Phosphoric acid, hexadecyl esters, potassium salts with cetyl alcohol and isostearyl isostearate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish embryo acute toxicity (FET)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From January 14, 2018 to January 19, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 236 (Fish embryo acute toxicity (FET) test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

To demonstrate that nominal exposure concentrations were being achieved the concentrations of test substance in the test vessels were measured using the high-performance liquid chromatography method.

Details on sampling:

At study start, samples were taken from excess test solutions and at study end from pooled test vessel replicates of the dilution water control and each test concentration.

Vehicle:

yes

Remarks:

Tetrahydrofuran

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The test organisms were newly fertilised embryos of unexposed wild-type Tübingen zebrafish, Danio rerio, obtained from continuous laboratory cultures held at Scymaris. The broodstock of zebrafish were free from infection and disease and had not undergone any pharmaceutical (acute or prophylactic) treatment for >2 months before spawning. The zebrafish brood stock was maintained in dechlorinated water, the same as the test dilution water, at a temperature of 26 ± 1°C. A photoperiod of 16 h light:8 h dark, with 20 minute transition periods was provided. Pairs were set up the night before the study in spawning chambers with dividers. Dividers were removed in the morning and eggs collected. To avoid genetic bias, eggs were collected from a minimum of three breeding pairs, mixed and impartially selected. The eggs were washed with dilution water after collection from the spawning chambers. The fertilisation rate of the eggs collected from 5 pairs was approximately 90 to 95%. The embryos were immersed in the test solutions within 90 minutes after the dividers were removed to ensure the embryos were exposed, at latest, by the 16 cell-stage.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

26 ± 1ºC

pH:

7.49 to 7.80

Dissolved oxygen:

8.46 to 9.25 mg/L

Nominal and measured concentrations:

Control and 0.3125, 0.625, 1.25 2.5 and 5.0 mg/L (nominal)
Control and 0, 0, 0.23, 0.40 and 0.71 mg/L (mean measured)
A 5.0 mg/L test concentration was selected as the highest concentration based on the dispersibility observed in a non-GLP solubility trial and range finding test

Details on test conditions:

The test vessels were 24-well plates, containing a nominal 2 mL of solution per well. Twenty replicates per test concentration were employed with four internal plate controls, the control consisted of twenty-four replicates. The well plates were covered with loose fitting lids. The positions of the treatments were randomly allocated within the test area.

The study was run with a dilution water control, solvent control and positive control together with nominal exposure concentrations of 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/L. Levels were based on the level of achievable solubility.

Reference substance (positive control):

yes

Remarks:

3,4-dichloroaniline (Supplier: Acros organics, Lot/batch number: A0336829, Purity: 99.6%, Certificate of Analysis re-test date: January 2021, Sample storage: Room temperature

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.71 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Calculation method: Direct observation from data

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.71 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Calculation method: Direct observation from data

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 0.71 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Calculation method: Direct observation from data

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 0.71 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Calculation method: Direct observation from data

Results with reference substance (positive control):

The positive control had 100% mortality at 96 h.

Sublethal observations / clinical signs:

Results

Analytical data

The limit of quantification (LOQ) of test substance in thisstudy was 0.4 mg/L for all concentrations. The instrument LOQ was 0.2 mg/L but during analysis, samples from the control and each test concentration were diluted ×2, doubling the LOQ. All analytical values are quoted to two significant figures and percentages to the nearest integer. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R² value of 0.995. The maximum percentage difference from nominal concentration for standards at the LOQ is less than 20% and less than 15% at levels greater than the LOQ. All analytical values are quoted to two significant figures and percentages to the nearest integer. The measured concentration at the start of the study ranged from <LOQ - 36% of nominal. The measured concentration at the end of the study were all <LOQ. On the basis of theanalytical data the mean measured concentrations were used for the calculation and reporting of results.

