1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C8-18(even numbered) acyl derivs., hydroxides, inner salts

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
see "General Justification for Read-Across" attached to IUCLID section 13

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Mutual read across from the AAPBs to one another is justified:

a) Based on the information given in section 1, it can be concluded that all AAPBs mentioned above are similar in structure, since they are manufactured from similar resp. identical precursors under similar conditions and all contain the same functional groups. Thus a common mode of action can be assumed.
b) The content of minor constituents in all products are comparable and differ to an irrelevant amount.
c) The only deviation within this group of substances is a minor variety in their fatty acid moiety, which is not expected to have a relevant impact on intrinsic toxic or ecotoxic activity and environmental fate. Potential minor impact on specific endpoints will be discussed in the specific endpoint sections.

The read-across hypothesis is based on structural similarity of target and source substances. Based on the available experimental data, including key physico-chemical properties and data from toxicokinetic, acute toxicity, irritation, sensitisation, genotoxicity and repeated dose toxicity studies, the read-across strategy is supported by a quite similar toxicological profile of all five substances.
The respective data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see "General Justification for Read-Across" attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see "General Justification for Read-Across" attached to IUCLID section 13

4. DATA MATRIX
see "General Justification for Read-Across" attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Duration:

21 d

Dose descriptor:

NOEC

Remarks:

geometric mean of all available studies

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Reported statistics and error estimates:

Steel-Test

Conclusions:

Three of the Daphnia magna reproduction tests were conducted in reconstituted dilution water resulting in 21 d NOEC values of 0.03, 0.32, and 0.932 mg a.i./L. Based on the test results using 2/3 reconstituted dilution water and 1/3 pond water (21 d NOEC=0.9 mg a.i./L), it can be assumed that the presence of pond water has little or no influence, as the results are comparable to those obtained in the newly performed study in which analytical monitoring of the test substance concentration was performed using state-of-the-art equipments and techniques and which seems to be the most reliable study.
The very low 21 d NOEC and LOEC of 0.03 and 0.1 mg a.i./L determined in one test could not be confirmed by the other studies and based on the available data this discrepancy could not be resolved. Therefore a weight of evidence approach seems to be justified taking into account the 21 d NOEC values of 0.03, 0.32, 0.9, and 0.932 mg a.i./L. The calculation yielded a 21 d NOEC of 0.3 mg a. i./L as the geometric mean with respect to reproduction.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2006-03-08-2006-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- fresh media on day 0, 2 and 14; old media on day 2, 5 and 16

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared (100 mg test item/L). The test concentrations were obtained by dilution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: waterflea (Daphnia magna)
- Strain/clone: Daphnia magna STRAUS (clone 5)
- Source: origin: WaBoLu; breeder: Dr. U. Noack-Laboratorien, Käthe-Paulus-Str. 1, Sarstedt (Germany)
- Culture: 2-3 L glass vessels with 1.8 L culture medium (Elendt M4, modified to a total hardness of 160 to 180 mg CaCO3/l) at 20 °C (incubator), 16 h illumination (20 µE/m2s)
- Age of parental stock (mean and range, SD): 2-24 h old
- Feeding during test: 5 days per week (Desmodesmus subspicatus (0.35-1.20 ml) and Chlorella vulgaris (algae cell density > 1000000 cells/ml; 0.365-1.65 ml) corresponding to 0.1-0.2 mg C per daphnid and day

ACCLIMATION
- Acclimation conditions (same as test or not): yes
- Health during acclimation (any mortality observed): not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

160 - 190 mg/l as CaCO3

Test temperature:

19.2 - 22 °C

pH:

7.38 - 8.97

Dissolved oxygen:

8.07 - 10.51 mg/l

Nominal and measured concentrations:

0.1, 0.32, 1.02, 3.28 and 10.5 mg product/l
corresponding to 0.0284, 0.091, 0.290, 0.932 and 2.98 mg active substance/l

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): not reported
- Material, size, headspace, fill volume: glass/100 mL/-/50 mL
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): test conducted under semi-static conditions
- Renewal rate of test solution (frequency/flow rate): 3x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water Elendt M4 modified to a total hardness of 160 to 180 mg CaCO3/l
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH, dissolved oxygen, total hardness and water temperature were measured once a week in fresh and old media, in one replicate of the control and the highest concentration of the test item

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: max. 20 µE/m² x s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Reproduction rate, adult survival, first appearance, intrinsic rates of natural increase, length and dry weight

