Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

27 Jun 2017 to 28 Jul 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study is conducted to support notification works in other worldwide jurisdictions.  As such, no study proposal was made, as this data is deemed to be “existing”.  It is provided in support of the REACH registration as existing data.

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Version / remarks:

OECD Guideline 215. “Fish, Juvenile Growth Test - 28 days”, 2000.

Deviations:

yes

Remarks:

see "Any other information" for details

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from all test concentrations and the solvent-control.
Frequency: Before the start of exposure to check the functioning of the flow-through system. At the start of the test and on days 7, 14, 21 and 28.
Volume: 1.0 mL
Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
Additionally, single reserve samples of 1.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

yes

Details on test solutions:

The standard test procedures required generation of test solutions, which should contain completely dissolved test item concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. precipitate or a film of the test item on the water surface).
No correction was made for the purity/composition of the test item.
Stock solutions were prepared in Methanol (≥99.5%; Merck, Darmstadt, Germany). The concentrations in these stocks were a factor 20,000 higher than the target concentrations in water (0.13 to 5.0 mg/mL). No special treatment was needed to fully dissolve these stocks in methanol. The stocks were all clear and the colour ranged between colourless and slightly yellow.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, the Netherlands.
Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 112

Holding
Quarantine/Acclimatisation: At least 14 days after delivery.
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality parameters: Were kept within the optimum limits for the respective fish species.
Feeding: Daily with pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands).
Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

No post exposure observation period specified.

Hardness:

180 mg CaCO3 per litre

Test temperature:

During the study, temperature was maintained between 21.5 and 22.2°C and variation between the different test chambers was always within 1°C at any one time during the test. Temperature continuously measured in a control vessel showed that temperature was maintained between 22 and 24°C. Hence, temperature was also maintained within the limits prescribed by the study plan (20-24°C, constant within 2°C).

pH:

The pH ranged between 7.4 and 8.0 and was within the limits prescribed by the study plan (pH 6.0-8.5).

Dissolved oxygen:

Oxygen concentration was generally found to be > 6 mg O2/L for all measurements and was also within the set requirements (> 5 mg/L, i.e. > 60% of air saturation). On day 24 oxygen concentrations tended to decrease below 6 mg/L and an additional measurement at the end of the day did indicate this happening. Subsequently, aeration was introduced in all test vessels.

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations: Target 6.4, 16, 40, 100 and 250 μg/L.

Details on test conditions:

Test duration: 28 days.
Test vessels: 35 litres, containing 30 litres of test solution, consisting of glass plates and covered by a removable Perspex plate.
Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
Loading: At the start of the test: 0.05 g fish/L/day.
Number of fish: 16 per test group.
Illumination: 16 hours photo-period daily.
Aeration: The test solutions were not aerated during the 1st 23 days of the test. From day 24 to day 28 test solutions were aerated.
Feeding: Daily with pelleted fish food at a rate of 4% of initial body weight per vessel.
Ration: The daily ration was generally divided into two equal portions and given to the fish in two feeds per day, except on weekend days and on days 0 and 3. After 14 days of exposure the ration was recalculated based on the fish weights then determined. Fish were not fed 24 hours prior to weighing.

Measurements and recordings
Mortality and clinical signs: Daily
Fish length and weight: After 14 days all surviving fish were weighed and at the end of the test all surviving fish were weighed and measured individually (the fish were not individually identified). The fish were measured from the front to the caudal peduncle.
Oxygen concentration and pH: In all vessels twice a week.
Temperature of medium: Continuously, in one control vessel and twice a week in all vessels.

TERMINAL PROCEDURES
Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

69 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

187 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Measured Concentrations
Analyses of samples taken from the target concentrations on a weekly basis showed that measured concentrations were generally somewhat below the target concentrations with some fluctuations. The highest fluctuations were observed at the lowest concentrations tested. It was unclear why at certain times very low concentrations were measured. The flow-through system worked without any hick-ups and as such the right amounts of stock and dilution water were mixed. Possibly, the low concentrations might be due to adsorption to e.g. the food given to the fish that might have been supplied just before sampling and thus causing an occasional low recovery.
However, in this study only the two highest test groups were of importance regarding toxicity determinations and as such this did not hamper the evaluations. The range tested based on average measured concentrations was 3.4, 7.4, 22, 69 and 187 μg/L.

Mortality and other visible effects
No mortality or visible effects were observed in any of the tested groups. Hence, the NOEC for survival equalled the highest test group of 187 μg/L tested.

