Oxoaluminium, C16-C18-alkyl esters

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:

24 January 2013 to 25 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

See test material information

Analytical monitoring:

yes

Details on sampling:

- Total organic carbon and aluminium analysis: Samples were collected at 0 and 72 hours from the uninoculated and pooled treatment replicates and stored at -20°C until analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

- Test water: Prepared from reverse osmosis purified water, pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl and water hardness adjusted to 150 mg/L as CaCO3.
- Preparation: 250 mg aluminum, benzoate C16-18-fatty acids complexes was added to the surface of 2.5 L of culture medium, to give a loading rate of 100 mg/L, and stirred using a magnetic stirrer at a rate that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal and the WAFs removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the WAF.
- Controls: Test control: test medium only. Positive control: potassium dichromate at concentrations; 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
- Evidence of undissolved material: Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Source: Liquid cultures (strain CCAP 278/4) were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Scottish Marine Institute, Oban, Argylll, Scotland and maintained in the laboratory under constant aeration and illumination at 21°C.
- Method of cultivation: Prior to the start of the test, approximately 100 mL of culture media, with a cell density of approximately 10+03 cells/mL, were added to conical flasks which were plugged with polyurethane foam stoppers. The flasks were kept under constant agitation by orbital shaker at 100-150 rpm and constant illumination at 24 ± 1°C until algal cell density was approximately 10E+04 to 10E+05 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

At the request of the sponsor, the water hardness was changed from that of the culture medium of 15 mg/L to approximately 150 mg/L CaCO3.

Test temperature:

The temperature was maintained at 24 ± 1°C throughout the test.

pH:

The pH of the control cultures was 7.6 at 0 hours and 7.8 at 72 hours. For the treatment group the pH was 7.7 at 0 hours and 7.8 at 72 hours.

Dissolved oxygen:

No data reported

Salinity:

Not applicable

Nominal and measured concentrations:

- Preliminary test: The loading rates to be used in the definitive test were determined on the basis of a range finding study at 10 and 100 mg/L nominal loading rates showing no effects.
- Nominal concentration: The test was conducted as a limit test with a single nominal loading rate of 100 mg/L (WAF).
- Measured aluminium concentrations: The aluminium concentration was 0.13 mg/L at both 0 and 72 hours.
- Measured total organic carbon: Total Organic Carbon (TOC) analysis of the test preparation showed measured concentrations of less than the limit of quantitation (LOQ) (assessed to be 1.0 mg C/L for the method employed) at 0 hours and less than the control value at 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass conical flask.
- Type: Flask plugged with polyurethane foam bungs.
- Material, size, headspace, fill volume: 250 mL glass concical flask containing 100 mL of test preparation.
- Aeration: Not reported
- Initial cells density: 5.45 x 10E+03 cells/mL.
- Control end cells density: 4.94 x 10E+05
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared from reverse osmosis purified water, pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl and water hardness adjusted to 150 mg/L as CaCO3.
- Total organic carbon: < 1.0 mg C/L (- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the control and 100 mg/L loading rate WAF was determined at initiation of the test and after 72 hours exposure. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: No reported
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod: Continuous illumination
- Light intensity and quality: Approximately 7000 lux provided by warm white lighting (380 - 730 nm)
- Other conditions: Flask constantly shaken at approximately 150 rpm.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and cell density determined using a Coulter Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test at 100 mg/L (WAF).
- Range finding study: Yes
- Range finding test concentrations: 10 and 100 mg test item/L.
- Range findgin results used to determine the conditions for the definitive study: No effect seen during range finding study, therefore definitive test carried out as limit test at 100 mg/L (WAF).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accommodated Fraction

Basis for effect:

other: Growth rate and yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accommodated Fraction

Basis for effect:

other: Growth rate and yield

Details on results:

The growth rate and yield of the algae were not affected (P ≥ 0.05) by the presence aluminum, benzoate C16-18-fatty acids complexes over the 72 hour exposure period. Microscopic inspection at 72 hours showed no abnormalities in the test and control cultures. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L. Given that the toxicity cannot be attributed to a single component or a mixture of components but to aluminum, benzoate C16-18-fatty acids complexes as a whole, the results were based on nominal loading rates only.