VEHICLE CONTROL PERFORMED: no vehicle used

RANGE-FINDING STUDY
- Test concentrations: 1, 10, and 100 mg test item/L used in a 48 h test
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.932 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

2.98 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

RESULTS: EXPOSED
- Nominal/measured concentrations: nominal

- NOEC (adult survival) = 10.5 mg product/l)
- statistically significant difference in growth of parent animals at 10.5 mg/l

21-day NOEC (reproduction/first appearance/intrinsic rate) = 3.28 mg product/l
21-day LOEC (reproduction/first appearance/intrinsic rate) = 10.5 mg product/l

Assuming a content of 28.4% active substance:
21-day NOEC (reproduction/first appearance/intrinsic rate) = 0.932 mg product/l
21-day LOEC (reproduction/first appearance/intrinsic rate) = 2.98 mg product/l

Results with reference substance (positive control):

Potassium dichromate p.a. (Sigma), >99.5%, Batch No. 065K1349

RESULTS:
- Date: 14.-15.3.2006
- EC50(24h)=1.84 mg/l, within the prescribed concentration range

Reported statistics and error estimates:

STATISTICS:
- One way analysis of Variance, Bonferroni t-test (p<0.05)
- Kruskal-Wallis ANOVA on Ranks, Dunnet's method (p<0.05)

Reproductive Output
The mean number of offspring alive produced per parent animal surviving at the end of the test
was 97 juveniles in the control group . In the tested concentrations levels of 0.028 to 0.932 mg a.i./l
(0.1 to 3.28 mg product/l) the mean number of offspring alive was comparable to the control. At the
concentration 10.5 mg product/l the reproduction rate was significantly reduced when compared to the
control.

EC50 for the Reproduction
The reduction of reproduction in percent came to a maximum of 26 % when compared to the
control. The concentration that caused a 50 % reduction in reproduction (EC50
reproduction ) was not determinable in the tested concentration range (< 2.98 mg a.i./l /> 10.5 mg product/L).

Intrinsic Rate of Natural Increase (IR)
The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for 
generation time and offspring numbers were used for calculation of population growth and
maintenance. The mean IR of the surviving daphnids of the treatment group
2.98 mg a.i./l (10.5 mg product/l) was reduced about 22.1 % in comparison to the control group. The mean IR of the
other tested concentrations levels of 0.028 to 0.932 mg a.i./l (0.1 to 3.28 mg product/L) were comparable to the control.

Stillborn Juveniles and Aborted Eggs 
The percentage number of dead juveniles (sum of stillborn juveniles and aborted eggs) was related to the 
total number of produced juveniles (alive, stillborn juveniles and aborted eggs) of the surviving daphnids. 
Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was ≤ 3 % 
at 0.09 to 2.98 mg a.i./l (0.32 to 10.5 mg product/l). In the control and at the concentration level of 0.028 mg a.i./l
(0.1 mg product/l) no stillborn juveniles or aborted eggs occurred.

First Appearance of Juveniles 
The first day of appearance of juveniles at all test item and control groups producing juveniles was between day 8 and day 17.
The days of first appearance of living juveniles of the treatment groups were compared to the control by KRUSKAL-WALLIS ANOVA ON RANKS,
DUNN’S Method, p < 0.05. There was a statistically significant difference at the concentration
2.98 mg a.i./l (10.5 mg product/l) compared to control.

Presence of Males 
No males were present neither in the control nor in the test groups during the test. 

Occurrence of Ephippia (Winter Eggs) 
No ephippia occurred in the control or in the test groups during the test.

Immobilisation of the Parental
The test item did not induce significant mortality (> 20 %) of parent animals at any of the test
item concentrations and in the control.

Growth (Total Length and Dry Weight) of the Parent Animals
At the end of the test the total length and the dry weight of all living parent animals at each
concentration and control were determined (Table 9). The body length of the surviving daphnids
of the treatment groups were compared to the control by KRUSKAL-WALLIS ANOVA ON RANKS,
DUNN’S Method, p < 0.05. There was a statistically significant difference at 2.98 mg a.i./l (10.5 mg product/l).