Fish body weight
The mean body weights of fish increased equally in all test groups during the 28-day test period, i.e. by 172 to 187% below their respective start weights. Statistical analysis showed that the weights obtained for the two control groups were not statistically different and therefore further analyses of effects in the test item groups were compared with the pooled control group. The maximum reduction obtained was ~ 7% and therefore the EC10 for effects on growth exceeded 187 μg/L. Statistical analysis showed that the NOEC for growth (weight) was at 187 μg/L for both the 0-14 d and 0-28 d period.

Fish growth rates
Statistical analysis showed that the growth rates obtained for the two control groups were statistically different and therefore further analyses of effects in the test item groups were compared with the solvent-control group.
The maximum reduction obtained was 17% for the 0-14 d period and 7% for the 0-28 d test period at 187 μg/L. Hence, the EC10 for effects on growth rate for the total test period exceeded 187 μg/L. Statistical analysis (see Appendix 3) showed a statistically significant reduction of growth rate at 187 μg/L and the NOEC for growth rate was 69 μg/L for both the 0-14 d and 0-28 d period.

Fish body length
There appeared to be no treatment-related effects on body lengths. Mean lengths at the start ranged between 3.1 and 3.2 cm, while the mean lengths at the end of the test varied between 4.7 and 4.9 cm.

Experimental conditions
The pH ranged between 7.4 and 8.0 and was within the limits prescribed by the study plan (pH 6.0-8.5). Oxygen concentration was generally found to be > 6 mg O2/L for all measurements and was also within the set requirements (> 5 mg/L, i.e. > 60% of air saturation). On day 24 oxygen concentrations tended to decrease below 6 mg/L and an additional measurement at the end of the day did indicate this happening. Subsequently, aeration was introduced in all test vessels. During the study, temperature was maintained between 21.5 and 22.2 °C and variation between the different test chambers was always within 1 °C at any one time during the test. Temperature continuously measured in a control vessel showed that temperature was maintained between 22 and 24 °C. Hence, temperature was also maintained within the limits prescribed by the study plan (20-24 °C, constant within 2 °C).

Results with reference substance (positive control):

Not required

Reported statistics and error estimates:

Reported in table form - see "Overall remarks" for details

Target and measured concentrations

	Measured concentration (μg/L)					Average measured day 0-28 (μg/L)	Stand. Dev. (μg/L)
Target Conc. (μg/L)	Day 0	Day 7	Day 14	Day 21	Day 28
6.4	4.46	2.21	3.87	6.06	0.25	3.37	2.22
16	10.9	8.90	4.19	10.7	2.35	7.41	3.91
40	30.0	22.7	21.5	23.2	12.5	22.0	6.26
100	85.7	62.1	72.0	75.0	48.6	68.7	14.0
250	249	189	185	194	119	187	46.4

 

Mean fish weights recorded at the start of the study, after 14 days and at the end of the 28-day test period

	Mean fish weight (g):
Day of recording	Blank-Control	Solvent-Control	3.4 μg/L	7.4 μg/L	22 μg/L	69 μg/L	187 μg/L
Day 0	0.80	0.84	0.79	0.83	0.79	0.81	0.80
Day 14	1.10	1.15	1.08	1.14	1.08	1.12	1.05
Day 28	1.47	1.51	1.44	1.55	1.44	1.51	1.39
% increase on day 28	185	179	182	187	182	185	172

 

Percentage reduction of weight after 14 days

Treatment [μg/L]	Mean	Std. Dev.	n	%Reduction
Pooled Control 3.4 7.4 22 69 187	1.13 1.08 1.14 1.08 1.12 1.05	0.118 0.113 0.205 0.108 0.138 0.164	32 16 16 16 16 16	4.4 -1.3 4.5 1.1 7.2

 

Percentage reduction of weight after 28 days

Treatment [μg/L]	Mean	Std. Dev.	n	%Reduction
Pooled Control 3.4 7.4 22 69 187	1.49 1.44 1.55 1.44 1.51 1.38	0.160 0.174 0.273 0.160 0.207 0.228	32 16 16 16 16 16	3.6 -3.9 3.4 -1.4 7.0

 

Mean growth rates recorded for the intervals of 0-14 days and 0-28 days

	Mean growth rates:
Interval	Blank-Control	Solvent-Control	3.4 μg/L	7.4 μg/L	22 μg/L	69 μg/L	187 μg/L
Day 0-14	2.33	2.27	2.25	2.29	2.21	2.24	1.88
Day 0-28	2.20	2.09	2.15	2.23	2.14	2.20	1.94

 

Percentage reduction of specific growth rates after 14 days

Treatment [μg/L]	Mean	Std. Dev.	n	%Inhibition
Solvent-control 3.4 7.4 22 69 187	0.023 0.023 0.023 0.022 0.022 0.019	0.0028 0.0015 0.0016 0.0022 0.0020 0.0015	16 16 16 16 16 16	0.7 -1.0 2.6 1.1 17.0

