of fish Within 18 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH an

ACCLIMATION
- Quarantine/Acclimation period: At least 12 days after delivery
- Medium: water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition: CaCl2 .2H2O (211.5 mg/l), MgSO4 .7H2O (88.8 mg/l), NaHCO3 (46.7 mg/l), KCl (4.2 mg/l).
- Acclimation conditions (same as test or not):
- Type and amount of food: pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands). Amount not reported.
- Feeding frequency: daily
- Health during acclimation (any mortality observed): In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

EUTHANASIA:
- At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable.

Hardness:

80 mg/L CaCO3 and a pH of 7.7 ± 0.3

Test temperature:

20.9-22.1

pH:

7.5-7.7

Dissolved oxygen:

6.4-10.0 mg/L

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentration:
- Limit test: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 litres, all-glass, containing 9 litres of test solution.
- Aeration: The control group was aerated continuously, while the TDI-Urone test group was not aerated during the test.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.29 g fish/litre, i.e. 7 fish per 9 litres of test medium

TEST MEDIUM / WATER PARAMETERS
- Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality after 4h, 24h, 48h, 72h, and 96h. (No other effects were observed).

TEST CONCENTRATIONS
- Range finding study: yes (see section "Any other information on materials and methods incl. tables" for details)

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP)

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)

Key result

Duration:

96 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:

A) Range-finding test:
No mortality or clinical effects were observed at any of the test concentrations during the test period. Based on these results samples taken from nominal 100 mg/l were analysed. Analyses showed that the measured concentration was stable and in agreement with nominal during the test period (101-102%,). Therefore, the expected LC50 was above 100 mg/l.
All test conditions were maintained within the limits prescribed by the protocol.

B) Limit test:
1) Measured concentrations
Analysis of the samples taken from 100 mg/l at the start and the end of the test showed that the measured concentration was stable and in agreement with nominal (100%).
2) Mortality and other effects
The mortality data recorded during the limit test confirmed that TDI-Urone was not toxic for carp up to 100 mg/l. No effects were observed.

Results with reference substance (positive control):

96-hour acute toxicity study in the carp with pentachlorophenol (PCP); NOTOX Project 498549 (Batch K11-06):
The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, the procedures were designed to meet the test methods of the ISO International Standard 7346-1: Static method, 1996. (Start: 05 January 2012, End: 09 January 2012).

This reference test was carried out to check the sensitivity of the test system as used by NOTOX. The reference substance was pentachlorophenol (PCP, SIGMA, Art. P2604, Batch 06324ED-405).

Concentrations: 0.10, 0.22 and 0.46 mg/l in ISO-medium.

During the test the pH, oxygen concentration and the temperature of the medium were within the optimal ranges for fish.

Under the conditions of the present test with carp exposed to PCP, the 96h-LC50 was 0.25 mg/l based on nominal concentrations, with a 95% confidence interval between 0.20 and 0.41 mg/l. This effect was already reached within 24 hours of exposure.

The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/l based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.

Sublethal observations / clinical signs:

Acceptability of the test

1. No mortality was observed in the control at the end of the test.

2. Test conditions were maintained constant throughout the test.

3. The dissolved oxygen concentration has been at least 60% of the air saturation value throughout the test (>5 mg/l at 22°C).

4. The concentrations of the substance being tested have been at least 80% of the nominal concentration throughout the test.

Mortality and other effects

Table 1 shows the mortality data recorded during the limit test. The responses confirmed that TDI-Urone was not toxic for carp up to 100 mg/l. No effects were observed.

Table 1: Incidence of mortality and total mortality during the limit test

Concentration TDI-Urone (mg/l)	Initial number of fish	Cumulative mortality					Total mortality (%)
		4h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
100	7	0	0	0	0	0	0

Calibration curves

Calibration curves were constructed using five concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration² weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.

Procedural recovery samples

The mean recoveries of the procedural recovery samples fell within the criterion of 70-110%. It demonstrated that the analytical method was adequate for the determination of the test substance in the test samples.

Test samples

The results for the test samples are given in Table 2 and Table 3.

Table 2 Concentrations of the test substance in test medium - range-finding test

Time of sampling [hours]	Date of sampling [dd-mm-yy]	Date of analysis 1 [dd-mm-yy]	Concentration		Relative to nominal [%]	Relative to initial [%]
			Nominal [mg/l]	Analysed [mg/l]
0	09-01-12	16-01-12	100	101	101
24	10-01-12	16-01-12	100	101	101	99
96	13 -01 -2012	16 -01 -2012	100	102	102	101

1 Samples were stored in the freezer (≤ -15°C) until the day of analysis.

Table 3 Concentrations of the test substance in test medium - final test

Time of sampling [hours]	Date of sampling [dd-mm-yy]	Date of analysis 1 [dd-mm-yy]	Concentration		Relative to nominal [%]	Relative to initial [%]
			Nominal [mg/l]	Analysed [mg/l]
0	06-02-12	10-02-12	0 / 100	n.d. / 100	n.a. / 100
96	10-02-12	10-02-12	0 / 100	n.d. / 100	n.a. / 100	n.a. / 100

1 Samples were stored in the freezer (≤ -15°C) until the day of analysis.

n.d. Not detected.

n.a. Not applicable.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present test TDI-Urone induced no visible or lethal effects in carp at or below 100 mg/l (NOEC).
The 96h-LC 50 exceeded 100 mg/l based on analytically confirmed nominal concentrations.

Executive summary:

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.1, 2008 and the ISO International Standard 7346-1: Static method, 1996.

The batch of TDI-urone tested was a clear colourless powder with a purity of 97.5% active content (being a mixture of isomers) and completely soluble in test medium at the concentrations tested.

A limit test was performed based on the results obtained in a preceding range-finding test. Seven carp per test group were exposed to a control and a TDI-Urone concentration of 100 mg/l. The total test period was 96 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test period.

Analysis of the samples taken from 100 mg/l at the start and the end of the test showed that the measured concentration was stable and in agreement with nominal (100%).

The study met the acceptability criteria prescribed by the protocol and was considered valid.

TDI-Urone induced no visible or lethal effects in carp at or below 100 mg/l (NOEC).

The 96h-LC50 exceeded 100 mg/l based on analytically confirmed nominal concentrations.

Description of key information

TDI-Urone induced no visible or lethal effects in carp at or below 100 mg/l (NOEC).

The 96h-LC50 exceeded 100 mg/l based on analytically confirmed nominal concentrations.

Additional information

96-Hour acute toxicity study in carp with TDI-urone.

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.1, 2008 and the ISO International Standard 7346-1: Static method, 1996.

The batch of TDI-Urone tested was a clear colourless powder with a purity of 97.5% active content (being a mixture of isomers) and completely soluble in test medium at the concentrations tested.

A limit test was performed based on the results obtained in a preceding range-finding test. Seven carp per test group were exposed to a control and a TDI-Urone concentration of 100 mg/l. The total test period was 96 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test period.

Analysis of the samples taken from 100 mg/l at the start and the end of the test showed that the measured concentration was stable and in agreement with nominal (100%).

The study met the acceptability criteria prescribed by the protocol and was considered valid.

TDI-Urone induced no visible or lethal effects in carp at or below 100 mg/l (NOEC).

The 96h-LC50 exceeded 100 mg/l based on analytically confirmed nominal concentrations.