All quality criteria were met:
• Mortality of the parent animal in the control group did not exceed 20 % at the end of the test.
• The mean number of living neonates produced per parent animal surviving at the end of the
test was > 60 in the control group.
• The oxygen concentration was > 3 mg/L at the beginning and during the test.
• The pH-value was in the range of 6 - 9. Normally deviations of initial and final pH values
were < 1.5 units.
• The test temperature was within a range 18 - 22 °C, ± 2 °C constant.
• The photoperiod of the incubator was set to a light/dark cycle of 16/8 h.
• The EC50- value of reference item potassium dichromate after 24 h was within the prescribed
concentration range of 1.0 - 2.5 mg/L of quality criteria. (acc. to AQS P 9/2 and DIN 38412 L 11)

Validity criteria fulfilled:

yes

Conclusions:

The 21-day NOEC and LOEC based on reproductionof Daphnia magna were found to be 0.932 and 2.98 mg active substance/L nominal, respectively, in a study conducted according to OECD Guideline 211 (Daphnia magna Reproduction Test) and EU Method C.20 (Daphnia magna Reproduction Test).

Executive summary:

The 21-day-chronic toxicity of C8 -18 AAPB (28.4 % active matter) to Daphnia magna was investigated in a study conducted according to OECD Guideline 211 (Daphnia magna Reproduction Test) and EU Method C.20 (Daphnia magna Reproduction Test) under semi-static conditions. Daphnids were exposed to control, and test chemical at nominal concentrations of 0.0284, 0.091, 0.290, 0.932 and 2.98 mg active substance/L. Samples of all concentrations and control were analytically verified via LC-MS/MS on days 0, 2, 14 (fresh media, 0 h) and on days 2, 5, 16 (old media, 48/72 h). The recovery rates of fresh and old media of the biologically relevant concentration levels were > 80%. The 21-day NOEC and LOEC based on reproduction were 0.932 and 2.98 mg active substance/L nominal, respectively.

This study is regarded as reliable without restriction and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

Results Synopsis
Test Organism/Age (e.g. 1^st instar): Daphnia magna/<=24 h
Test Type: semi-static
21 d NOEC: 0.932 mg active substance/L nominal
21 d LOEC: 2.98 mg active substance/L nominal
Endpoint(s) Effected: reproduction

Reference 3

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07.11.1990-28.11.1990 (first experiment) and 01.03.1995-22.03.1995 (second experiment)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

cited as OECD Guideline 202, part 2 (Daphnia sp., Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the analysis of the test substance concentrations, a sample was taken from the freshly prepared stock solution (100 mg/l nominal) and duplicate samples from the freshly prepared test media of the nominal test concentrations 0.32, 1.0, and 3.2 mg test substance/l and the control at days 0, 12, and 16. The stability of the test substance was also investigated. To the analytical samples, immediately HgCl2 was added to prevent biodegradation. The samples were analyzed by VIS-spectrometry at 485 nm.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared (100 mg/L). The test concentrations were obtained by dilution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: waterflea (Daphnia magna)
- Strain/clone: Daphnia magna Straus clone 5
- Source: BASF AG, Ludwigshafen (Germany)
- Age of parental stock (mean and range, SD): <24 h
- Feeding during test
- Food type: green algae (Scenedesmus subspicatus)
- Amount: 0.48 x 10E7 -1.8 x 10E7 cells/80 mL
- Frequency: dahnids were fed on day 0, 1, 2, 5, 6, 7, 8, 9, 12-16 and 19-20

ACCLIMATION
- Acclimation period: not necessary
- Acclimation conditions (same as test or not): yes

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

19.8-20.8°C

pH:

initial: 7.9-8.0
at the end: 8.2-8.9

Dissolved oxygen:

initial: 8.5-10.3 mg/L
at the end: 8.6-10.6 mg/L

Nominal and measured concentrations:

0.1, 0.32, 1.0, 3.2, and 10 mg product/L nominal
0.032, 0.1, 0.32, 1.0 and 3.2 mg a.i./L nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): test vessels covered with watch glasses
- Material, size, headspace, fill volume: glass/100 mL/20 mL/80 mL
- Aeration: not during the test
- Type of flow-through (e.g. peristaltic or proportional diluter): test conducted under semi-static conditions
- Renewal rate of test solution (frequency/flow rate): every Monday, Wednesday, and Friday
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: test was conducted in reconstituted water according to Elendt (1990) with an initial pH of 7.9 +/- 0.3. Before use, the test water was aerated until oxygen saturation.
- Alkalinity: 0.9 mmol/L
- Hardness: 250 mg/L as CaCO3
- Conductivity: <0.1 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH and dissolved oxygen were measured in the lowest and highest test concentration and in the control at the beginning and at the end of each treatment period. The water temperature was measured in one control beaker at the start and at the end of each treatment period.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark
- Light intensity: ca. 400-700 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality of adults and number of young

RANGE-FINDING STUDY
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: survival and reproduction rate

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: survival and reproduction rate

Reported statistics and error estimates:

The NOEC and the LOEC of the mean reproduction rate were statistically evaluated by the Williams-Test, based on ANOVA.