 

Percentage reduction of specific growth rates after 28 days

Treatment [μg/L]	Mean	Std. Dev.	n	%Inhibition
Solvent-control 3.4 7.4 22 69 187	0.021 0.021 0.022 0.021 0.022 0.019	0.0013 0.0012 0.0013 0.0018 0.0018 0.0016	16 16 16 16 16 16	-2.4 -6.6 -2.1 -5.0 7.4

 

pH values

Test group (μg/L)	pH measured on day:
	0	3	7	10	14	17	21	24	28
Blank-control	8.0	7.6	8.0	7.8	7.8	7.9	7.6	7.9	8.0
Solvent-control	8.0	7.6	7.8	7.5	7.7	7.8	7.7	7.8	7.9
3.4	8.0	7.5	7.7	7.5	7.7	7.8	7.7	7.7	7.8
7.4	8.0	7.5	7.6	7.4	7.7	7.8	7.6	7.7	7.8
22	8.0	7.5	7.6	7.4	7.6	7.7	7.7	7.6	7.8
69	8.0	7.5	7.5	7.4	7.6	7.7	7.5	7.6	7.8
187	8.0	7.5	7.5	7.5	7.7	7.7	7.6	7.6	7.8

 

Dissolved oxygen concentrations (mg O₂/L)

Test group (μg/L)	Dissolved oxygen measured on day:
	0	3	7	10	14	17	21	24	28
Blank-control	8.5	7.9	8.2	7.8	8.3	7.8	7.5	7.5	8.4
Solvent-control	8.4	7.3	7.1	6.5	7.2	7.3	7.3	7.4	8.0
3.4	8.3	7.2	7.4	6.6	7.9	7.3	7.7	7.1	7.7
7.4	8.3	7.3	6.4	6.4	7.8	6.9	7.3	6.6	7.8
22	8.3	7.0	6.3	6.4	7.1	7.3	7.0	6.3*	7.6
69	8.3	7.3	6.7	6.8	7.7	6.8	7.1	6.3	7.6
187	8.3	7.6	6.8	7.0	8.1	7.0	7.6	6.9	7.9

*Oxygen measured at the end of the day showed a value of 5.9 mg/L. Subsequently, aeration was introduced in all vessels.

 

Temperature in °C

Test group (μg/L)	Temperature measured on day:
	0	3	7	10	14	17	21	24	28
Blank-control	21.7	21.6	21.8	21.7	21.6	21.5	21.5	21.7	21.7
Solvent-control	21.7	21.6	21.8	21.8	21.7	21.6	21.6	21.8	21.8
3.4	22.0	21.9	22.1	22.1	21.9	21.9	21.9	22.0	22.1
7.4	22.0	21.9	22.1	22.2	22.0	22.0	21.9	22.1	22.1
22	22.0	21.9	22.1	22.2	22.0	22.0	21.9	22.1	22.1
69	21.9	21.9	22.1	22.1	22.0	21.9	21.7	22.1	22.0
187	21.9	21.9	22.1	22.2	21.9	21.9	21.8	22.1	22.1
max	22.0	21.9	22.1	22.2	22.0	22.0	21.9	22.1	22.1
min	21.7	21.6	21.8	21.7	21.6	21.5	21.5	21.7	21.7
Dif	0.3	0.3	0.3	0.5	0.4	0.5	0.4	0.4	0.4

 

Validity criteria fulfilled:

yes

Conclusions:

This prolonged toxicity test investigated the possible effects of MLA-3202 (Naugalube OFM 3202) on survival and growth of carp exposed to various concentrations for a period of 28 days in a flow-through system.
MLA-3202 (Naugalube OFM 3202) induced no mortality or visible effects in carp at average measured concentrations up to and including 187 μg/L.
Exposure to MLA-3202 (Naugalube OFM 3202) resulted in a statistically significant reduction of growth rate at the highest tested concentration of 187 μg/L. The NOEC for growth rate was consequently determined to be 69 μg/L.

Executive summary:

This study is conducted to support notification works in other worldwide jurisdictions.  As such, no study proposal was made, as this data is deemed to be “existing”.  It is provided in support of the REACH registration as existing data.

The objective of the study was to assess the effects of MLA-3202 (Naugalube OFM 3202) on survival, appearance, swimming behaviour and growth of fish exposed to various concentrations for a period of 28 days in a system with continuous (flow-through) renewal of the test solutions. Exposed fish were observed at intervals during the test period to determine the lowest concentration at which effects of the test item could be observed for the first time (threshold level) and the highest concentration at which the substance still had no effect (NOEC).