Results with reference substance (positive control):

- Reference: Harlan study number 41205049 used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L under similar exposure conditions and data evaluation to the definitive test.
- Results: The 72 hour ErC50 was 1.1 mg/L with 95 % confidence limits of 1.0 to 1.3 mg/L and the EyC50 was 0.70 mg/L. It was not possible to calculate 95% confidence limits for the EyC50 value as the data generated did not fit the models available for the calculation of confidence limits. The 72 hour NOEC was 0.50 mg/L based on growth rate and 0.25 mg/L based on yield. The 72 hour LOEC was 1.0 mg/L based on growth rate and 0.50 mg/L based on yield. The results were within normal ranges for this reference item.

Reported statistics and error estimates:

A student's t-test incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure were carried out on the growth rate and yield data after 72 hours for the control and treatment group to determine any statistically signficiant differences.

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)			Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h		% Inhibition
Control	R1	0.063	-	4.75E+05		-
	R2	0.064		4.88E+05
	R3	0.066		5.57E+05
	R4	0.065		5.27E+05
	R5	0.064		4.80E+05
	R6	0.061		4.10E+05
	Mean	0.064		4.89E+05
	SD	0.002		5.03E+05
100	R1	0.065	[2]	5.51E+05
	R2	0.066	[3]	5.56E+05
	R3	0.066	[3]	5.79E+05
	R4	0.064	0	4.96E+05
	R5	0.063	2	4.74E+05
	R6	0.064	0	4.94E+05
	Mean	0.065	[1]	5.25E+05		[7]
	SD	0.001		4.22E+05

* In accordance with the OECD test guideline only the mean value for yield is calculated

R1 – R6 = Replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

Validity criteria fulfilled:

yes

Remarks:

All validity criteria met.

Conclusions:

The toxicity of aluminum, benzoate C16-18-fatty acids complexes to algal growth showed no effects at a water accommodated fraction nominal loading rate of 100 mg/L. Therefore, the EL50 is determined to be greater than 100 mg/L loading rate (WAF) and the NOELR is 100 mg/L loading rate (WAF).

Executive summary:

The toxicity of aluminum, benzoate C16-18-fatty acids complexes to algal growth showed no effects at a water accommodated fraction nominal loading rate of 100 mg/L. Therefore, the EL50 is determined to be greater than 100 mg/L loading rate (WAF) and the NOELR is 100 mg/L loading rate (WAF). The toxicity of aluminum, benzoate C16-18-fatty acids complexes to algae (Pseudokirchneriella subcapitata) was determined in a GLP-compliant, static growth inhibition study following OECD guideline 201 (Harlan 2013). Algae were exposed for 72 hours to a control and a single concentration of 100 mg/L loading rate WAF in media adjusted to a hardness of approximately 150 mg CaCO3/L. Observations of algal biomass were made at 24, 48 and 72 hours and the test solutions analysed for aluminium content and total organic carbon at test initiation and termination. The study is considered reliable and relevant for use for this endpoint.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

In accordance with the Regulation (EC) No 1907/2006, Annex XI, section 1.5, read-across to aluminum, benzoate C16-18-fatty acids complexes has been used to fulfil REACH information requirements where appropriate and is justified by the chemical structures and common physiological active moieties of the substances. The chemical structures of the target and read-across substances are very closely aligned. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances. The key difference is that read-across substance contains a benzoate moiety linked to the aluminium cation, which is absent from the target substance. Benzoic acid and benzoates have been well characterized (eco)toxicologically, but in this case generating experimental data on the aluminium salt containing benzoate would be expected to demonstrate a ‘worst case’ hazard profile when compared to the target substance. Since no intrinsic toxicity could be demonstrated from any of the Annex VII or VIII endpoints with the benzoate-containing aluminium salt, then these results can be read across to the target substance without restriction.

