Glutens, hydrolyzates, reaction products with lauroyl chloride, sodium salts

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 24 November 2008 and 23 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Sponsor's identification: LCE08082
Description: extremely pale yellow liquid
Date received: 5 September 2008
Storage conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Total Organic Carbon (TOC) analysis was performed on samples taken from the test
preparations at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old
media)

Vehicle:

yes

Details on test solutions:

Information provided by the Sponsor indicated that the test material was a complex mixture of amino acids.

The test material was dissolved directly in dechlorinated tap water.
An amount of test material (4000 mg) was dissolved in reverse osmosis purified water with the aid of ultrasonication and the volume adjusted to 1 litre to give a 4000 mg/L stock solution.
Aliquots (160, 280 and 500 mL) of this stock solution were each separately dispersed in 20 litres (final volume) of dechlorinated tap water, and stirred using a flat bladed mixer to approximately 1 minute, to give the required test concentrations.
The stock solution was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Strain: not stated in the report
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation (length definition, mean, range and SD): a mean standard length of 4.7 cm (sd = 0.4), at the end of the test
- Weight at study initiation (mean and range, SD): a mean weight of 1.31 g (sd = 0.34) at the end of the definitive test.
- Method of breeding: Not stated
- Feeding during test: No
- Food type: Not applicable
- Amount: Not applicable
- Frequency: Not applicable


ACCLIMATION
- Acclimation period: Fish were acclimatised to test conditions from 07 October 2009 to 19 October 2009.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: The stock fish were fed commercial trout pellets which were discontinued approximately
48 hours prior to the start of the definitive test. The protocol stated that feeding was to
be discontinued 24 hours prior to the start of the definitive test. However, in order to
comply with other studies conducted at the same time, the feeding was discontinued
approximately 48 hours prior to the start of the test. This deviation was considered not to
have affected the outcome or validity of the test given that no mortalities or adverse
effects were observed in the control and test fish during the test and that a 48-Hour
starvation period is recommended in some test guidelines for example US EPA guideline
OPPTS 850.1010.
- Feeding frequency: not stated in report
- Health during acclimation (any mortality observed): There was zero mortality in the 7 days prior to the start of the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/l as CaCO3

Test temperature:

12°C to 14°C in the initial range-finding test and approximately 14°C in the main test. Some of the temperatures in the initial range-finding test were measured to be outside the 14 ± 1°C range given in the protocol. This was considered not to have affected the validity or integrity of the tests as no adverse effects of exposure were observed in the control fish throughout the duration of the tests.

pH:

The pH was measured using a WTW pH/Oxi 340I.

Dissolved oxygen:

The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The pH and the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Salinity:

Not stated

Nominal and measured concentrations:

Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 32, 56 and 100 mg/l (Nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass 20 litre
- Aeration: Yes
- Type of flow-through (e.g. peristaltic or proportional diluter): Not stated
- Renewal rate of test solution (frequency/flow rate): Daily renewal
- No. of organisms per vessel: 7 fish/vessel
- No. of vessels per concentration (replicates): 1 vessel/xconcentration
- No. of vessels per control (replicates):1 vessel/concentration
- No. of vessels per vehicle control (replicates): 1 vessel/concentration
- Biomass loading rate: 0.53 g bodyweight/litre (static volume)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener)
- Total organic carbon: 0.81 - 1.38 mg/l
- Particulate matter: not stated
- Metals: various
- Pesticides: No
- Chlorine: Total 0.03 - 0.55 mg/l, Free: 0.08 - 0.61mg/l
- Alkalinity: Not stated
- Ca/mg ratio: Not stated
- Conductivity: 251,000 - 542,000 micro.S at 20 deg C
- Culture medium different from test medium: No
- Intervals of water quality measurement: Varied


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Light intensity: Not stated


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.
- Results used to determine the conditions for the definitive study: The results showed no mortalities at the test concentration of 10 mg/l. However, mortalities were observed at 100 mg/l. Mortalities were observed at 1.0 mg/l during the initial range-finding test however, this was considered to be due to natural causes and/or handling stress given that no mortalities were observed at a test concentration of 10 mg/l in either range-finding test.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the definitive test.

Reference substance (positive control):

no

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: 95% CL

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL

Details on results:

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 100 mg/I, the lowest test concentration resulting in 100% mortality to be greater than 100 mg/I and the No Observed Effect Concentration (NOEC) to be 100 mg/L. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.

These results were slightly different to those obtained in the range-finding and initial tests. In the definitive test seven fish per test concentration were used and hence this was a more robust test than the range-finding tests where only three fish per concentration were used. This may account for the differences observed between the range-finding and definitive tests. In the initial tests, measured concentrations were shown to be variable and hence the effects observed could not be attributed solely to test material exposure concentrations. It is therefore considered that the results of the definitive test provide a true estimate of the toxicity to the test material. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Reported statistics and error estimates:

None stated in report

Sublethal observations / clinical signs:

Range-finding Tests:

In the initial range-finding test, mortalities were observed at 1.0 mg/l, however given that no mortalities were observed at 10 mg/l these mortalities were considered to be due to natural causes andtor handling stress and hence were considered not to be significant.