The following are the results of the analysis in Table 1:

Nominal concentration of test substance (mg/L)	Measured concentration of test substance (mg/L)				Mean measured concentration (mg/L)	Mean measured concentration (%)
	0 h		96 h
	(mg/L)	% of nominal	(mg/L)	% of nominal
Control	<LOQ	-	<LOQ	-	0	-
0.3125	<LOQ	-	<LOQ	0	0	0
0.625	<LOQ	-	<LOQ	0	0	0
1.25	0.46^a	36	<LOQ^b	0	0.23	18
2.5	0.79	32	<LOQ	0	0.40	16
5.0	1.4	28	<LOQ	0	0.71	14

a = Triplicate analyses: 0.46, 0.45, 0.46 mg/L

b = Triplicate analyses: <LOQ, <LOQ, <LOQ mg/L

Biological data

The numbers of zebrafish mortalities after 24, 48 72 and 96 h are given in Table 2:

Table 2: Embryo mortality and hatching observations

Time (h)	Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Number of mortalities per treatment	Total number tested	Percentage mortality	Percentage hatching
24	Control	0	1	24	4	0
	0.3125	0	1	20	5	0
	0.625	0	1	20	5	0
	1.25	0.23	1	20	5	0
	2.5	0.40	1	20	5	0
	5.0	0.71	0	20	0	0
48	Control	0	1	24	4	17
	0.3125	0	1	20	5	0
	0.625	0	2	20	10	0
	1.25	0.23	2	20	10	6
	2.5	0.40	1	20	5	5
	5.0	0.71	1	20	5	5
72	Control	0	1	24	4	96
	0.3125	0	1	20	5	95
	0.625	0	2	20	10	94
	1.25	0.23	2	20	10	72
	2.5	0.40	1	20	5	100
	5.0	0.71	2	20	10	94
96	Control	0	2	24	8	100
	0.3125	0	1	20	5	100
	0.625	0	2	20	10	100
	1.25	0.23	2	20	10	100
	2.5	0.40	1	20	5	100
	5.0	0.71	2	20	10	100

The results obtained (based on mean measured concentrations of test substance) were:

Time	LC50 (mg/L)
48 h	>0.71
96 h	>0.71

 

Based on mortality compared to the control (p <0.05) the 96 h No Observed Effect Concentration (NOEC) was determined to be 0.71 mg/L and the Lowest Observed Effect Concentration (LOEC) was >0.71 mg/L. Comparisons were made using CETIS. There was no mortality observed in the internal plate controls. The control had 8% mortality which is acceptable within the test Guideline. The positive control had 100% mortality at 96 h.

 

Validity criteria

The OECD 236 Guideline details the following performance criteria for the test validity:

- The overall fertilisation rate of all eggs collected should be ≥70% in the batch tested.

- The water temperature should be maintained at 26 ± 1 deg C in the test chambers at any time during the test.

- Overall survival of embryos in the dilution water control and where relevant, in the solvent control should be ≥90% until the end of the 96 h exposure.

- Exposure to the positive control (4 mg/L 3,4-dichloroaniline) should result in a minimum mortality of 30% at the end of the 96 h exposure.

- Hatching rate in the dilution water control and solvent control if appropriate, should be ≥80% at the end of the 96 h exposure.

- At the end of the 96 h, the dissolved oxygen concentration in the dilution water control and the highest test concentration should be ≥80% of saturation.

All validity criteria were met during the study.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, similar LC50 and NOEC values of >0.71 mg/L and 0.71 mg/L respectively can be expected for the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate'.

Executive summary:

A study was conducted to determine the acute toxicity potential of the read across substance, 'mono- and di- C16 PSE, K+ and H3PO4' (100%), to Zebrafish (Danio rerio), according to OECD Guideline 236 (Fish Embryo Toxicity (FET)), in compliance with GLP. The test was initiated by the addition of 1 impartially selected Zebrafish embryo, to each 24 well. The embryos were pre-exposed in petri dishes and sorted prior to addition to the well plates within 90 minutes of fertilisation. Fertilised eggs, undergoing cleavage and showing no obvious irregularities during cleavage or injuries of the chorion were selected. Loading of embryos to the well plates was completed less than 3 h post fertilisation. The test organism were exposed to dilution water control and positive control (3,4-dichloroaniline) together with nominal test substance exposure concentrations of 0.3125, 0.625, 1.25, 2.5 and 5.0 mg/L. Levels were based on the dispersibility observed in a non-GLP solubility trial and range finding test. To demonstrate that nominal exposure concentrations were being achieved the concentrations of test substance in the test vessels were measured using the high-performance liquid chromatography method. The mean measured concentrations of the test substance were determined to be 0, 0, 0.23, 0.40 and 0.71 mg/L. An assessment of the response of the Zebrafish embryos was made at 24, 48, 72 and 96 h after the commencement of the test using a low power binocular microscope, with bright field illumination. Any positive outcome of the four observations (coagulation of the embryo, lack of somite formation, non-detachment of the tail, lack of heartbeat) meant that the zebrafish embryo was dead. The 96 h and 48 h LC50 (median lethal concentration) were determined to be >0.71 mg/L (measured). Based on mortality compared to the solvent control (p <0.05) the 48 h NOEC was determined to be 0.71 mg/L (measured) and the LOEC was >0.71 mg/L (measured). All validity criteria were met during this study (Scymaris, 2018). Based on the results of the read across study, similar LC50 and NOEC values can be expected for the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate'.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From Janaury 14, 2013 to February 27, 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Institut für Biologische Analytik und Consulting, IBACON GmbH, Arheilger Weg 17, 64380 Rossdorf, Germany

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test medium of the only test concentration and the control were taken at the start of the test. For the determination of the stability of the test substance under the test conditions and of the maintenance of the test substance concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 96 h) from the approximate centre of the aquaria.

Vehicle:

no

Details on test solutions:

Preparation and application of test solution
- Method:
The test substance was not well soluble in test water. Therefore, a stock suspension of 100 mg test substance/L was prepared by suspending 1.0002 g of test substance in 10.0002 L test water for preparing the test concentration. The stock suspension was stirred for 24 h to dissolve as much test substance as possible. Then, the undissolved test substance was separated by filtration (0.45 µm cellulose acetate nitrate filter). The test media were prepared just before introduction of the test fish (= start of the test).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test organism
- Common name: Rainbow trout
- Source: The test fish were obtained from Forellenzuchtbetrieb Störk, 88348 Bad Saulgau, Germany
- Age at study initiation: Juveniles
- Length at study initiation: 4.83 cm ± 0.30 cm
- Weight at study initiation: 1.12 ± 0.23 g
- Feeding during test: no

Acclimation
- Acclimation period: All fish were obtained and held in the laboratory for at least 12 d before the start of the test.
- Acclimation conditions: same as test conditions
- Feeding frequency: three times per week or daily until 24 h before the test was started
- Health during acclimation: During the last 7 d prior to the start of the test no fish died in the test fish batch. Therefore the mortalities in the fish batch were below 5 % and the fish batch was accepted.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

2.5 mmol/L (= 250.0 mg/L) as CaCO3

Test temperature:

15°C

pH:

7.8 to 8.2

Dissolved oxygen:

93 to 98 % of the air saturation value

Nominal and measured concentrations:

A filtrate of a supersaturated stock suspension of nominal 100 mg/L. No measured concentrations are available (below LOD).

Details on test conditions:

Test system
- Test vessel: 12 L glass aquaria with 10 L test medium
- Aeration: The test media were slightly aerated during the test.
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1

Test medium / water parameters
- Chlorine: 2.0 mmol/L (= 294.0 mg/L)
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4: 1 (based on molarity)
- Conductivity: < 10 µScm-1
- Culture medium different from test medium: no

Other test conditions
- Adjustment of pH:
- Photoperiod: 16 h light: 8 h dark; 30 min dawn/dusk period was provided
- Light intensity: 780 to 810 lux

Test concentrations
- Test concentrations:
The test substance was not well soluble in test water. Therefore, a stock suspension of 100 mg test substance/L was prepared by suspending 1.0002 g of test substance in 10.0002 L test water for preparing the test concentration. The stock suspension was stirred for 24 h to dissolve as much test substance as possible. Then, the undissolved test substance was separated by filtration (0.45 µm cellulose acetate nitrate filter). The test media were prepared just before introduction of the test fish (= start of the test).