 

The study procedures described in this report were based on the OECD guideline for Testing of Chemicals: Guideline No. 215, 2000. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

 

The batch of MLA-3202 (Naugalube OFM 3202) tested was a clear amber-red liquid that was completely soluble in test medium at the concentrations tested. The test was performed using a flow-through system with target concentrations of 6.4, 16, 40, 100 and 250 μg/L. A blank and solvent-control (methanol) were also included.

 

A total of sixteen fish were exposed per test group. Stock solutions were dosed via a computer-controlled system and entered a mixing chamber separately from the dilution water supply (flow-rates 10 L/h). The functioning of the system was checked daily during the 28-day test period. Fish were fed daily at 4% of the body weight per vessel. Fish were observed daily for possible mortality and clinical signs. Specific growth rates were based on the body weights measured at the start of the study and after 14 and 28 days of exposure. Samples for chemical analysis of the actual MLA-3202 (Naugalube OFM 3202) concentrations were taken before the start of the exposure, at the start, at weekly intervals and at the end of the test.

 

Analyses of samples taken from the target concentrations on a weekly basis showed that the range tested was based on average measured concentrations of 3.4, 7.4, 22, 69 and 187 μg/L.

 

The mean weight of the fish in the control groups had increased by an average of > 150% above their initial weight over 28 days and all survived the test period. Oxygen concentration was maintained > 5 mg O₂/L in all vessels during the test, while temperature was maintained between 22 and 24 °C and variation between the different test chambers was always within 1 °C at any one time during the test. The study therefore met the acceptability criteria prescribed by the study plan and was considered valid.

 

This prolonged toxicity test investigated the possible effects of MLA-3202 (Naugalube OFM 3202) on survival and growth of carp exposed to various concentrations for a period of 28 days in a flow-through system.

 

MLA-3202 (Naugalube OFM 3202) induced no mortality or visible effects in carp at average measured concentrations up to and including 187 μg/L.

 

Exposure to MLA-3202 (Naugalube OFM 3202) resulted in a statistically significant reduction of growth rate at the highest tested concentration of 187 μg/L. The NOEC for growth rate was consequently determined to be 69 μg/L.

Description of key information

This study is conducted to support notification works in other worldwide jurisdictions.  As such, no study proposal was made, as this data is deemed to be “existing”.  It is provided in support of the REACH registration as existing data.

MLA-3202 (Naugalube OFM 3202) induced no mortality or visible effects in carp at average measured concentrations up to and including 187 μg/L.

 

Exposure to MLA-3202 (Naugalube OFM 3202) resulted in a statistically significant reduction of growth rate at the highest tested concentration of 187 μg/L. The NOEC for growth rate was consequently determined to be 69 μg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

69 µg/L

Additional information

The objective of the study was to assess the effects of MLA-3202 (Naugalube OFM 3202) on survival, appearance, swimming behaviour and growth of fish exposed to various concentrations for a period of 28 days in a system with continuous (flow-through) renewal of the test solutions. Exposed fish were observed at intervals during the test period to determine the lowest concentration at which effects of the test item could be observed for the first time (threshold level) and the highest concentration at which the substance still had no effect (NOEC).

 

The batch of MLA-3202 (Naugalube OFM 3202) tested was a clear amber-red liquid that was completely soluble in test medium at the concentrations tested. The test was performed using a flow-through system with target concentrations of 6.4, 16, 40, 100 and 250 μg/L. A blank and solvent-control (methanol) were also included.

 

A total of sixteen fish were exposed per test group. Stock solutions were dosed via a computer-controlled system and entered a mixing chamber separately from the dilution water supply (flow-rates 10 L/h). The functioning of the system was checked daily during the 28-day test period. Fish were fed daily at 4% of the body weight per vessel. Fish were observed daily for possible mortality and clinical signs. Specific growth rates were based on the body weights measured at the start of the study and after 14 and 28 days of exposure. Samples for chemical analysis of the actual MLA-3202 (Naugalube OFM 3202) concentrations were taken before the start of the exposure, at the start, at weekly intervals and at the end of the test.

 

Analyses of samples taken from the target concentrations on a weekly basis showed that the range tested was based on average measured concentrations of 3.4, 7.4, 22, 69 and 187 μg/L.

 

The mean weight of the fish in the control groups had increased by an average of > 150% above their initial weight over 28 days and all survived the test period. The study therefore met the acceptability criteria prescribed by the study plan and was considered valid.

MLA-3202 (Naugalube OFM 3202) induced no mortality or visible effects in carp at average measured concentrations up to and including 187 μg/L.

 

Exposure to MLA-3202 (Naugalube OFM 3202) resulted in a statistically significant reduction of growth rate at the highest tested concentration of 187 μg/L. The NOEC for growth rate was consequently determined to be 69 μg/L.