The substances consist of aluminium salts of fatty acids, with the source substance having additionally a benzoate moiety linked to the aluminium cation as well. As such, both substances have common functional groups and as the fatty acid moieties are considered not to be hazardous, since they are an endogenous part of living cells and are a dietary requirement for many organisms, the toxicity of the substances will be driven by the presence of the aluminium species (and additionally the benzoate for the source substance). The substances have common starting materials and methods of manufacture, principally the reaction of aluminium with organic acids, with the target substance often being used as a pre-cursor reacted with benzoic acid in the manufacture of the source substance. The substances have common breakdown products and, in the environment, the substances will dissociate and biodegrade into inorganic aluminium species and fatty acids then carbon dioxide and water.

2. CHEMICAL(S)

Source chemical: Aluminum, benzoate, C16-18 fatty acids complexes (EC: 303-385-6, CAS: 94166-87-7)

See robust study summaries for further details on the identity of the tested substances and IUCLID dataset for further information on the substance identity and the data to support the read across justification.

3. ANALOGUE APPROACH JUSTIFICATION
The literature search identified no ecotoxicological data for the substance and no experimental ecotoxicity data are available for the substance. The hazard testing was conducted on the substance prepared as a 50% w.w. concentration in pharmaceutical white oil. The presence of the oil restricts the solubility of the substance and reduces its bioavailability to the aquatic environment therefore, no testing was carried out on the substance as no meaningful results would be achieved. Instead, in order to provide a worst-case scenario for the aquatic ecotoxicity potential for the substance, data have been read across from a structural analogue which was tested in in isolated form (i.e. extracted from base oil).

No novel aquatic ecotoxicity testing on the substance was carried out but novel proprietary data were read across from aluminum, benzoate C16-18-fatty acids complexes. This substance is considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. The chemical structures of the target and read-across substances are very closely aligned; both substances consist of aluminium salts of fatty acids. The aluminium cation, a long chain fatty acid, and the –Al=O (-AlOH in aqueous solution) moieties are identical in both substances.

The fatty acids present in both substances are the same, consisting of a mixture of C16 and C18 chain lengths at approximately a 1:2 ratio. The C16 and C18 fatty acid moieties are derived from natural fatty materials, or substances which are chemically indistinguishable from natural fatty acids. The fatty acid moieties are considered not to be hazardous to humans as they are natural constituents of the human body and essential components of a balanced human nutrition. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances.

Fatty acids are an endogenous part of living cells and are an essential dietary requirement for many organisms. They are absorbed, digested and transported in animals and humans. When taken up by tissues they can either be stored as triglycerides or can be oxidised via the ß-oxidation and tricarboxylic acid pathways. The ß-oxidation uses a mitochondrial enzyme complex for a series of oxidation and hydration reactions, resulting in a cleavage of acetate groups as acetyl CoA. Acetyl CoA is used mainly to provide energy but also to provide precursors for numerous biochemical reactions. Alternative minor oxidation pathways can be found in the liver and kidney (ω-oxidation and ω-1 oxidation) and in peroxisomes for ß-methyl branched fatty acids (α-oxidation). The metabolic products can then be incorporated for example into membrane phospholipids.

The read across substance also contains a benzoate moiety linked to the aluminium cation, which is absent from the target substance. Although it also contains a coordinated benzoate ion, under environmental relevant conditions the benzoate ion has a LC or EC50s of > 100 mg/L for fish (OECD SIDS 2001) and therefore does not contribute any additional toxicity to the substance. Benzoic acid and benzoates have been well characterized (eco)toxicologically, but in this case generating experimental data on the aluminium salt containing benzoate would be expected to demonstrate a ‘worst case’ hazard profile when compared to the target substance as this substance is tested in isolated form (i.e. outside of the base oil). Since no intrinsic toxicity could be demonstrated from any of the Annex VII or VIII endpoints with the benzoate-containing aluminium salt, then these results can be read across to the target substance without restriction.

Comparison of the data for the two substances indicates that they are expected to have similar properties. Neither the target or read-across substance meets the criteria for classification for physico-chemical, environmental or human health endpoints, based on the available data.