Reference substance (positive control):

no

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)

Remarks on result:

other: up to the water solubility of the test substance no mortality of the test animals occurred.

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: up to the water solubility of the test substance no mortality of the test animals occurred.

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

Results and discussion

Validity criteria of the study

Control: In the control no fish died until the end of the test.

Dissolved Oxygen Concentration: The dissolved oxygen concentration in the test media did not fall below 93 % of air saturation value during the test.

 

Biological results

Sublethal Effects: In the control and the only test concentration of nominal 100 mg test substance/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time.

96 h LC50: > 100 mg test substance/L (nominal)

95 % Confidence Interval: Not determinable

96 h LC10: > 100 mg test substance/L (nominal)

95 % Confidence Interval: Not determinable

96 h LC0: 100 mg test substance/L (nominal)

96 h LC100: > 100 mg test substance/L (nominal)

96 h NOEC: 100 mg test substance/L (nominal)

96 h LOEC: > 100 mg test substance/L (nominal)

Table: Mortality and sublethal effects

Exposure time (h)	Effects	Nominal concentration (mg/L)
		Control	100
0 h	#Mortality	0	0
	#Sublethal effects	0	0
2 h	#Mortality	0	0
	#Sublethal effects	0	0
24 h	#Mortality	0	0
	#Sublethal effects	0	0
48 h	#Mortality	0	0
	#Sublethal effects	0	0
72 h	#Mortality	0	0
	#Sublethal effects	0	0
96 h	#Mortality	0	0
	#Sublethal effects	0	0

Analytical Results

Determination of the test substance: Based on the results of LC-MS/MS measurements the concentration of the test substance was determined using a calibration curve.

Calibration range: 0.005 to 0.15 mg test substance/L

Linearity of response: Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression

Regression coefficient (r2): 0.9994

Calibration curve: y = 12112 * x + 10276 (see also Figure 1)

Limit of detection: 1.6 μg test substance/L

Limit of quantification: 18.83 μg test substance/L

The Limit of quantification was calculated according to DIN 32645 from the calibration curve. A preparation of fortified samples was not possible, since the test substance is not soluble in the test medium of the aquatic test.

Mean Recovery in the Fortified

Samples: 60 % (n = 10, RSD 16 %)

The measurements showed unsatisfying recoveries. This might be caused by the bad solubility of the test substance in test medium of the aquatic test.

As the solubility of the test substance in the test medium is low, the concentrations of dissolved test substance were below the limit of detection. However, all reported results refer to nominal concentrations.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 96 h LC50 and NOEC for the test substance, can be considered to be >100 mg/L (nominal) and 100 mg/L (nominal), respectively.

Executive summary:

A study was conducted to determine the acute toxic potential of read across substance, mono- and di- C16 PSE, K+ (purity: ca. 85%) to rainbow trout (Oncorhynchus mykiss), according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Seven juvenile fish were exposed to the test substance at nominal loading rate of 100 mg/L for 96 h under static conditions. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 h test duration for sublethal effects and mortality. As the test substance is poorly water soluble, the water-accommodated fraction (WAF) was tested. The quantification of the test substance was performed in duplicates using liquid chromatography (LC-MS/MS-method). The concentrations of dissolved test substance were found to be below the Limit of Detection (1.6 μg test substance/L). Therefore, all results were presented in terms of nominal loading rates. In the control and the only test concentration of nominal 100 mg test substance/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LC50 and NOEC for the read across substance were determined to be >100 mg/L (nominal) and 100 mg/L (nominal), respectively (Kuhl and Wydra, 2013). Based on the results of the read across study, similar 96 h LC50 and NOEC values can be expected for the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate'.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

not specified

Vehicle:

yes

Remarks:

Tetrahydrofuran

Details on test solutions:

Vehicle, solvent: Tetrahydrofuran
Concentration of vehicle, solvent: 100 uL/L

Dilution water
Source: Dechlorinated laboratory tap water
Aeration: Test vessels aerated via narrow bore glass tubes

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Strain: Oncorhynchus mykiss
Supplier: Donnington Fish Farm, Upper Swell, Gloucestershire, UK
Weight: 1.20 g
Feeding: Commercial trout pellets
Pretreatment: Acclimatised to test conditions for 1 week prior to test
Feeding during test: None
Control group: 1 control and 1 solvent control group
Photoperiod: 16 h light and 8 h darkness
Renewal of test solution: Daily
Exposure vessel type: 20 L glass vessels
Number of replicates: 2
Fish per replicate: 10

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Mortalities and adverse reactions to exposure were recorded at 3, 6, 24, 48, 72 and 96 h

Hardness:

136 mg/L CaCO3

Test temperature:

13-14 deg C

pH:

7.4 - 7.9

Dissolved oxygen:

9.2 - 10.1 mg O2/L

Salinity:

80 mg/L

Conductivity:

405 uS/cm

Nominal and measured concentrations:

0.4 mg/L (nominal, limit of solubility)

Details on test conditions:

Number of replicates: 2
Fish per replicate: 10

Reference substance (positive control):

not specified

Key result

Duration:

96 d

Dose descriptor:

LC50

Effect conc.:

> 0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks on result:

other: i.e., equivalent to >0.38 mg a.i./L

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks on result:

other: i.e., equivalent to 0.38 mg a.i./L

Validity criteria fulfilled:

not specified

Conclusions:

Under the study conditions, the 96 h LC50 and NOEC for the test substance were determined to be >0.4 mg/L (i.e., >0.38 mg a.i./L) and 0.4 mg/L (i.e., 0.38 mg a.i./L) (nominal), respectively.

Executive summary:

A study was conducted to determine the acute toxic potential of test substance, hexadecan-1-ol (purity: >95%) to Salmo gairdneri (Oncorhynchus mykiss), according to OECD Guideline 203, in compliance with GLP. Twenty Salmo gairdneri were exposed to the test substance at nominal concentration of 0.4 mg/L for 96 h under semi-static conditions. The solubility of the test substance was about 0.01 mg/L. Analytical monitoring data was not specified in the study. The test fish were observed for mortality and other adverse reactions after 3, 6, 24, 48, 72 and 96 h test duration. In the control and the only test concentration of nominal 0.4 mg/L, all fish survived until the end of the experiment and showed no sub lethal effects during the exposure time. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LC50 and NOEC values of the test substance were determined to be >0.4 mg/L (i.e., >0.38 mg a.i./L) and 0.4 mg/L (i.e., 0.38 mg a.i./L) (nominal), respectively (OECD SIDS, 2006).

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From April 20, 1998 to April 24, 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Two stock solutions were prepared in water at nominal 1 and 100 mg/L. applying magnetic stirring for ca. 23 h to achieve maximum soluble fraction of the test substance in the test medium. Subsequently, these stock solutions were transferred to a separating funnel and left tot stabilize for ca. 4 h. The water phase in the middle of the separating funnel was used for testing. Both solutions were clear and colourless.
- Eluate: no
- Differential loading: yes
- Controls: yes, water control

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: carp
- Source: Obtained from Bio International, Roermond, The Netherlands (F1 from a single parent-pair nred om UV-treated water)
- Length at study initiation: 2.09 +/- 0.10 cm
- Weight at study initiation: 0.22 +/- 0.04 g
- Feeding during test: none

ACCLIMATION
- Acclimation period: At least 12 days after delivery.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Commercial fish feed "Trouvit"
- Feeding frequency: daily
- Health during acclimation (any mortality observed): Mortality seven days prior to the start of the test was less than 5%.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

250 mg CaCO3/L

Test temperature:

20.1 - 20.6 °C

pH:

7.8 - 8.1

Dissolved oxygen:

6.9 - 9.3 mg O2/L

Nominal and measured concentrations:

Nominal: 0, 10 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass vessels
- Material, size, headspace, fill volume: all glass, 4 L, headspace: 1.5 L, fill volume: 2.5 L
- Aeration: no
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.77 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO-medium, formulated using Milli-RO water (tap water purified by reverse osmosis) with the following composition: Ca2+ 80 mg/L, Mg2+ 12 mg/L, Na+ 15 mg/L, K+ 3 mg/L, Cl- 145 mg/L, SO4 2- 49 mg/L, HCO3 47 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Oxygen, pH, nitrate and nitrite and ammonia concentration were measured once a week.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark

EFFECT PARAMETERS MEASURED: Mortality was observed after 2, 24, 48, 72 and 96 h.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accommodated Fraction (WAF)

Basis for effect:

mortality (fish)

Details on results:

- Mortality of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: A thin test substance film was observed on the surface after 24 h for the 10 mg/L test concentration after 24 h. A test substance film was observed on the surface for the 100 mg/L test concentration after 24 h.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Mortality: yes
- LC50: Between 0.10 - 0.15 mg/L

Validity criteria fulfilled:

not specified

Conclusions:

Based on the results of the read across study, similar LC50 value of >100 mg/L (nominal) can be expected for the test substance.

Executive summary:

A study was conducted to determine the acute toxic potential of read across substance, ‘octyldodecyl isooctadecanoate’ (purity: assumed to be 100%) to Cyprinus carpio, according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Three replicates of seven fish were exposed to the read across substance at nominal loading rate concentrations of 0, 10 and 100 mg/L for 96 h under static conditions. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 h test duration for mortality. As the read across substance is poorly water soluble, the water-accommodated fraction (WAF) was tested. No chemical analysis was performed, as it was not possible to detect the read across substance concentrations in the test medium using the available analytical techniques.No mortality was observed in the control and the test groups during the exposure period. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LL50 was determined to be >100 mg/L (nominal) (Notox, 1998). Based on the results of the read across study, similar LC50 and NOEC values can be expected for the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate'.

Description of key information

Based on the available weight of evidence from studies on the main constituents, a similar absence of toxicity up to highest soluble test concentrations can be expected for the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate'. However, as a conservative approach, the lowest 96 h LC50 value of >0.4 mg/L (nominal), from the study with alcohol, ‘hexadecan-1-ol’, has been considered further for hazard/risk assessment.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.4 mg/L

Additional information

In absence of short-term toxicity to fish with the test substance, the endpoint has been assessed based on studies for substances representative of the main constituents, which can be categorised as phosphate esters (PSE i.e., mono- and di C16 PSE, K+), alcohol (i.e., hexadecanol) and isoalkylester (i.e., isostearyl isostearate). The results are presented below:

Constituent: PSE - read across studies:

Study 1: A study was conducted to determine the acute toxicity potential of the read across substance, ‘mono- and di- C16 PSE, K+ and H3PO4’ (100%), to Zebrafish (Danio rerio), according to OECD Guideline 236 (Fish Embryo Toxicity (FET)), in compliance with GLP. The test was initiated by the addition of 1 impartially selected Zebrafish embryo, to each 24 well. The embryos were pre-exposed in petri dishes and sorted prior to addition to the well plates within 90 minutes of fertilisation. Fertilised eggs, undergoing cleavage and showing no obvious irregularities during cleavage or injuries of the chorion were selected. Loading of embryos to the well plates was completed less than 3 h post fertilisation. The test organism were exposed to dilution water control and positive control (3,4-dichloroaniline) together with nominal test substance exposure concentrations of 0.3125, 0.625, 1.25, 2.5 and 5.0 mg/L. Levels were based on the dispersibility observed in a non-GLP solubility trial and range finding test. To demonstrate that nominal exposure concentrations were being achieved the concentrations of test substance in the test vessels were measured using the HPLC method. Except for the measured values of the top 3 test concentrations at 0 h (i.e., 0.46, 0.79 and 1.4 mg/L), the remaining measurements at 0 and 96 h were found to be below the limit of quantification (LOQ). Therefore, the test concentrations were presented as mean measured concentrations i.e., 0, 0, 0.23, 0.40 and 0.71 mg/L. An assessment of the response of the Zebrafish embryos was made at 24, 48, 72 and 96 h after the commencement of the test using a low power binocular microscope, with bright field illumination. Any positive outcome of the four observations (coagulation of the embryo, lack of somite formation, non-detachment of the tail, lack of heartbeat) meant that the zebrafish embryo was dead. The 96 h and 48 h LC50 (median lethal concentration) were determined to be >0.71 mg/L (measured). Based on mortality compared to the solvent control (p <0.05) the 48 h NOEC was determined to be 0.71 mg/L (measured) and the LOEC was >0.71 mg/L (measured). All validity criteria were met during this study. Under the conditions of the study, the 96 h LC50 and NOEC values of the read across substance were determined to be >0.71 mg/L and 0.71 mg/L respectively (measured) (Scymaris, 2018).

