1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-C12-14 acyl derivs., hydroxides, inner salts

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 december 2008 - 30 january 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed according to an international guideline and according to GLP. The validity criteria were fulfilled except the lack of data relating to the analytical monitoring showing that the concentrations of the test item were maintained during the test. However, the test has been performed under semi-static conditions and according to the long-term toxicity to Daphnia, the substance is stable over 48 hours. Thus, we can consider that in a short-term study, the substance is also stable.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Complementary information on test material: aqueous solution at 42% w/w of active content (= surface-active fraction, i.e. alkylamidopropylhydroxysultaine).

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION :
- Method : the test solutions were prepared without using of any organic solvent
- Stock solution : 0.01006 g test item in 1000 ml treated water in order to obtain a nominal concentration of 10.06 mg/L
- Controls : treated seawater without test item
- Evidence of undissolved material (e.g; precipate, surface film, etc) : No

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name:Skeletonema costatun
- Culture number : C5B2A
- Method of cultivation: pre-cultures in the exponential growth phase were prepared from stock laboratory cultures by inoculating treated seawater with nutrient medium (culture medium) to a cell density of approximately 2 x 10E3 to 10E4 cells per millilitre. The pre-cultures were incubated at approximately 20 +/- 2°C under constant illumination for 3 days +/6 1 day, and where uses as the iniculum source for the test.

ACCLIMATION
- Acclimation period: not stated
- Culturing media and conditions: see method of cultivation above
- Any deformed or abnormal cells observed: data not available

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

At 0h : 20.2 - 21.4
At 24h : 19.1 - 21.0
At 48h : 19.9 - 21-3
At 72h : 19.7 - 21.6

pH:

- pH during the test in the control : 8.11 - 9.10
- pH at pre 0h/0h in the culture medium and the test material stocks : 8.14 - 8.16 (see table 2 below)

Dissolved oxygen:

No data

Salinity:

36 (salinity of diluent water at 0h)

Nominal and measured concentrations:

1.0 - 1.8 - 3.2 - 5.6 - 10.06 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: Borosilicate glass conical flasks
- Material, size, headspace, fill volume: 100-mL flasks, filled with 80mL of algal suspension
- Aeration: During the test, the test solutions were continuously stirred by orbital shaker.
- Type of flow-through (e.g. peristaltic or proportional diluter): none (static test)
- Renewal rate of test solution (frequency/flow rate): a static, non-renewal exposure system was used
- Initial cells density: 10000 algal cells per mL of test medium
- Control end cells density:
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS :
Culture medium is prepared from natural seawater supplied by pump from Scapa Flow, Orkney. All seawater was UV sterilised and filtered to 0.2 µm. The filtered treated seawater was enriched with nutrients and vitamins in accordance with ISO Guidelines. The salinity of the enriched natural seawater at àh was 36 °/oo +/- 4°/oo.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: at 0h for the test concentration of 10 mg/L
- Photoperiod: continuously illuminated
- Light intensity and quality: the measured light intensity (7160 - 8900) was achieved by four 40W tubes mounted at a distance of approximatively 40 cm directly above the test area. Light intensity values were measured daily during the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Counts of algal cell numbers were carried out daily by fluorometer measurements. Three readings were performed on each test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not stated
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes
- Test concentrations: 1 - 10 - 100 - 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits = 4.99 - 5.84 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.26 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.76 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Results with reference substance (positive control):

For evaluation of the quality of the aquatic organisms and the experimental conditions, 3,5-dichlorophenol is tested as a positive control.
72h EC50 = 2.19 mg/L (with 95% confidence limits : 2.03 - 2.34 mg/l)
(See results on tables 2 and 5 below)

Reported statistics and error estimates:

The raw data for each duplicate vessel and time period were averaged, to give values for each concentration of celle volume. Growth rate calculated on the basis of these measurements. Daily intrinsic growth rate was calculated for each duplicate for each time period using an exponential model :
Nt = N0.ekt where :
Nt : volume or number at time t
N0 = volume or number at beginning of test
t = time in days
k = growth rate (d -1)

The average value of k for each time interval was calculated for each concentration. Since the criterion of effect was the concentration causing 50% reduction in growth rate with respect to the controls, the response for each concentration was estimated from . Effect = 1- (control k/treatment k).

The resulting values represent proportional reduction in growth rate. The EC50 for each time interval and the 72h EC90 and NOEC values were calculating using an appropriate statistical method from the ToxCalc Version 5 software.

