Reaction products of amines, polyethylenepoly-, triethylenetetramine fraction and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

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 22 October 2017 and 27 October 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:

2018

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:

EDA-BADGE-EDA

Cas Number:

854009-15-7

Molecular formula:

C25H40N4O4

IUPAC Name:

EDA-BADGE-EDA

Constituent 2

Reference substance name:

EDA-BADGE-EDA-BADGE-EDA

Molecular formula:

C48H72N6O8

IUPAC Name:

EDA-BADGE-EDA-BADGE-EDA

Constituent 3

Reference substance name:

EDA-BADGE α-glycol

Molecular formula:

C23H34N2O5

IUPAC Name:

EDA-BADGE α-glycol

Constituent 4

Reference substance name:

reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

IUPAC Name:

reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

Constituent 5

Reference substance name:

Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

IUPAC Name:

Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

Test material form:

liquid: viscous

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine
Physical state/Appearance: Yellow/brown extremely viscous liquid
Batch: BBF01102V1
Purity: Not provided
Expiry Date: 01 January 2021
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Total Organic Carbon (TOC) analysis was performed on the test solutions at 0, 24, 72 and 96 hours

Test solutions

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

A nominal amount of test item (20 mg) was added to a glass slide and suspended in the water column of 20 liters of test water to give a 1.0 mg/L loading rate. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0 mg/L loading rate WAF from which a dilution was performed in test water to give the test concentration of 0.16 mg/L loading rate WAF.
Total Organic Carbon (TOC) analysis was performed on the test solutions at 0, 24, 72 and 96 hours

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 12 September 2017. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from
16 October 2017 to 23 October 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 14”C to 15”C with a dissolved oxygen content of greater than or equal to 9.4 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 5.0 cm (sd = 0.30) and a mean weight of
1.01 g (sd = 0.18) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.35 g bodyweight/liter.
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 - 16°C

pH:

7.1 - 8.1

Nominal and measured concentrations:

Total Organic Carbon Analysis
Total Organic Carbon (TOC) analysis of the test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) (see Annex 3) showed no significant differences in the amount of carbon present within the 0.16 mg/L loading rate WAF test vessel when compared to the control vessel. Therefore, given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantification of the analytical method.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Details on test conditions:

Experimental Design and Study Conduct
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances, in the WAF.

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 EL50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL50 is greater than the threshold loading rate. The EL50 value obtained from the Algal Growth Inhibition study (Envigo Study Number BW18HH) was the lowest of these two EL50 values and hence the test was conducted at a single loading rate of 0.16 mg/L.

Experimental Preparation
A nominal amount of test item (20 mg) was added to a glass slide and suspended in the water column of 20 liters of test water to give a 1.0 mg/L loading rate. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0 mg/L loading rate WAF from which a dilution was performed in test water to give the test concentration of 0.16 mg/L loading rate WAF.
Total Organic Carbon (TOC) analysis was performed on the test solutions at 0, 24, 72 and 96 hours

Exposure Conditions
In the definitive test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each 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 14”C to 16”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.

Vortex depth measurements
The vortex depth was recorded at the start and end of each mixing period.

Total Organic Carbon analysis
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control and the 0.16 mg/L loading rate WAF test group at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media). Duplicate samples were taken and stored frozen for further analysis if necessary.

Data Evaluation

Statistical Analysis
An estimate of the LL50 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 ≥60% of Air Saturation Values (ASV) in the control and test vessels.

Reference substance (positive control):

no

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:

Validation of Mixing Period
Preliminary investigational work indicated that there was no significant increase in the amount of dissolved total organic carbon when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Definitive Test
Total Organic Carbon Analysis
Total Organic Carbon (TOC) analysis of the test preparations at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) (see Table 1) showed no significant differences in the amount of carbon present within the 0.16 mg/L loading rate WAF test vessel when compared to the control vessel. Therefore, given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantification of the analytical method.
The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates 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 0.16 mg/L loading rate WAF for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h) LL50 (mg/L Loading Rate WAF)
1 >0.16
3 >0.16
6 >0.16
24 >0.16
48 >0.16
72 >0.16
96 >0.16

The No Observed Effect Loading rate (NOEL) was considered to be 0.16 mg/L loading rate WAF.

Sub-Lethal Effects
Sub-lethal effects of exposure were observed in the 0.16 mg/L loading rate WAF after 1-Hour exposure. This response was swimming on the bottom of the tank and was considered to be due to natural causes as the observation did not last past 1-Hour.

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 ≥60% of ASV in the control and test vessels.

Water Quality Criteria
The results of the water quality measurements are given in Table 3. Temperature was maintained at 14”C to 16”C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAF.
At the start of the mixing period the 1.0 mg/L loading rate was observed to be a clear colorless water column with test item adhered to a microscope slide suspended in the water column. After 23 hours stirring and a 1-Hour standing period the 1.0 mg/L loading rate was observed to be a clear colorless water column with a microscope slide suspended in the water column. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
Throughout the duration of the test, the control and 0.16 mg/L loading rate were observed to be clear colorless solutions.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 2       Cumulative Mortality Data in the Definitive Test 

Nominal Loading Rate (mg/L)	Cumulative Mortality (Initial Population = 7)							% Mortality
	1 Hour	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0	0
0.16	0*	0	0	0	0	0	0	0

*5 out of the 7 fish observed to be swimming at the bottom.

 

Table 3            Water Quality Measurements

 

Nominal Loading Rate (mg/L)	Time (Hours)
	24 Hours (Fresh Media)			48 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.3	9.9	16	7.4	9.6	15
0.16	7.4	9.8	15	7.5	9.5	15

 

Nominal Loading Rate (mg/L)	Time (Hours)
	48 Hours (Fresh Media)			72 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.5	9.8	15	7.1	9.4	15
0.16	7.6	9.9	14	7.2	9.2	15

 

Nominal Loading Rate (mg/L)	Time (Hours)
	72 Hours (Fresh Media)			96 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.5	10.2	16	8.1	9.9	15
0.16	7.5	9.7	15	8.0	9.4	15

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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 ≥60% of ASV in the control and test vessels.

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated using the threshold approach and gave a 96-Hour LL50 value of greater than 0.16 mg/L loading rate WAF. The No Observed Effect Loading rate was 0.16 mg/L loading rate WAF.

Executive summary:

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

 Methods…….

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA.  Using this approach the lowest EL₅₀value from either the Algal Growth Inhibition study or Acute Toxicity toDaphnia magnastudy is set as the threshold loading rate and a “Limit test” is conducted at this threshold loading rate.  If no mortalities are observed this indicates that fish are not the most sensitive species and that the LL₅₀is greater than the threshold loading rate. The EL₅₀value obtained from the Algal Growth Inhibition study (Envigo Study Number BW18HH) was the lowest of these two EL₅₀values and hence the test was conducted at a single loading rate of 0.16 mg/L.

Seven fish were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 0.16 mg/L for a period of 96 hours at a temperature of 14°C to 16 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

Samples of the control and the 0.16 mg/L loading rate WAF were taken at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media) for Total Organic Carbon (TOC) analysis. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantification of the analytical method.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of rainbow trout to the test item gave LL₅₀values of greater than 0.16 mg/L loading rate WAF. The No Observed Effect Loading Rate was 0.16 mg/L loading rate WAF.