[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 30 January 2017 and 06 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study was conducted according to OECD TG 203, without deviations that influence the quality of the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

EC Number:

289-348-4

EC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Cas Number:

87788-32-7

Molecular formula:

C10H22B2F6N2

IUPAC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

impurity 1

Reference substance name:

{3-[(amino-N)methyl]-3,5,5-trimethylcyclohexanamine}(trifluoro)boron

Molecular formula:

C10H22BF3N2

IUPAC Name:

{3-[(amino-N)methyl]-3,5,5-trimethylcyclohexanamine}(trifluoro)boron

additive 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

Test material form:

liquid

Specific details on test material used for the study:

Identification: (μ(5-amino-1,3,3-trimethylcycloihexylamine-N,N')hexafluorodiboron
Physical state/Appearance: Clear colorless liquid
Batch: AEF0009100
Purity: Not provided
Expiry Date: 29 August 2017
Storage Conditions: Room temperature in darkness

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0 and 72 hours, and of the old media at 24 and 96 hours

Test solutions

Vehicle:

no

Details on test solutions:

Test Water
The test water used for the definitive test was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

At 0, 24, 48 and 72 hours, a nominal amount of test item (1000 mg) was dissolved in 1000 mL of test water to give a 1000 mg/L stock solution. An aliquot (940 mL) of this stock solution was diluted in 20 liters of test water to give the 47 mg/L test concentration.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 16 January 2017. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 23 January 2017 to 30 January 2017. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at approximately 14”C with a dissolved oxygen content of greater than or equal to 9.2 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.3 cm (sd = 0.2) and a mean weight of 0.65 g (sd = 0.08) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 1.5 g bodyweight/liter (static volume).
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Test temperature:

14 °C

pH:

7.4 - 8.0

Nominal and measured concentrations:

Chemical analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations to be near nominal, and there was not a decline in the measured concentrations of the aged test preparations at 24 and 96 hours. Therefore, it was considered justifiable to estimate the LC50 values in terms of the nominal test concentration only.

Details on test conditions:

Experimental Design and Study Conduct
Definitive Test
In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. The EC50 value obtained from the Algal Growth Inhibition study (Envigo Study Number YM13LN) was the lower of these two EC50 values and hence the test was conducted at a single concentration of 47 mg/L.

Experimental Preparation
At 0, 24, 48 and 72 hours, a nominal amount of test item (1000 mg) was dissolved in 1000 mL of test water to give a 1000 mg/L stock solution. An aliquot (940 mL) of this stock solution was diluted in 20 liters of test water to give the 47 mg/L test concentration.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0 and 72 hours, and of the old media at 24 and 96 hours (see Table 1).

Exposure Conditions
In the definitive test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for the control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14”C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions, but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.

Assessments
Test Organism Observations
Any mortalities and sub-lethal effects of exposure were recorded at 1, 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.
Water Quality Criteria
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
Verification of Test Concentrations
Water samples were taken from the control and 47 mg/L test vessel at 0, 24, 48 and 72 hours from fresh media and at 24, 48, 72 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.
Duplicate samples at 0 (fresh media), 24 (old media), 72 (fresh media) and 96 hours (old media) were taken and stored frozen for further analysis if necessary.

Data Evaluation
Statistical Analysis
An estimate of the LC50 values was given by inspection of the mortality data.

Data Validation
The results of the test are considered valid if the following criteria are met:
• In the control, not more than one of the fish should die or show signs of stress during the 96 hours.
• The dissolved oxygen concentration at the end of the test should be greater than or equal to 60% of Air Saturation Values (ASV) in the control and test vessels.

Reference substance (positive control):

no

Remarks:

CuSO4 · 5H2O

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Definitive Test
Verification of Test Concentrations
Chemical analysis of the fresh test preparations at 0 and 72 hours (see Table 1) showed measured test concentrations to be near nominal, and there was not a decline in the measured concentrations of the aged test preparations at 24 and 96 hours. Therefore, it was considered justifiable to estimate the LC50 values in terms of the nominal test concentration only.

Mortality Data
Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 2.
There were no mortalities in 7 fish exposed to a test concentration of 47 mg/L for a period of 96 hours.

Sub-Lethal Effects
There were no sub-lethal effects of exposure observed in 7 fish exposed to a test concentration of 47 mg/L for a period of 96 hours.

Validation Criteria
The test was considered to be valid given that none of the control fish died or showed signs of stress during the test, and that the oxygen concentration at the end of the test was greater than 60% of ASV (approximately 6.1 mg O2/L under test conditions) in the control and test vessels.

Water Quality Criteria
Temperature was maintained at approximately 14”C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Observations on Test Item Solubility
At the start and throughout the test, the control and 47 mg/L test concentration were observed to be clear colorless solutions.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 1       Cumulative Mortality Data in the Definitive Test

 

Nominal conc (mg/mL)	Cumulative Mortality (Initial Population = 7)							% Mortality
	1 h	3 h	6 h	24 h	48 h	72 h	96 h	96 hours
Control	0	0	0	0	0	0	0	0
17	0	0	0	0	0	0	0	0

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

None of the control fish died or showed signs of stress during the test, and that the oxygen concentration at the end of the test was greater than 60% of ASV (approximately 6.1 mg O2/L under test conditions) in the control and test vessels

Conclusions:

The 96-h LC50 value is <47 mg/L in rainbow trout (Oncorhynchus mykiss). The No Observed Effect Concentration (NOEC) was 47 mg.mL

Executive summary:

In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA.  Using this approach the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration.  If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. The EC50 value obtained from the Algal Growth Inhibition study (Envigo Study Number YM13LN) was the lower of these two EC50 values, and hence the test was conducted at a single concentration of 47 mg/L.

Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 47 mg/L for a period of 96 hours at a temperature of 14 ºC under semi-static test conditions.  The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure, and then daily throughout the test until termination after 96 hours.

Results

Chemical analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations to range from 87% to 90% of the nominal concentration, and did not decline in the aged test preparations at 24 and 96 hours. Therefore, the results are based on the nominal test concentration only.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentration of greater than 47 mg/L. The No Observed Effect Concentration was 47 mg/L.