Table 4 - Fluorescence measurements (ppb) in test vessels after 24, 48 and 72h

Concentration (mg/L)	24h	48h	72h
1.00a	2.195	25.606	48.091
1.00a	2.170	25.802	48.660
1.00a	2.265	25.702	49.169
1.00b	3.307	24.599	47.314
1.00b	2.309	24.662	47.756
1.00b	2.256	24.586	48.137
1.80a	1.697	20.980	40.686
1.80a	1.651	20.832	41.037
1.80a	1.647	21.025	41.390
1.80b	2.090	23.245	48.156
1.80b	2.133	23.268	48.455
1.80b	2.102	23.333	48.818
3.20a	0.623	6.235	32.901
3.20a	0.603	6.265	32.724
3.20a	0.607	6.255	32.876
3.20b	0.536	7.087	34.907
3.20b	0.573	6.967	35.041
3.20b	0.578	6.961	35.008
5.60a	0.140	0.313	3.955
5.60a	0.132	-	3.911
5.60a	0.127	-	3.821
5.60b	0.145	0.530	4.617
5.60b	0.148	-	4.647
5.60b	0.130	-	4.630
10.0a	0.003	0.000	0.129
10.0a	0.002	-	-
10.0a	0.008	-	-
10.0b	0.009	-0.004	0.108
10.0b	0.013	-	-
10.0b	0.002	-	-

Table 5 - Fluorescence measurements (ppb) in control and referencesubstance (3,5 -DCP) after 72h

Concentrations (mg/L)	72h			Rep growth	Mean growth
	Measurement 1	Measurement 2	Measurement 3
Control 1	47.406	47.489	44.259	1.00	1.00
Control 2	44.461	44.858	45.081	1.03
Control 3	55.560	55.454	55.601	0.98
Control 4	49.561	49.809	49.812	0.99
1.0a	42.755	50.684	51.023	1.00	0.99
1.0b	46.574	46.256	46.355	0.99
1.8a	13.181	13.169	13.138	0.71	0.69
1.8b	11.148	11.116	11.037	0.67
3.2a	0.608	0.585	0.566	0.02	0.01
3.2b	0.473	0.479	0.478	0.00

 

Table - Coefficient of variation at 24, 48 and 72h (%)

24 h control CV %	7.59
48 h control CV %	1.53
72 h control CV %	2.10

Table - Summary of compliance guideline criteria and test values recorded

Validity criteria fulfilled:

yes

Remarks:

See "executive summary" and remarks "Rationale for reliability incl. deficienties"

Executive summary:

The influence of the test item on the growth of a marine unicellular algae Skeletonema costatum was investigated in a 72 hour static test according to ISO 10253 (2006).

The nominal test item concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were tested in parallel to a control and a positive substance (3,5 DCP)

The 72-hour EC50 of the test item was determinated to be 5.38 mg test item/L (2.26 mg active ingredient/L)

The 72-hour NOEC (highest concentration tested without toxic effects after the test period of 72 hours) of the test item was determined to be 1.80 mg test item/L (0.76 mg active ingredient/L) .

The 72-hour EC50 of the positive control was determinated to be 2.19 mg/L (with 95% confidence limits 2.03 - 2.34 mg/L).

According to available data, the validity criteria were fulfilled except the lack of data relating to the analytical monitoring showing that the concentrations of the test item were maintained during the test. However, the test has been performed under semi-static conditions and according to the long-term toxicity to Daphnia, the substance is stable during 48 hours. Thus, we can considered that in a short-term study, the substance is also stable.

Validity criteria fulfilled :

- Reference toxicant 72h EC50 was 2.0 -4.0 mg/L

- the control cell density had increased by a factor of more than 16 in 72h. this increase corresponds to a specific growth rate of 0.9 d-1

- the variation coefficient of the control specific growth rates didn't exceed 7%

- the control pH haven't increased by more than 1.0 during the test

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source substance, C8-C18 AAPHS, has the same functional groups and general composition as the target C12-14 substance. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3).
Target chemical = C12-14 cocamidopropyl hydroxysultaine (EC 293-878-1).

3. ANALOGUE APPROACH JUSTIFICATION
Cf. attaced Read-Across Justification Document (§13 Assessment reports).

4. DATA MATRIX
Cf. attached Read-Across Justification Document (§13 Assessment reports).

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

Aqueous solution at 42% w/w of active content (= surface-active fraction, i.e. alkylamidopropylhydroxysultaine) is being used as a read across for 1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-C12-14 acyl derivs., hydroxides, inner salts

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits = 4.99 - 5.84 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.26 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.76 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Reported statistics and error estimates:

The raw data for each duplicate vessel and time period were averaged, to give values for each concentration of celle volume. Growth rate calculated on the basis of these measurements. Daily intrinsic growth rate was calculated for each duplicate for each time period using an exponential model :
Nt = N0.ekt where :
Nt : volume or number at time t
N0 = volume or number at beginning of test
t = time in days
k = growth rate (d -1)

The average value of k for each time interval was calculated for each concentration. Since the criterion of effect was the concentration causing 50% reduction in growth rate with respect to the controls, the response for each concentration was estimated from . Effect = 1- (control k/treatment k).

The resulting values represent proportional reduction in growth rate. The EC50 for each time interval and the 72h EC90 and NOEC values were calculating using an appropriate statistical method from the ToxCalc Version 5 software.