RESULTS: EXPOSED
- Nominal/measured concentrations: nominal
- Effect data:
  survival:
  control/0.1/0.32/1.0/3.2/10 mg product/l
  100/90/100/40/20/20 % surviving on day 21
  reproduction:
  control/0.1/0.32/1.0/3.2/10 mg product/l
  100/89.4/75.1/33.7/20.0/9.1 % of control total number of alive young  Daphnia
  reproduction/surviving adult:
  control/0.1/0.32/1.0/3.2/10 mg product/l
  100/95.9/75.1/79.2/60.5/37.7 % of control

  21-day NOEC = 0.1 mg product/l 
  21-day LOEC = 0.32 mg product/l

Assuming a content of 32.1% active substance:
  21-day NOEC=0.03 mg active substance/l
  21-day LOEC=0.10 mg active substance/l

ANALYTICAL MONITORING:
The analytically determined concentrations of cocamidopropyl betaine in  
the freshly prepared test media of the analyzed test concentrations of  
nominal 0.32, 1.0, and 3.2 mg/l varied from 101 to 124% of the nominal  
concentrations, the mean measured values of all measurements amounted  
from 104 to 118% of the respective nominal value. In the test media  
samples, taken out of the actual test, the test concentrations could not  
be quantified due to disturbance of the measurements by the algae.  
Additionally stability controls showed that the test substance was  
sufficiently stable in the test media (test media without algal food and  
Daphnia) during the longest test medium renewal periods of 72 h.

Validity criteria fulfilled:

yes

Conclusions:

The 21-day NOEC and LOEC of C8-18 AAPB were determined to be 0.03 mg active substance/L and 0.10 mg active substance/L (nominal each), respectively, in a study conducted according to OECD Guideline 211 and using Daphnia magna as test organism.

Executive summary:

The 21 d toxicity of C8 -18 AAPB to Daphnia magna was investigated in a study conducted according to OECD Guideline 211 (Daphnia magna Reproduction Test; in the Report cited as OECD Guideline 202, part 2 Daphnia sp., Reproduction Test). Daphnids were exposed to nominal concentrations of 0.1, 0.32, 1.0, 3.2, and 10 mg test substance/L under semi-static conditions. The analytically determined concentrations of C8 -18 AAPB in the freshly prepared test media of the analysed test concentrations of 0.32, 1.0, and 3.2 mg/L varied from 101 to 124% of the nominal concentrations, the mean measured values of all measurements amounted from 104 to 118% of the respective nominal value. In the test media samples taken out of the actual biological test (including algal food), the test substance concentration could not be quantified due to disturbance of the measurements by the algae. However, additional stability controls showed that C8 -18 AAPB is sufficiently stable in the test media (but without algal food and Daphnia) during the longest test medium renewal periods of 72 h. The 21 d NOEC and LOEC based on survival and reproduction were determined to be 0.03 and 0.1 mg a.i./L nominal, respectively.

This study is regarded as valid without restriction and satisfies the requirements of the guideline.

Results Synopsis
Test Organism/Age (e.g. 1^stinstar): Daphnia magna/<24 h
Test Type: semi-static
21 d NOEC: 0.03 mg active substance/L nominal
21 d LOEC: 0.1 mg active substance/L nominal
Endpoint(s) Effected: reproduction, survival

Reference 4

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31.10.1990-21.11.1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

cited as OECD Guideline 202, part 2 (Daphnia sp., Reproduction Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

For the analysis, duplicate samples from a low (0.75 mg/l), medium (3.0 mg/l) and high (12 mg/l) test concentration and from the control were taken. From the additional test vessel containing 12 mg/l test substance (without feed and daphnids), samples were taken at day 0, 2, and 21. From the stock solution (50 mg/l nominal) subsamples of 10 ml were taken at day 0. The stability of the test substance was also investigated.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of 50 mg C8-18 AAPB was made up to 1000 mL with test medium. This stock solution was freshly prepared on each treatment day. The test concentrations were obtained by dilution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: waterflea (Daphnia magna)
- Strain/clone: not reported
- Source: RCC own culture
- Age of parental stock (mean and range, SD): <24 h
- Feeding during test
- on days 0, 2, 5: 200 000 cells/ml yeast, 200 000 cells/ml algae; on days 7, 9, 12: 300 000 cells/ml yeast, 300 000 cells/ml algae; on days 14, 16, 19: 600 000 cells/ml yeast, 600 000 cells/ml algae

