Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

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:

25th February to 29 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

EC Number:

269-358-5

EC Name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Cas Number:

68227-46-3

Molecular formula:

C21H35N3O4.C3H6O3

IUPAC Name:

2-hydroxypropanoic acid 2-(dimethylamino)ethyl N-[5-({[(2-ethylhexyl)oxy]carbonyl}amino)-2-methylphenyl]carbamate

Details on test material:

- Name of test material (as cited in study report): WRS-2390TX
- Physical state: Vicous liquid
- Analytical purity: 71.2% in water
- Expiration date of the lot/batch: 15th November 2004
- Storage condition of test material: The test item was stored at ambient laboratory temperature in the dark for the duration of the study.
- Sponsor's information on this batch detailed in Notebook No. 02-181-126.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Based on the results of the range finding test and discussions with the Sponsor, a static exposure definitive test was conducted over 48 h with an untreated control at the following concentrations of WRS-2390TX:
0.64, 1.4, 3.0, 6.4, 14 mg/L
These concentrations were equivalent to the following concentrations when corrected for the purity of the test item:
0.46, 1.0, 2.14, 4.56, 9.97 mg/L
- Sampling method:
Duplicate ca 20 ml samples were taken from freshly prepared test item and control media for chemical analysis at test initiation (0 h) and from pooled samples from all replicates at 48 h in both test phases of the study. In the range finding test, only samples from the highest and lowest concentration plus controls were taken. In the definitive test samples from all test and control solutions were taken.
The test solutions were analysed according to the procedures established and validated under Inveresk Study No. 342753.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Test solutions were prepared by parallel dilution of a 100 mg/L stock solution. The stock solution was prepared by adding a weighed amount of WRS-2390TX (25.1 mg) to Elendt M4 and bringing to volume in a 250 ml volumetric flask. This was then placed in an ultrasonic bath for ca 10 min to aid dissolution.
Four vessels were prepared for each treatment group and control, each containing 100 ml of media.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: In-house laboratory culture
- Age at study initiation (mean and range, SD): <24 hrs old
- Method of breeding: They were bred within the laboratory by acyclical parthenogenesis.
- Feeding during test: No
Daphnia cultures were fed on a diet of Chlorella vulgaris. Daphnia were not fed during the test. Daphnia cultures were maintained in a synthetic medium (Elendt M4) and all testing was conducted using this medium

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Water hardness was determined in a sample of the medium used to prepare the test solutions at initiation of the definitive test as 270 mg CaCO3/L.

Test temperature:

19.6-21.0°C

pH:

7.6-7.7

Dissolved oxygen:

80.7-93.2% ASV (air saturation value)

Salinity:

nda

Nominal and measured concentrations:

Based on the results of the range finding test and discussions with the Sponsor, a static exposure definitive test was conducted over 48 h with an untreated control at the following concentrations of WRS-2390TX:
0.64, 1.4, 3.0, 6.4, 14 mg/L
These concentrations were equivalent to the following concentrations when corrected for the purity of the test item:
0.46, 1.0, 2.14, 4.56, 9.97 mg/L

Samples were taken for chemical analysis at initiation and completion of the exposure phase. The corresponding mean measured concentrations were 0.36, 0.79, 1.72, 3.77, 8.88 mg/L, which was equivalent to 78, 79, 80, 83 and 89% of the nominal values, corrected for purity. Results are based on mean measured concentration but the concentrations at which effects were observed were closest to nominal and within 20% of the expected concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel:
The test was conducted using glass crystallising dishes (ca 200 ml), covered with perspex lids to prevent dust contamination and evaporation loss. Each vessels contained 100 ml of media.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
Water quality parameters (pH, temperature, conductivity and dissolved oxygen concentration) remained within acceptable limits over the definitive test: pH 7.6-7.7, temperature 19.6-21.0C; conductivity 652-687 μS/cm and dissolved oxygen 80.7-93.2% ASV (air saturation value).
Water hardness was determined in a sample of the medium used to prepare the test solutions at initiation of the definitive test as 270 mg CaCO3/L. .
- Intervals of water quality measurement:
Water quality parameters were measured in one vessel for each treatment group at 24 h intervals during the definitive test, using a Sentron Argus X pH meter, a Jenway 470 conductivity meter and a YSI 550A dissolved oxygen meter.


OTHER TEST CONDITIONS
Test vessels were maintained within a temperature-controlled laboratory with the aim of achieving a temperature in the range 18-22 °C. The temperature did not deviate by more than 2 °C throughout the test. A light cycle of 16 h light and 8 h dark was in operation throughout the test and provided by artificial daylight fluorescent tubes. Vessels were not aerated during the test.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Test vessels were observed for any immobile Daphnia at 24 and 48 h after test initiation. Daphnia were recorded as immobile if no movement was observed within 15 s following gentle agitation of the test vessel.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations:
A range finding test was conducted over 48 h, at the following nominal concentrations of WRS-2390TX without adjustment for purity:
0.1, 1.0, 10, and 100 mg/L, with an untreated control.
Test solutions were prepared by the serial dilution of a 100 mg/L WRS-2390TX stock solution with Elendt M4 Daphnia medium. The 100 mg/L solution was prepared by adding a weighed amount of WRS-2390TX (100.7 mg) to Elendt M4 and bringing to volume in a 1000 ml volumetric flask. This was then placed in an ultrasonic bath for ca 10 min to aid dissolution. Duplicate vessels were prepared for each treatment group, each containing 100 ml of test solution. Five Daphnia neonates <24 h old were then added to each vessel.
- Results used to determine the conditions for the definitive study:
Analysis of samples at 0 h in the range finding indicated that samples had been accurately prepared. At 48 h samples showed a loss of measured test item at both the high and low concentrations analysed. It is unclear why concentrations were not maintained over 48 h in the range finding test. It was noted that the 100 mg/L solution was opaque white but all other test solutions were clear and colourless.
One replicate vessel of the control treatment Daphnia neonates were immobilised. The reason for this is not known. However this did not impede the selection of a suitable test range for the definitive test. After 24 h 100% of neonates at 100 mg/L were immobile and after 48 h 100% of neonates at 10 mg/L were immobile. No immobile neonates were observed at 1 mg/L.
In the 0.1 mg/L group, 3/10 neonates were immobile at 48 h. This was not regarded as a test item effect as it did not follow a normal concentration response.
Based on the results of the range finding test and discussions with the Sponsor, a static exposure definitive test was conducted over 48 h with an untreated control at the following concentrations of WRS-2390TX: 0.64, 1.4, 3.0, 6.4, 14 mg/L.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 8.88 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Highest concentration tested.