Table 4 - Fluorescence measurements (ppb) in test vessels after 24, 48 and 72h

Concentration (mg/L)	24h	48h	72h
1.00a	2.195	25.606	48.091
1.00a	2.170	25.802	48.660
1.00a	2.265	25.702	49.169
1.00b	3.307	24.599	47.314
1.00b	2.309	24.662	47.756
1.00b	2.256	24.586	48.137
1.80a	1.697	20.980	40.686
1.80a	1.651	20.832	41.037
1.80a	1.647	21.025	41.390
1.80b	2.090	23.245	48.156
1.80b	2.133	23.268	48.455
1.80b	2.102	23.333	48.818
3.20a	0.623	6.235	32.901
3.20a	0.603	6.265	32.724
3.20a	0.607	6.255	32.876
3.20b	0.536	7.087	34.907
3.20b	0.573	6.967	35.041
3.20b	0.578	6.961	35.008
5.60a	0.140	0.313	3.955
5.60a	0.132	-	3.911
5.60a	0.127	-	3.821
5.60b	0.145	0.530	4.617
5.60b	0.148	-	4.647
5.60b	0.130	-	4.630
10.0a	0.003	0.000	0.129
10.0a	0.002	-	-
10.0a	0.008	-	-
10.0b	0.009	-0.004	0.108
10.0b	0.013	-	-
10.0b	0.002	-	-

Table 5 - Fluorescence measurements (ppb) in control and referencesubstance (3,5 -DCP) after 72h

Concentrations (mg/L)	72h			Rep growth	Mean growth
	Measurement 1	Measurement 2	Measurement 3
Control 1	47.406	47.489	44.259	1.00	1.00
Control 2	44.461	44.858	45.081	1.03
Control 3	55.560	55.454	55.601	0.98
Control 4	49.561	49.809	49.812	0.99
1.0a	42.755	50.684	51.023	1.00	0.99
1.0b	46.574	46.256	46.355	0.99
1.8a	13.181	13.169	13.138	0.71	0.69
1.8b	11.148	11.116	11.037	0.67
3.2a	0.608	0.585	0.566	0.02	0.01
3.2b	0.473	0.479	0.478	0.00

 

Table - Coefficient of variation at 24, 48 and 72h (%)

24 h control CV %	7.59
48 h control CV %	1.53
72 h control CV %	2.10

Table - Summary of compliance guideline criteria and test values recorded

Validity criteria fulfilled:

yes

Executive summary:

The influence of C12 -14 cocamidopropyl hydroxysultaine (C12 -14 AAPHS), on the growth of a marine unicellular algae Skeletonema costatum was estimated based on read-across from a study testing C8-18 cocamidopropyl hydroxysultaine (C8-18 AAPHS). Given that C12 -14 AAPHS and C8 -18 AAPHS share the same functional groups and general composition, the main variable being alkyl chain distribution, C8-18 AAPHS was considered appropriate for the purposes of read-across to C12 -14 AAPHS.

The influence of the test item on the growth of a marine unicellular algae Skeletonema costatum was investigated in a 72 hour static test according to ISO 10253 (2006). The nominal test item concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were tested in parallel to a control and a positive substance (3,5 DCP).

According to available data, the validity criteria were fulfilled except the lack of data relating to the analytical monitoring showing that the concentrations of the test item were maintained during the test. However, the test has been performed under semi-static conditions and according to the long-term toxicity to Daphnia, the substance is stable during 48 hours. Thus, we can consider that in a short-term study, the substance is also stable.

Validity criteria were fulfilled :

- Reference toxicant 72h EC50 was 2.0 -4.0 mg/L

- the control cell density had increased by a factor of more than 16 in 72h. this increase corresponds to a specific growth rate of 0.9 d-1

- the variation coefficient of the control specific growth rates didn't exceed 7%

- the control pH haven't increased by more than 1.0 during the test

The 72-hour EC50 of C8 -18 AAPHS was determined to be 5.38 mg test item/L (2.26 mg C8-18 AAPHS /L)

The 72-hour NOEC (highest concentration tested without toxic effects after the test period of 72 hours) of the test item was determined to be 1.80 mg test item/L (0.76 mg C8-18 AAPHS /L).

The 72-hour EC50 of the positive control was determined to be 2.19 mg/L (with 95% confidence limits 2.03 - 2.34 mg/L).

C8-18 AAPHS is considered appropriate for read-across purposes to C12 -14 AAPHS.

Description of key information

The 72-hour EC50 of the test item (C8 -18 cocamidopropyl hydroxysultaine) was determined to be 5.38 mg test item/L (equivalent to 2.26 mg C8 -18 AAPHS /L) and the 72 -hour NOEC was determined to be

1.80 mg test item/L (equivalent to 0.76 mg C8 -18 AAPHS /L), and are considered appropriate for read-across purposes to C12 -14 AAPHS.

Key value for chemical safety assessment

EC50 for marine water algae:

2.26 mg/L

EC10 or NOEC for marine water algae:

0.76 mg/L