ACCLIMATION
- Acclimation period: not necessary
- Acclimation conditions (same as test or not): yes

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Hardness of water: 15.2-16.2 dH° (measured once a week)

Test temperature:

18.7-20.5°C

pH:

7.8 - 8.7

Dissolved oxygen:

8.0 - 12.6 mg/l (before use, test water was aerated until oxygen saturation, no aeration during the test)

Nominal and measured concentrations:

0.036 (0.047*), 0.188, 0.75, 3.0, and 12 mg product/l (nominal)
0.011 (0.014*), 0.056, 0.22, 0.9, 3.6 mg active substance/l (nominal)

*At test start and during test the lowest concentration was about 76.5% of nominal concentration. Therefore the lowest concentrations has to be corrected to 0.036 mg product/l and 0.011 mg active substance/l. The other concentrations were within 80% of nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): not reported
- Material, size, headspace, fill volume: glass/250 mL/-/200 mL and glass/100 mL/-50 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): test conducted under semi-static conditions
- Renewal rate of test solution (frequency/flow rate): every Monday, Wednesday and Friday during the test
- No. of organisms per vessel: 10 (after observation of eggs (on day 7): 10 daphnids with eggs were transferred individually in 50 ml glass-beakers for reproduction; unhealthy and damaged animals were excluded from the reproduction test; the remaining daphnids were kept at 5 animals per beaker, they were included in assessing the mortality rates)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: test water: reconstituted water (2/3) and pond water (1/3, Anwiler Teich) with an initial pH of 7.8-8.2
- Conductivity: 606 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH and temperature of the test samples (controls, lowest and highest test concentration) were controlled at all treatment periods at the beginning and end of the respective periods.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark
- Light intensity: 500-2000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality of adults and number of young

RANGE-FINDING STUDY
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: The results of a previous study (48 h EC50=21.5 mg/L) were used to determine the test concentration range

Reference substance (positive control):

not specified

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

RESULTS: EXPOSED
- Nominal/measured concentrations: nominal

- Effect data:
survival:
control/0.047/0.188/0.75/3.0 / 12 mg product/l
100 / 100 / 100 / 95 / 100/ 35 % surviving on day 21

reproduction/surviving adult:
control/0.047/0.188/ 0.75 / 3.0 / 12 mg product/l
100 />100 / 99 / >100 / >100 / 91 % of control

21-day NOEC = 3 mg product/l
21-day LOEC = 12 mg product/l

Assuming a content of 30% active substance:
21-day NOEC = 0.9 mg active substance/l
21-day LOEC = 3.6 mg active substance/l

Reported statistics and error estimates:

Steel-Test

The 21-day-chronic toxicity of C8 -18 AAPB (30% active matter) to Daphnia magna was studied under semi-static conditions. Daphnids were exposed to control, and test chemical at concentrations of 0.011, 0.056, 0.22, 0.9, 3.6 mg active substance/L (nominal). The 21-day NOEC based on reproduction was 0.9 mg a.i./L nominal. The effects included were mortality of adults and number of young.

Validity criteria fulfilled:

yes

Conclusions:

The 21-day NOEC and LOEC based on reproduction were found to be 0.9 and 3.6 mg active substance/L nominal in a study conducted according to OECD Guideline 211.

Executive summary:

The 21-day-chronic toxicity of C8 -18 AAPB (30% active matter) to Daphnia magna was investigated in a study conducted according to OECD Guideline 211 (Daphnia magna Reproduction Test; in the report cited as OECD Guideline 202, part 2 Daphnia sp., Reproduction Test) under semi-static conditions. Daphnids were exposed to control, and test chemical at concentrations of 0.011, 0.056, 0.22, 0.9, 3.6 mg active substance/L (nominal). The test was performed in a mixture of 2/3 reconstituted water and 1/3 pond water. Per concentration 20 daphnids (ten per beaker) were kept in bulk culture. After 7 d 10 daphnids with eggs in the brood pouch were separated and kept individually. The remaining daphnids were observed for mortality rates (unhealthy and damaged daphnids were excluded). At the start of the treatment periods the measured concentrations (photometric) ranged from 76.7 to 117% of the nominal values and at the end of the exposure period from 76.3 to 96.9%. Additionally performed stability controls showed that the test substance was sufficiently stable in the test media during the renewal periods. The 21-day NOEC and LOEC based on reproduction were 0.9 and 3.6 mg active substance/L nominal.