On the basis of the physico-chemical results, the substances are not flammable and have similar densities. The low vapour pressure results indicates that hazards associated with the atmospheric compartment or inhalation routes of toxicity are not expected to be relevant. The substances show similar water solubility, without surface active properties, indicating that they are likely to have similar behaviour in the aquatic environment.

Although the read-across substance met the criteria for ready biodegradability and the target substance did not, neither substance was inhibitory to micro-organisms at the concentration tested. The difference in biodegradation results is expected to derive from the presence of the base oil in the target substance sample, which is designed to minimise leaching of the grease thickener, and therefore less of the grease thickener would have been available for degradation by the micro-organisms.

There are no results available for the ecotoxicity of the target substance and therefore comparison of the effect concentrations against the read-across substance is not possible. However, leaching studies on grease thickeners in base oils have been used to assess the potential bioavailability of the grease components. The bioavailability potential of the water accommodated fractions (WAFs) of metal (lithium and calcium) soap complex based grease thickeners was assessed using a solid-phase micro-extraction (SPME) method combined with gas chromatography (GC). This approach was complemented with metal ion analysis to determine whether the metal leaches out of the base grease during WAF preparation and the ecotoxicity of WAFs was also monitored using an in vitro Microtox assay. The SPME-GC data confirmed that there was negligible leaching of the thickeners from base oils in the samples tested, with measurements for calcium and lithium below the limit of detection (<0.1 mg/L) and the screening ecotoxicity data also showed a lack of toxicity of the greases.

The results of the bioavailability potential of the WAFs, the metal ion analysis and the screening ecotoxicity of lithium and calcium based complexes have been read across to aluminium based thickeners. All of these metal salts of fatty acids are expected to behave in a very similar manner when entrained within a grease matrix, with high temperature stability indicating that the thickener structure is robust and resistant to diffusion out of the oil. Dissolution of grease thickeners from grease into water is very unlikely as the thickeners are poorly water soluble and the thickeners are embedded in the hydrophobic grease matrix and thus unlikely to leach out. Therefore, although there are no data on the ecotoxicity of the target substance, no effects are expected based on the lack of bioavailability of the thickener.

These data on the potential for leaching of other metal salt complex based grease thickeners have been read across to both the target and read across substances. On the basis of these results, it is expected that neither the target nor the read across substance would leach from the base oil in which they are typically marketed and therefore neither substance would be bioavailable. Thus, reading across data from the source substance tested in its isolated form is considered robust as it provides a worst-case conclusion for the target substance which is only manufactured in an inert carrier, typically base oil. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies on the target and read-across substances themselves. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.

The available mammalian toxicity data show that neither the target nor read-across substance would be classified as irritating to skin or eyes and would not be classified for acute oral toxicity, with LD50 values of >2000 mg/kg. Although no other data are available for comparison of the potential mammalian toxicity of the two substances, the target and read-across substances are expected to behave in a very similar manner. As grease thickeners are entrained within grease matrices which are robust and resistant to diffusion out of the oil, neither substance is expected to be bioavailable. In order to provide further evidence for the lack of bioavailability, it is proposed to undertake leaching studies in fed state simulated intestinal fluid (FeSSIF) on the target and read-across substances. Dependent on the results, the two studies would then be used to show the similarity in the bioavailability of the two substances and provide further weight of evidence for the read-across approach.

For the ecotoxicity of the substances, read across from the source to the target substance is considered justified as both substances have very low solubility in water, would not leach when in situ in base oil during use as grease thickeners and are not expected to be bioavailable. The substances, in the environment, would dissociate and degrade into inorganic aluminium species and firstly, fatty acids (plus benzoic acid for the source substance), then ultimately carbon dioxide and water. As the fatty acid components are biodegradable and are not expected to be hazardous (and the benzoate component of the source substance is not expected to be hazardous), the toxicity is expected to be driven by the aluminium component, so would be the same in both the source and target substances. As such, read across from the source substance, tested in its isolated form, is considered to provide a worst-case scenario for the target substance, which would have even more limited bioavailability as it is manufactured in situ in an inert carrier, typically base oil.

