Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish embryo acute toxicity (FET)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 02, 2018 to January 07, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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, solvent 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 groups, mixed and randomly selected. The eggs were washed with dilution water after collection from the spawning chambers. The fertilisation rate of the eggs collected from four 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 ± 1 deg C

pH:

7.74 to 7.94

Dissolved oxygen:

8.75 to 8.96 mg/L

Nominal and measured concentrations:

Control, Solvent Control and 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/L (nominal)
Control, Solvent Control and 0.026, 0.036, 0.074, 0.16 and 0.38 mg/L (mean measured)
Test concentrations were based on the level of achievable solubility.

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.:

> 0.38 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.38 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

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 0.01 mg/L.The instrument LOQ was 0.005 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 0.99. The maximum percentage difference from nominal concentration for standards at the LOQ was less than 30% and less than 20% 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 83 - 98% of nominal. The measured concentration at the end of the study for the nominal 0.0625 mg/L test solution was <LOQ and other test solutions ranged 9 - 16% of nominal.

On the basis of the analytical 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	-
Solvent Control	<LOQ	-	<LOQ	-	0	-
0.0625	0.052	83	<LOQ	0	0.026	42
0.125	0.11^a	87	0.012^b	9	0.036	28
0.25	0.24	96	0.023	9	0.074	30
0.5	0.49	98	0.051	10	0.16	32
1.0	0.90	90	0.16	16	0.38	38

a = Triplicate analyses: 0.11, 0.11, 0.11 mg/L

b = Triplicate analyses: 0.011, 0.012, 0.012 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 hatched*
24	Control	0	4	24	17	0
	Solvent Control	0	0	20	0	0
	0.0625	0.026	0	20	0	0
	0.125	0.036	1	20	5	0
	0.25	0.074	0	20	0	0
	0.5	0.16	0	20	0	0
	1.0	0.38	0	20	0	0
48	Control	0	4	24	17	0
	Solvent Control	0	0	20	0	5
	0.0625	0.026	0	20	0	0
	0.125	0.036	1	20	5	0
	0.25	0.074	0	20	0	0
	0.5	0.16	0	20	0	0
	1.0	0.38	0	20	0	0
72	Control	0	4	24	17	80
	Solvent Control	0	0	20	0	70
	0.0625	0.026	0	20	0	85
	0.125	0.036	1	20	5	95
	0.25	0.074	0	20	0	80
	0.5	0.16	0	20	0	75
	1.0	0.38	1	20	5	74
96	Control	0	4	24	17	100
	Solvent Control	0	0	20	0	100
	0.0625	0.026	0	20	0	100
	0.125	0.036	1	20	5	100
	0.25	0.074	1	20	5	100
	0.5	0.16	0	20	0	100
	1.0	0.38	1	20	5	100

* Percentage hatched based on surviving embryos

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

Time	LC50(mg/L)
48 h	>0.38
96 h	>0.38

 

Based on mortality compared to the solvent control (p <0.05) the 96 h No Observed Effect Concentration (NOEC) was determined to be 0.38 mg/L and the Lowest Observed Effect Concentration (LOEC) was >0.38 mg/L. There was no mortality observed in the solvent control or the internal plate controls. The control had 17% mortality. Due to this mortality, statistical comparisons were made as treated groups versus the solvent control. 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 this study with exception of the control mortality. However, the study is deemed valid in this case as the solvent control and all test concentrations had a >90% survival thus showing no effects.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the test substance 96 h LC50 and NOEC were determined to be >0.38 mg/L and 0.38 mg/L respectively.

Executive summary:

A study was conducted to determine the acute embryo toxicity of the test substance, C16-18 AMP, to Zebrafish (Danio rerio), according to the OECD Guideline 236 (Fish Embryo Toxicity (FET)), in compliance with GLP. The test was initiated by the addition of one impartially selected Zebrafish embryo to each of 24 wells. 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, solvent control (tetrahydrofuran) and positive control (3,4-dichloroaniline) together with nominal test substance concentrations of 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/L. Levels were based on achievable solubility. Analytical dose verification was conducted via HLPC. The mean measured concentrations of the test substance were determined to be 0.026, 0.036, 0.074, 0.16 and 0.38 mg/L. An assessment Zebrafish embryo response 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 and 48 h LC50 (median lethal concentration) were determined to be >0.38 mg/L (measured). Based on mortality compared to the solvent control (p <0.05), the 48 h NOEC was determined to be 0.38 mg/L (measured) and the LOEC was >0.38 mg/L (measured). All validity criteria were met during this study, with exception of the control mortality. However, the study is deemed valid in this case as the solvent control and all test concentrations had a >90% survival. Under the study conditions, the 96 h LC50 and NOEC values for the test substance were >0.38 and 0.38 mg/L, respectively (Scymaris, 2018).

Description of key information

Based on the results of an FET study, the 96 h LC50 and NOEC values of the test substance for toxicity to fish embryo were determined to be >0.38 and 0.38 mg/L (measured), respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.38 mg/L

Additional information

A study was conducted to determine the acute embryo toxicity of the test substance, C16-18 AMP, to Zebrafish (Danio rerio), according to the OECD Guideline 236 (Fish Embryo Toxicity (FET)), in compliance with GLP. The test was initiated by the addition of one impartially selected Zebrafish embryo to each of 24 wells. 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, solvent control (tetrahydrofuran) and positive control (3,4-dichloroaniline) together with nominal test substance concentrations of 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/L. Levels were based on achievable solubility. Analytical dose verification was conducted via HLPC. The mean measured concentrations of the test substance were determined to be 0.026, 0.036, 0.074, 0.16 and 0.38 mg/L. An assessment Zebrafish embryo response 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 and 48 h LC50 (median lethal concentration) were determined to be >0.38 mg/L (measured). Based on mortality compared to the solvent control (p <0.05), the 48 h NOEC was determined to be 0.38 mg/L (measured) and the LOEC was >0.38 mg/L (measured). All validity criteria were met during this study, with exception of the control mortality. However, the study is deemed valid in this case as the solvent control and all test concentrations had a >90% survival. Under the study conditions, the 96 h LC50 and NOEC values for the test substance were >0.38 and 0.38 mg/L, respectively (Scymaris, 2018).

The FET study according to the official ECHA recommendations (ECHA report, 2016), is to be used within a weight of evidence approach (Annex XI, Section 1.2 to the REACH Regulation) together with other independent, adequate, relevant and reliable sources of information leading to the conclusion that the substance has or does not have a particular dangerous property. No further testing or read across is required for the test substance, as based on the available data, fish has not been identified as the most sensitive species.