This study is classified as reliable without restriction and satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates.

Results Synopsis
Test Organism/Age (e.g. 1^st instar): Daphnia magna/<24 h
Test Type: semi-static
21 d NOEC: 0.9 mg a.i./L
21 d LOEC: 3.6 mg a.i./L

(all values refer to nominal concentrations)
Endpoint(s) Effected: reproduction

Description of key information

The long-term toxicity of C8-18 AAPB and Coco AAPB was investigated in four studies conducted according to OECD Guideline 211, in some reports cited as OECD Guideline 202, part II Daphnia sp., Reproduction Test. It was considered appropriate to evaluate the studies using a weight of evidence approach.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.3 mg/L

Additional information

Adequate and reliable long-term toxicity data to aquatic invertebrates are available for C8-18 AAPB as well as for the closely related source substance C8-18 and C18 unsatd. AAPB (Coco AAPB). A justification for read-across is given below.

 

In the first study the 21 d toxicity of C8-18 AAPB toDaphnia magna was investigated according to OECD Guideline 211 (Daphnia magna Reproduction Test) under semi-static conditions. The analytically determined concentrations of C8-18 AAPB in the freshly prepared test media of the analysed test concentrations of 0.32, 1.0, and 3.2 mg/L varied from 101 to 124 % of the nominal concentrations, the mean measured values of all measurements amounted from 104 to 118 % of the respective nominal value. In the test media samples taken out of the actual biological test (including algal food), the test substance concentration could not be quantified due to disturbance of the measurements by the algae. However, additional stability controls showed that C8-18 AAPB was sufficiently stable in the test media (but without algal food and Daphnia) during the longest test medium renewal periods of 72 h. The 21 d NOEC and LOEC based on survival and reproduction were determined to be 0.03 and 0.1 mg a.i./L nominal, respectively. These very low values could not be confirmed in the other studies. Based on the available data this discrepancy could not be resolved.

 

In the second study the 21 d toxicity of Coco AAPB to Daphnia magna was investigated according to OECD Guideline 211 (Daphnia magna Reproduction Test) under semi-static conditions. The 21 d NOEC and LOEC values were determined to be 0.32 and 0.56 mg a. i./L in respect to reproduction. The most sensitive endpoint was release of the first brood (21 d NOEC=0.18 mg a. i./L; 21 d LOEC=0.32 mg a. i./L nominal each).

 

In the third study the 21-day-chronic toxicity of C8-18 AAPB (30 % active matter) to Daphnia magna was investigated according to OECD Guideline 211 (Daphnia magna Reproduction Test) under semi-static conditions. The test was performed in a mixture of 2/3 reconstituted water and 1/3 pond water. Per concentration 20 daphnids (ten per beaker) were kept in bulk culture. After 7 d 10 daphnids with eggs in the brood pouch were separated and kept individually. The remaining daphnids were observed for mortality rates (unhealthy and damaged daphnids were excluded). At the start of the treatment periods the measured concentrations (photometric) ranged from 76.7 to 117 % of the nominal values and at the end of the exposure period from 76.3 to 96.9 %. Additionally performed stability controls showed that the test substance was sufficiently stable in the test media during the renewal periods. The 21-day NOEC and LOEC based on reproduction were 0.9 and 3.6 mg active substance/L nominal.

 

In the newly performed 21-day-chronic, semi-static reproduction tests conducted according to OECD Guideline 211 (Daphnia magna Reproduction Test) and EU Method C.20 (Daphnia magna Reproduction Test), the influence of natural ingredients in environmental aquatic systems on the chronic toxicity of C8-18 AAPB (28.4 % active matter) to Daphnia magna was investigated. Daphnids were exposed to control, and test chemical at nominal concentrations of 0.0284, 0.091, 0.290, 0.932 and 2.98 mg a.i./L (dilution water according to Elendt). Samples of all concentrations and control were analytically verified via LC-MS/MS on days 0, 2, 14 (fresh media, 0 h) and on days 2, 5, 16 (old media, 48/72 h). The recovery rates of fresh and old media of the biologically relevant concentration levels were > 80 %. The 21-day NOEC and LOEC based on reproduction were 0.932 and 2.98 mg active substance/L nominal, respectively (CEFIC/CESIO [ICCA Initiative] Alkylamidopropyl Betaines Consortium (2006a))

 

An additional study with same study design was conducted with natural river water. The study is described and compared to the study with reconstituted water as follows in the SIAR CAPB 2006.