Study 2: A study was conducted to determine the acute toxic potential of read across substance, ‘mono- and di- C16 PSE, K+’ (purity: ca. 85%) to rainbow trout (Oncorhynchus mykiss), according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Seven juvenile fish were exposed to the test substance at nominal loading rate of 100 mg/L for 96 h under static conditions. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 h test duration for sublethal effects and mortality. As the test substance is poorly water soluble, the water-accommodated fraction (WAF) was tested. The quantification of the test substance was performed in duplicates using liquid chromatography (LC-MS/MS-method). The concentrations of dissolved test substance were found to be below the Limit of Detection (1.6 μg test substance/L). Therefore, all results were presented in terms of nominal loading rates. In the control and the only test concentration of nominal 100 mg test substance/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LC50 and NOEC for the read across substance were determined to be >100 mg/L (nominal) and 100 mg/L (nominal), respectively (Kuhl and Wydra, 2013).

Constituent: Alcohol:

A study was conducted to determine the acute toxic potential of hexadecan-1-ol (purity: >95%) to Salmo gairdneri (Oncorhynchus mykiss), according to OECD Guideline 203, in compliance with GLP. Twenty Salmo gairdneri were exposed to the test substance at nominal concentration of 0.4 mg/L for 96 h under semi-static conditions. The solubility of the test substance was about 0.01 mg/L. Analytical monitoring data was not specified in the study. The test fish were observed for mortality and other adverse reactions after 3, 6, 24, 48, 72 and 96 h test duration. In the control and the only test concentration of nominal 0.4 mg/L, all fish survived until the end of the experiment and showed no sub lethal effects during the exposure time. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LC50 and NOEC values of the read across substance were determined to be >0.4 mg/L (i.e., >0.38 mg a.i./L) and 0.4 mg/L (i.e., 0.38 mg a.i./L) (nominal), respectively (OECD SIDS, 2006).

Constituent: Isoalkyl ester – read across study:

A study was conducted to determine the acute toxic potential of read across substance, ‘octyldodecyl isooctadecanoate’ (purity: assumed to be 100%) to Cyprinus carpio, according to OECD Guideline 203 and EU Method C.1, in compliance with GLP. Three replicates of seven fish were exposed to the read across substance at nominal loading rate concentrations of 0, 10 and 100 mg/L for 96 h under static conditions. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 h test duration for mortality. As the read across substance is poorly water soluble, the water-accommodated fraction (WAF) was tested. No chemical analysis was performed, as it was not possible to detect the read across substance concentrations in the test medium using the available analytical techniques. No mortality was observed in the control and the test groups during the exposure period. The pH and the oxygen values were in normal ranges. Under the study conditions, the 96 h LL50 was determined to be >100 mg/L (nominal).

Overall, based on the available weight of evidence information from studies for substances representative of the main constituents, similar absence of toxicity up to highest soluble test concentrations can be expected for the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate'. As a conservative approach, the lowest 96 h LC50 value of >0.4 mg/L, from the study with alcohol, ‘hexadecan-1-ol’, has been considered further for hazard/risk assessment.