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits could not be calculated as the probit model did not have a good fit to the observed data.

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.72 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: No mortality
At 48 h 10% immobilisation (2 Daphnia) was observed at the lowest concentration tested, 0.36 mg/L. This is not regarded as an effect of the test item, as this is within the permitted effect levels for controls and does not follow a standard concentration response.

Effects were observed after 24 h at the highest concentration only, 8.88 mg/L measured, where 20% (4 Daphnia) were immobilised. After 48 h, 100% immobilisation (20 Daphnia) was observed at the 8.88 mg/L treatment with 20% immobilisation (4 Daphnia) at the 3.77 mg/L treatment.
At 48 h 10% immobilisation (2 Daphnia) was observed at the lowest concentration tested, 0.36 mg/L. This is not regarded as an effect of the test item, as this is within the permitted effect levels for controls and does not follow a standard concentration response.

From these observations the following results were determined:
- The 24 h EC50 was >8.88 mg/L, the highest concentration tested in the definitive test.
- The 48 h EC50 was 4.2 mg/L. As there were limited intermediate levels of effect, the probit model was not a good fit to the observed data, so the estimated value should be regarded with caution and 95% confidence limits were not calculated.
- The 48 h No Observed Effect Concentration (NOEC) was 1.72 mg/L (measured), under the conditions of the test.

Results with reference substance (positive control):

nda

Reported statistics and error estimates:

Statistical analysis of the observed immobilisation data at each time point compared to the nominal concentration was conducted by probit (Finney 1971, 1978) transformation applied to the mortality data.
A Pearson Chi-square test on the sum of squares for each data point indicated low heterogeneity in the data. The probit transformed data were subjected to a regression procedure against logarithmically transformed concentrations where appropriate. The Davidon-Fletcher-Powell maximum likelihood algorithm was used to obtain parameter estimates but there was poor fit to the model as the number of intermediate values was limited. The EC50 value was estimated from the fitted model but was not reliable and no confidence limits were presented.

Any other information on results incl. tables

 Immobilisation Observed in the Definitive Test

Time (h)	Vessel Replicate	Mean Measured Concentration of WRS-2390TX (mg/L)
		Control	0.36	0.79	1.72	3.77	8.88
24	I	0	0	0	0	0	0
	II	0	0	0	0	0	0
	III	0	0	0	0	0	2
	IV	0	0	0	0	0	2
	% ImmobileDaphnia	0	0	0	0	0	20
48	I	0	0	0	0	1	5
	II	0	1	0	0	0	5
	III	0	1	0	0	2	5
	IV	0	0	0	0	1	5
	% ImmobileDaphnia	0	10^	0	0	20	100

^Not regarded as test item related

The results indicate that test solutions were either equal to or greater than 80% of nominal (corrected for purity) for the highest three concentrations at preparation. The lower concentrations were 78 and 79% of nominal. However results from 48 h show that the concentrations were stable over the test period. Mean measured concentrations for the exposure period were 0.36, 0.79, 1.72, 3.77 and 8.88 mg/L, which were between 78 and 89% of nominal. As the mean measured test item concentrations deviated by >20% from nominal, test results will be based on mean measured concentration.

All prepared test solutions were clear and colourless at test initiation. No change was noted at 48 h.

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

The 24 h EC50 for WRS-2390TX to Daphnia magna was >8.88 mg/L (measured), the highest concentration tested.
The 48 h EC50 for WRS-2390TX to Daphnia magna was estimated as 4.2 mg/L (measured), though 95% confidence limits could not be calculated as the probit model did not have a good fit to the observed data.
The 48 h NOEC for WRS-2390TX to Daphnia magna was 1.72 mg/L.
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability & repeatability).

Executive summary:

In a Determination of Acute Toxicity study to Daphnia (Inveresk report number: 24139) WRS-2390TX was found to have a 48 h NOEC of 1.72 mg/L.

The 24 h EC50 for WRS-2390TX to Daphnia magna was >8.88 mg/L (measured), the highest concentration tested.

The 48 h EC50 for WRS-2390TX to Daphnia magna was estimated as 4.2 mg/L (measured), though 95% confidence limits could not be calculated as the probit model did not have a good fit to the observed data.

As the mean measured test item concentrations deviated by >20% from nominal, test results were based on mean measured concentration.

All prepared test solutions were clear and colourless at test initiation. No change was noted at 48 h.

Effects were observed after 24 h at the highest concentration only, 8.88 mg/L measured, where 20% (4 Daphnia) were immobilised. After 48 h, 100% immobilisation (20 Daphnia) was observed at the 8.88 mg/L treatment with 20% immobilisation (4 Daphnia) at the 3.77 mg/L treatment.

The study was conducted according to OECD (1984) Guideline 202 Part I and EC (2001) Guideline C2 and is in compliance with the OECD principles of Good Laboratory Practice.