4. DATA

T = target substance (tests were undertaken on a sample prepared as a 50% w.w. concentration in medicinal white oil unless otherwise indicated)
RA = read-across substance

- State: Liquid (T), Solid (RA)
- Melting point: 21°C (T), 224°C (RA)
- Relative density: 0.933 (T), 1.08 (RA)
- Vapour pressure: 0.00015 Pa (T), 0.000044 Pa (RA)
- Surface tension: 72.5 mN/m (T), 72.6 mN/m (RA)
- Water solubility: ≤0.00015 g/L (T), ≤0.00026 g/L (RA)
- Flash-point: 159°C (T), No data available for RA
- Flammability: No data available for T, Not flammable (RA)
- Self-ignition temperature: 374°C (T), 383°C (RA)
- Viscosity: 174.3 mm2/s at 100°C (T), No data available for RA
- Biodegradation: Not readily biodegradable (31%) (T), Readily biodegradable (79%) (RA)
- Acute aquatic invertebrates: No data available for T, EL50 (48 h): > 100 mg/L (RA)
- Algae: No data available for T, EL50 (72 h): > 100 mg/L and NOELR (72 h): 100 mg/L (RA)
- Aquatic microorganisms: NOEC (28 d): 6.7 mg/L (T), NOEC (28 d): 15.4 mg/L (RA)
- Acute fish: No data available for T, LL50 (96 h): > 100 mg/L (RA)
- Skin irritation: Not irritating (T), Not irritating (RA)
- Eye irritation: Not classified (T). Not classified (RA)
- Skin sensitisation: No data available for T, Not sensitising (RA)
- In vitro gene mutation in bacteria: No data available for T, Negative (RA)
- Acute toxicity, oral route: LD50: > 2000 mg/kg (T, test undertaken on solid (isolated) form of the substance), LD50 >2000 mg/kg (RA)
- Acute toxicity, dermal route: No data available for T, LD50 >2000 mg/kg (RA)
- In vitro cytogenicity: No data available for T, Negative (RA)
- In vitro gene mutation in mammalian cells: No data available for T, Negative (RA)
- Short-term repeated dose toxicity, oral route: No data available for T, NOAEL: > 225 mg/kg (RA)
- Reproductive toxicity: No data available for T, NOAEL (P): > 225 mg/kg (RA)
- Developmental toxicity: No data available for T, NOAEL (F1): > 225 mg/kg (RA)

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accommodated Fraction

Basis for effect:

other: Growth rate and yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accommodated Fraction

Basis for effect:

other: Growth rate and yield

Details on results:

The growth rate and yield of the algae were not affected (P ≥ 0.05) by the presence aluminum, benzoate C16-18-fatty acids complexes over the 72 hour exposure period. Microscopic inspection at 72 hours showed no abnormalities in the test and control cultures. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L. Given that the toxicity cannot be attributed to a single component or a mixture of components but to aluminum, benzoate C16-18-fatty acids complexes as a whole, the results were based on nominal loading rates only.

Results with reference substance (positive control):

- Reference: Harlan study number 41205049 used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L under similar exposure conditions and data evaluation to the definitive test.
- Results: The 72 hour ErC50 was 1.1 mg/L with 95 % confidence limits of 1.0 to 1.3 mg/L and the EyC50 was 0.70 mg/L. It was not possible to calculate 95% confidence limits for the EyC50 value as the data generated did not fit the models available for the calculation of confidence limits. The 72 hour NOEC was 0.50 mg/L based on growth rate and 0.25 mg/L based on yield. The 72 hour LOEC was 1.0 mg/L based on growth rate and 0.50 mg/L based on yield. The results were within normal ranges for this reference item.

Reported statistics and error estimates:

A student's t-test incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure were carried out on the growth rate and yield data after 72 hours for the control and treatment group to determine any statistically signficiant differences.