In the second range-finding test, no mortalities were observed at the test concentration of 10 mg/l. However, mortalities were observed at 100 mg/l

Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the initial tests.

Definitive Test

Mortality data

There were no mortalities in fish exposed to a test concentration of 100 mg/l for a period of 96 hours. Inspection of the mortality data gave the following results:

Time (h)	LC50(mg/l)	95% Confidence limits (mg/l)
3	>100	-
24	>100	-
48	>100	-
72	>100	-
96	>100	-

Sub-lethal effect

There were no sub-lethal effects of exposure observed in 14 fish exposed to a test concentrations of 100 mg/l. for a period of 96 hours.

Observations on test material solubility

The freshly prepared test concentrations were observed to be clear colourless solutions, however the old test media was observed to have a clear colourless water column with particles of test material precipitate dispersed throughout. A similar effect was observed in the Acute Toxicity to Daphnia magna study conducted on this test material (Harlan

Project Number 2580-0007) at test concentrations of 32 mg/l and higher. Solubility determinations showed the test material was readily soluble in reverse osmosis purified deionised water, however at high concentrations in dechlorinated tap water a cloudy dispersion was formed. It was considered likely that the test material was forming insoluble calcium andlor magnesium salts in dechlorinated tap water and reconstituted water and this was the cause of the test material precipitate observed in the definitive

test. Given that the test media in the definitive test was replaced each 24 hours (semi-static test) it was considered that the test organisms were exposed to the highest possible dissolved test material concentration, within the confines of the maximum recommended test concentration given in the OECD Test Guidelines, and hence the presence of the test material precipitate had no adverse effects on the results of the study.

Total organic carbon analysis

Total Organic Carbon (TOC) analysis of the triplicate 500 mgll stock solutions showed the test material contained approximately 12.41% carbon (see Appendix 2 attached). The results from both the filtered and centrifuged test samples gave similar results to those analysed without any pre-treatment and as such it was considered that the test material was fully

dissolved. Total Organic Carbon (TOC) analysis of the freshly prepared test media at 0, 24 and 48 hours showed measured concentrations to range from 56% to 104% of nominal values with the lower percentage of nominal values being obtained from the lowest test concentrations (see Appendix 2 attached). This effect was considered to be due to the inherent variability in the TOC analyses at concentrations close to the Limit of Quantitation (LOQ) of the method (1.0 mg C/I) which was magnified by the low percentage carbon content of the test material (12.41%). Given that the mean measured test concentration in the 56 and 100 mgtl test concentrations, where the TOC was significantly higher than the LOQ, was 88% and 90% of nominal values, it was considered that the test system was correctly dosed. A decline in measured concentrations was observed in all the old test media samples in the range of 9% to 84% of nominal values. As TOC analysis is non-stability indicating this decline in measured concentrations was considered to be due to possible adsorption of the test material to the glassware andtor biological matter present. Due to an instrument malfunction, the samples taken from the freshly prepared test media at 72 hours were not analysed until approximately 24 hours after sampling. The results obtained from these samples were in line with other 24-Hour old test media samples thereby indicating that the test system had been correctly dosed at 72 hours. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole the results were based on nominal test concentrations only.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 value of greater than 100 mg/l. The No Observed Effect Concentration was 100 mg/l.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in 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.

Information provided by the Sponsor indicated that the test material was a complex mixture of amino acids. Pre-study solubility work conducted indicated that the test material was readily water soluble and as such it was considered that the most

suitable method of preparation for this material was as a direct solution in test medium. Method development work conducted for the determination of dissolved test material concentrations using HPLC-MS analysis gave a good response in single ion mode with m/z 511.4. However, as the test material is a mixture of various amino acid reaction products it was considered that chemical analysis was not suitable for detecting the total dissolved test material concentration present. As such it was considered appropriate to determine the dissolved test material concentration present in the test samples by Total Organic Carbon (TOC) analysis.

Following preliminary range-finding and initial tests, fish were exposed, in groups of seven, to an aqueous solution of the test material over a range of concentrations of 32, 56 and 100 mg/l for a period of 96 hours at a temperature of approximately 14°C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results.

The 96-Hour LCso based on nominal test concentrations was greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l. Total Organic Carbon (TOC) analysis of the freshly prepared test media at 0, 24 and 48 hours showed measured concentrations to range from 56% to 104% of nominal values with the lower percentage of nominal values being obtained from the lowest test concentrations. This effect was considered to be due to the inherent variability in the TOC analyses at concentrations close to the Limit of Quantitation (LOQ) of the method (1.0 mg G/I) which was magnified by the low percentage carbon content of the test material (12.41%). Given that the mean measured test concentration in the 56 and 100 mg/I test concentrations, where the TOC was significantly higher than the LOQ, was 88% and 90% of nominal values, it was considered that the test system was correctly dosed. A decline in measured concentrations was observed in all the old test media samples in the range of 9% to 84% of nominal values. As TOC analysis is non-stability indicating this decline in measured concentrations was considered to be due to possible adsorption of the test material to the glassware and/or biological matter present. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole the results were based on nominal test concentrations only.

Description of key information

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 value of greater than 100 mg/l.  The No Observed Effect Concentration was 100 mg/l.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L