“The test with natural river water resulted in a NOEC on reproduction of ≥ 2.98 mg active substance/l and a LOEC greater than 2.98 mg active substance/l (21 d-NOEC ≥ 10.5 mg product/l; 21 d-LOEC > 10.5 mg product/l). In these two studies additional parameters were investigated (e. g. first appearance of juveniles, intrinsic rate of natural increase). In the study with natural river water weak (but statistically significant) effects on the intrinsic rate were observed at all concentrations ≥ 0.32 mg product/L, showing no concentration effect relationship. Only at 10.5 mg/L a significant decrease in intrinsic rate and a significant effect on the first appearance of juveniles was determined. With reconstituted water the effect levels of reproduction rate, intrinsic rate, and first appearance were comparable. The test substance concentration was sufficiently stable at the relevant concentrations. Thus, humic acids and other natural ingredients have only a slight influence on the toxicity of C8-18 AAPB to Daphnia magna. The variation in the effect values described above could not be clarified.”

 

Conclusion

Three of the Daphnia magna reproduction tests were conducted in reconstituted dilution water resulting in 21 d NOEC values of 0.03, 0.32, and 0.932 mg a.i./L. Based on the test results using 2/3 reconstituted dilution water and 1/3 pond water (21 d NOEC=0.9 mg a.i./L), it can be assumed that the presence of pond water has little or no influence, as the results are comparable to those obtained in the newly performed study in which analytical monitoring of the test substance concentration was performed using state-of-the-art equipments and techniques and which seems to be the most reliable study.

As already mentioned above, the very low 21 d NOEC and LOEC of 0.03 and 0.1 mg a.i./L determined in one test could not be confirmed by the other studies and based on the available data this discrepancy could not be resolved. Therefore a weight of evidence approach seems to be justified taking into account the 21 d NOEC values of 0.03, 0.32, 0.9, and 0.932 mg a.i./L. The calculation yielded a 21 d NOEC of 0.3 mg a. i./L as the geometric mean with respect to reproduction.

 

 

Justification for read-across

For details on substance identity and detailed (eco)toxicological profiles, please refer also to the general justification for read-across given at the beginning of the CSR and attached as pdf document to IUCLID section 13.

 

This read-across approach is justified based on structural similarities. All AAPBs contain the same functional groups. Thus a common mode of action can be assumed.

The only deviation within this group of substances is a minor variety in their fatty acid moiety (chain length and degree of unsaturation), which is not expected to have a relevant impact on intrinsic ecotoxicological properties.

 

a. Structural similarity and functional groups

Alkylamidopropyl betaines (AAPBs) are – with the exception of C12 AAPB - UVCB substances (Substances of Unknown or Variable composition, Complex reaction products or Biological materials), which are defined as reaction products of natural fatty acids or oils with dimethylaminopropylamine and further reaction with sodium monochloroacetate. AAPBs are amphoteric surfactants, which are characterized by both acidic and alkaline properties.

 

Their general structure is:

 

R-C(O)-NH-(CH₂)₃-(N(CH₃)₂)⁺-CH₂-C(O)O^-

R = fatty acid moiety

 

The fatty acids have a mixed, slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 may be included. Consequently, the AAPBs differ by their carbon chain length distribution and the degree of unsaturation in the fatty acid moiety. However, Lauramidopropyl betaine (C12 fatty acid derivate) is the major ingredient of all AAPBs covered by this justification as listed in table 1 “Substance identities” of the general justification for read-across.

 

The substances under evaluation share structural similarities with common functional groups (quaternary amines, amide bonds and carboxymethyl groups), and fatty acid chains with differences in chain length and degree of saturation.

 

b. Differences

Differences in ecotoxicity of the AAPBs could potentially arise from the following facts:

-Different amounts of different carbon chain lengths (carbon chain length distribution):

Higher amounts of higher chain lengths and corresponding lower amounts of lower chain length could result in a rising average lipophilicity. However, the main component for all AAPBs is C12 AAPB. Relevant effects on ecotoxicity are not to be expected.

- Different amounts of unsaturated fatty ester moieties:

Effects may be expected for e.g. physical state, but are not considered to be of relevance for ecotoxicity.