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)			Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h		% Inhibition
Control	R1	0.063	-	4.75E+05		-
	R2	0.064		4.88E+05
	R3	0.066		5.57E+05
	R4	0.065		5.27E+05
	R5	0.064		4.80E+05
	R6	0.061		4.10E+05
	Mean	0.064		4.89E+05
	SD	0.002		5.03E+05
100	R1	0.065	[2]	5.51E+05
	R2	0.066	[3]	5.56E+05
	R3	0.066	[3]	5.79E+05
	R4	0.064	0	4.96E+05
	R5	0.063	2	4.74E+05
	R6	0.064	0	4.94E+05
	Mean	0.065	[1]	5.25E+05		[7]
	SD	0.001		4.22E+05

* In accordance with the OECD test guideline only the mean value for yield is calculated

R1 – R6 = Replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

Validity criteria fulfilled:

yes

Remarks:

All validity criteria met.

Conclusions:

The toxicity of aluminum, benzoate C16-18-fatty acids complexes to algal growth showed no effects at a water accommodated fraction nominal loading rate of 100 mg/L. Therefore, the EL50 is determined to be greater than 100 mg/L loading rate (WAF) and the NOELR is 100 mg/L loading rate (WAF).

Executive summary:

The hazard testing was conducted on the substance as a 50% w.w. concentration in pharmaceutical white oil.The presence of the oil restricts the solubility of the substance and reduces its bioavailabilty to the aquatic environment therefore, no testing was carried out on the substanceas no meaningful results would be achieved.Instead proprietary data are read across from aluminum, benzoate C16-18-fatty acids complexes. This substance is considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. Although it also contains a coordinated benzoate ion, under environmental relevant conditions the benzoate ion has a 72 hour ErC50 of > 100 mg/L for algal growth (OECD SIDS 2001) and therefore does not contribute any additional toxicity to the substance.Aluminum, benzoate C16-18-fatty acids complexes was tested in the form of an isolated solid and showed no toxic effects at a water accommodated fraction loading rate of 100 mg/L (Harlan 2013).Therefore, the 72 hour ErL50 is determined to be > 100 mg/L (WAF) and the 72 hour NOErLR is 100 mg/L (WAF).

The toxicity of aluminum, benzoate C16-18-fatty acids complexes to algal growth showed no effects at a water accommodated fraction nominal loading rate of 100 mg/L. Therefore, the EL50 is determined to be greater than 100 mg/L loading rate (WAF) and the NOELR is 100 mg/L loading rate (WAF). The toxicity of aluminum, benzoate C16-18-fatty acids complexes to algae (Pseudokirchneriella subcapitata) was determined in a GLP-compliant, static growth inhibition study following OECD guideline 201 (Harlan 2013). Algae were exposed for 72 hours to a control and a single concentration of 100 mg/L loading rate WAF in media adjusted to a hardness of approximately 150 mg CaCO3/L. Observations of algal biomass were made at 24, 48 and 72 hours and the test solutions analysed for aluminium content and total organic carbon at test initiation and termination. The study is considered reliable and relevant for use for this endpoint.

Description of key information

Based on read across, the acute toxicity to algae is expected to show no effects at a water accommodated fraction nominal loading rate of 100 mg/L. Therefore, the 72 hour ErL50 is determined to be > 100 mg/L (WAF) and the 72 hour NOErLR is 100 mg/L (WAF).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The hazard testing was conducted on the substance prepared as a 50% w. w. concentration in pharmaceutical white oil. The presence of the oil restricts the solubility of the substance and reduces its bioavailability to the aquatic environment therefore, no testing was carried out on the substance as no meaningful results would be achieved. Instead proprietary data are read across from aluminum, benzoate C16-18-fatty acids complexes. This substance is considered suitable for read-across as it contains a fatty acid moiety coordinated to an aluminium atom. Although it also contains a coordinated benzoate ion, under environmental relevant conditions the benzoate ion has a 72 hour ErC50 of > 100 mg/L for algal growth (OECD SIDS 2001) and therefore does not contribute any additional toxicity to the substance. Aluminum, benzoate C16-18-fatty acids complexes was tested in the form of an isolated solid and showed no toxic effects at a water accommodated fraction loading rate of 100 mg/L (Harlan 2013). Therefore, the 72 hour ErL50 is determined to be > 100 mg/L (WAF) and the 72 hour NOErLR is 100 mg/L (WAF).

The toxicity to algal growth was determined in GLP-compliant, static, freshwater test (Harlan 2013) following OECD guideline 201. As no effects were observed in the preliminary range finding test, the definitive toxicity to algal growth was conducted as a limit test at a nominal loading rate of 100 mg/L alongside a blank control. Algae (Pseudokirchneriella subcapitata) were exposed to water accommodated fraction (WAF) of aluminum, benzoate C16-18-fatty acids for 72 hours in standard ecotoxicity media adjusted to a hardness of approximately 150 mg/L CaCO₃. Observations of algal biomass were made at 24, 48 and 72 hours and the test solutions analysed for aluminium content and total organic carbon at test initiation and termination. As no inhibition to algal growth was observed, the 72 hour ErL50 is determined to be > 100 mg/L (WAF) and the 72 hour NOErLR is 100 mg/L (WAF).

The organic moieties of the substance, and the structural analogue Aluminum, benzoate C16-18-fatty acids complexes, are namely stearic acid, palmitic acid, benzoic acid and isopropyl alcohol (2-propanol). The organic moieties are known to have low toxicity to aquatic organisms and are not classified for the environment. Therefore, it is assumed that any potential for long-term ecotoxicity would be due to the Aluminium (Al) component of thesubstances.

Long term ecotoxicity data for Al are presented in IUCLID for algae (Raphidocelis subcapitata formally Pseudokirchneriella subcapitata), invertebrates (Ceriodaphnia dubia and Daphnia magna) and fish (Pimephales promelas). The toxicity of Al varies to aquatic organisms due to changes in environmental parameters such as pH, hardness and dissolved organic carbon (DOC). The data reported in IUCLID covers effects on standard test organisms taking into consideration changes in the relevant environmental parameters. The data covers a circumneutral pH range of 6-8 which would cover most natural surface waters in Europe. The data have been reported based on total Al concentration because the aquatic toxicity results should cover both the dissolved phase and also the presence of Al solids which can cause adverse physical effects (Gensemer et al. 2017).

The available data show that, under reasonable, normal environmental conditions, Al will not cause a concern for chronic toxicity at concentrations below 74.4 μg/L. The ERGTC have generated data to show that the substance has a low water solubility of ≤ 12.5 µg/L at 20°C based on total Al concentration and, therefore, is unlikely to occur in water at concentrations high enough to cause a toxicity concern.

Furthermore, the substance is not used in isolated form but as a grease thickener within base oil. The ERGTC have conducted leaching studies on the substance in deionised water which show that, when present within a grease base, the grease thickeners are not bioaccessible. The leaching studies were conducted using Water Accommodated Fractions (WAFs) at a loading rate of base grease (thickener in a base oil to form a grease matrix) of 1000 mg/L, with the substance being present at 50% in medicinal white oil. In the leaching studies, no Al was detected in water at the analytical method (ICP-MS) limit of detection (LOD) of 5 µg/L. On the basis of the information presented, the substance would not be expected to cause a concern for chronic toxicity to aquatic organisms. Therefore, experimental testing to satisfy REACH data requirements for these endpoints are waived.

This substance has been registered by a Member of the European REACH Grease Thickeners Consortium (ERGTC). A number of decisions have been made in the dossier with regard to the approach taken for registering the substance including the testing strategy and the justification for waiving certain endpoints. Several of the decisions reflect the technical difficulties of testing the substance and the relevance of data with regard to the potential for exposure, given that the substance typically occurs in situ in base oil. A face to face meeting between the ERGTC and ECHA was held in Helsinki on 8th September 2016 which discussed many of these topics and a copy of the minutes from the meeting are attached to the dossier (See section 13 of IUCLID). Therefore, if there are any queries or concerns which arise when the dossier is reviewed, it is requested that the reviewer discuss these with the ERGTC ([e-mail]) as there may be background information and previously discussions between the ERGTC and ECHA available which are relevant.

Reference

OECD (2001). SIDS, Benzoate, SIDS Initial Assessment Report for 13th SIAM, Bern 7^th– 9^thNovember 2001, UNEP Publications.