 

Comparison of long-term toxicity data to aquatic invertebrates

 

Endpoints	Target substance	Source substance
	C8-18 AAPB	C8-18 and C18 unsatd. AAPB
Long-term toxicity to aquatic invertebrates	WoE.Long-term toxicity to aquatic invertebrates: 97862-59-4_9.1.5_IUA_1995_OECD 202   Weight of evidence   OECD Guideline 211 (Daphnia magna Reproduction Test) (cited as OECD Guideline 202, part 2 (Daphnia sp., Reproduction Test)) Daphnia magna, semi-static, freshwater 21 d NOEC = 0.032 mg/Lact. ingr. (nominal) based on: survival and reproduction rate 21 d LOEC = 0.1 mg/Lact. ingr. (nominal) based on: survival and reproduction rate   Reliability: 1 (reliable without restriction), GLP	WoE.Long-term toxicity to aquatic invertebrates: 61789-40-0_9.1.5_Unilever Research_1990   Weight of evidence   OECD Guideline 211 (Daphnia magna Reproduction Test) (cited as OECD Guideline 202, part 2 (Daphnia sp., Reproduction Test)) Daphnia magna, semi-static, freshwater   21 d NOEC = 0.32 mg/Lact. ingr. (nominal) based on: reproduction 21 d LOEC = 0.56 mg/Lact. ingr. (nominal) based on: reproduction 21 d NOEC = 0.56 mg/Lact. ingr. (nominal) based on: mortality 21 d LOEC = 1 mg/Lact. ingr. (nominal) based on: mortality   Reliability: 2 (reliable with restrictions), GLP
	WoE.Long-term toxicity to aquatic invertebrates: 97862-59-4_9.1.5_THG_1991d_OECD 202   Weight of evidence   OECD Guideline 211 (Daphnia magna Reproduction Test) (cited as OECD Guideline 202, part 2 (Daphnia sp., Reproduction Test)) Daphnia magna, semi-static, freshwater 21 d NOEC = 0.9 mg/Lact. ingr. (nominal) based on: reproduction 21 d LOEC = 3.6 mg/Lact. ingr. (nominal) based on: reproduction   Reliability: 1 (reliable without restriction), GLP
	WoE.Long-term toxicity to aquatic invertebrates: 97862-59-4_9.1.5_CEFIC_CESIO_2006a_OECD 211   OECD Guideline 211 (Daphnia magna Reproduction Test) Daphnia magna, semi-static, freshwater   21 d NOEC = 0.932 mg/Lact. ingr. (nominal) based on: reproduction 21 d LOEC = 2.98 mg/Lact. ingr. (nominal) based on: reproduction   Reliability: 1 (reliable without restriction), GLP

 

Three of the reproduction tests were conducted in reconstituted dilution water resulting in 21 d NOEC values of 0.03, 0.32, and 0.932 mg a.i./L. Based on the test results using 2/3 reconstituted dilution water and 1/3 pond water (21 d NOEC=0.9 mg a. i./L), it can be assumed that the presence of pond water has little or no influence, as the results are comparable to those obtained in the newly performed study in which analytical monitoring of the test substance concentration was performed using state-of-the-art equipments and techniques and which seems to be the most reliable study.

The very low 21 d NOEC and LOEC of 0.03 and 0.1 mg a.i./L determined in one test could not be confirmed by the other studies, and based on the available data this discrepancy could not be resolved. Therefore a weight of evidence approach was applied calculating the geometric mean of the available NOEC. This resulted in a21 d NOEC of 0.3 mg a.i./L with respect to reproduction.

 

Quality of the experimental data of the analogues:

The available data are adequate and sufficiently reliable to justify the read-across approach.

The available studies were conducted according to OECD Guideline 211 and were reliable or reliable with restrictions (RL1-2, GLP).

The test materials used in the respective studies represent the source substance as described in the hypothesis in terms of substance identity and minor constituents.

Overall, the study results are adequate for the purpose of classification and labelling and risk assessment.

 

Conclusion

Based on structural similarities of the target and source substances as presented above and in more detail in the general justification for read across, it can be concluded that the available data from the source substance C8-18 and C18 unsatd. AAPB are also valid for the target substance C8-18 AAPB.

A weight of evidence approach was applied taking into account the 21 d NOEC values of 0.03, 0.32, 0.9, and 0.932 mg a.i./L. The calculation yielded a 21 d NOEC of 0.3 mg a.i./L as the geometric mean with respect to reproduction.