1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone

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:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

06 April 2022 to 07 April 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

The test item falls within the applicability domain of the models except for the descriptor domain. Therefore, both predicted TOXICITY TO ALGAE (72-HOUR ErC50) and TOXICITY TO ALGAE (72-HOUR NOECr) are considered as extrapolations. These results can be considered as reliable with restrictions (descriptor domain).

Justification for type of information:

1. SOFTWARE
iSafeRat® – in Silico Algorithms For Environmental Risk And Toxicity

2. MODEL (incl. version number)
iSafeRat® algErC50 v1.9
iSafeRat® algNOEC v1.2

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CCc1cccc(C)c1Nc2ccc(Nc3c(C)cccc3CC)c4C(=O)c5ccccc5C(=O)c24

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached Study Report and QPRF in Annex

6. ADEQUACY OF THE RESULT
See attached Study Report and QPRF in Annex

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

not applicable

Remarks:

QSAR model

Principles of method if other than guideline:

The TOXICITY TO ALGAE (72-HOUR ErC50 and NOECr) was determined using iSafeRat® algEC50 and iSafeRat® algNOEC, two validated QSAR models for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). The QSAR models provide in silico predictions for the 72-hour ErC50 and NOECr values that can effectively be used in place of an experimentally derived results. The QSAR models are based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 and 32 chemicals derived from 72-hour NOECr test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

GLP compliance:

no

Remarks:

QSAR model

Analytical monitoring:

no

Remarks:

QSAR model

Details on sampling:

not applicable

Vehicle:

no

Remarks:

QSAR model

Details on test solutions:

not applicable

Test organisms (species):

other: Pseudokirchneriella subcapitata, Desmodesmus subspicatus, Scenedesmus quadricauda

Details on test organisms:

No difference in terms of toxic mechanism of action between algae (or indeed other) aquatic species is expected. Any observed differences may be attributed to lifestyle related parameters and relative duration of study versus cell size rather than to a specific toxic mechanism causing species differences.

Test type:

other: QSAR model

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Results from a test duration of 72 hours only were used for this algorithm.

Post exposure observation period:

not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The temperatures varied from approximately 20 to 25 °C depending on the species used to construct the models. This small difference is not expected to significantly contribute to the variability of the values found in experimental data.

pH:

Test results were preferably taken from studies with measured pHs between 6 - 9. However it is recognized that in some cases (due to high luminosity) the pH may increase in the control and lower concentrations (which do not cause significant effect over the study period). This pH increase did not generally disqualify the study from being used in the test and validation set for non-polar chemicals.

Dissolved oxygen:

The temperatures varied from approximately 20 to 25 °C depending on the species used to construct the algorithm. This small difference is not expected to contribute to the variability of the toxic values found in experimental data.

Salinity:

not applicable

Conductivity:

not applicable

Nominal and measured concentrations:

Studies were used only where sufficient evidence was presented to determine that the stubstance was stable under test conditions (i.e. maintened within ± 20 % of the nominal or measured initial concentration throughout the test) or, if not, the result was based on measured concentrations as geometric mean.

Details on test conditions:

Following the guideline OECD 201, all studies were from a static test design. For suspected volatile substances only tests performed in closed vessels were accepted unless accompanying analytical monitoring proved such a design was not necessary.

Reference substance (positive control):

not required

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 0.003 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.003 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Details on results:

The test item falls within the applicability domain of the models except for the descriptor domain. Therefore, both predicted TOXICITY TO ALGAE (72-HOUR ErC50) and TOXICITY TO ALGAE (72-HOUR NOECr) are considered as extrapolations. These results can be considered as reliable with restrictions (descriptor domain).

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

95% confidence interval (α = 0.05) for 72h-ErC50: not applicable
95% confidence interval (α = 0.05) for 72h-NOECr: not applicable

Applicability Domain

Descriptor domain

The Subcooled Liquid Water Solubility value (< -6.749 in log10 (mol/L)) given as the input to the iSafeRat® algErC50 model falls within the intermediate domain of the model between a Subcooled Liquid Water Solubility of -9.34 to -4.38 in log10 (mol/L) where baseline toxicity cannot be experimentally measured accurately. In this intermediate domain, the toxicity may to be greater than the water solubility limit. For confirmation, a statistical k-NN approach (k = 3) is performed on the data of substances found to be in the intermediate domain of the model. The toxicity of the three closest neighbours based on the solubility are considered. Based on these data, either the toxicity of the test item is expected to be greater than the limit of solubility, or the toxicity is estimated by the geometric mean between the toxicity value predicted using the regression line and the solubility cut-off line. According to this analysis, the toxicity of the test item is estimated as greater than the water solubility limit.

 

The Subcooled Liquid Water Solubility value (< -6.749 in log10 (mol/L)) given as the input to the iSafeRat® algNOECr model does not fall within the descriptor domain of the model between a Subcooled Liquid Water Solubility of -5.154 to 0.490. Therefore, the prediction is considered as an extrapolation.

Structural fragment domain

The variability of structure in the training set is not considered as a relevant domain since the model is based on a mechanistic approach (mechanism of action). Since the MechoA is related to the molecular structure, the following list of chemical moieties can give an overview of the structural domain:

• Alcohol


• Alkane


• Alkene


• Anthraquinones


• Aromatic hydrocarbons and polycyclic aromatic hydrocarbon


• Ester


• Ether


• Halogenated hydrocarbons


• Ketone

The test item as an anthraquinone can be taken into account by the model.

Mechanistic domain
The iSafeRat® algErC50 model can reliably predict the aquatic toxicity for chemicals with the following mechanisms of action of toxicity (MechoA):
• non-polar narcosis (MechoA 1.1)
• polar narcosis of alkyl-/alkoxy-phenols (MechoA 1.2)
• polar narcosis of aliphatic amines (MechoA 1.2)
• cationic narcosis of quaternary ammoniums (MechoA 1.3)
• mono-/poly-esters whose hydrolysis products are narcotics (MechoA 2.1)
• hard electrophile reactivity (MechoA 3.1)
• RedOx cycling of primary thiols (MechoA 4.4)
• Proton release of carboxylic acids (MechoA 5.2)

The iSafeRat® algNOECr model can only reliably predict the aquatic toxicity for chemicals with the mechanism of action of non-polar narcosis (MechoA 1.1).

The MechoA of molecules is predicted directly from the structure. The test item as an  aminoanthraquinone is expected to exert a MechoA 1.1 & m4.3: Non-polar narcosis for all species & metabolisation into nitroso generating protein and DNA adducts, oxidative stress, DNA adducts, cancer development for mammals only (Bauer et al., 2018). Therefore, the test item can be taken into account by the model in order to predict toxicity to non-mammal aquatic organisms.

Validity criteria fulfilled:

yes

Conclusions:

The test item falls within the applicability domain of the models except for the descriptor domain. Therefore, both predicted TOXICITY TO ALGAE (72-HOUR ErC50) and TOXICITY TO ALGAE (72-HOUR NOECr) are considered as extrapolations. These results can be considered as reliable with restrictions (descriptor domain).
The TOXICITY TO ALGAE (72-HOUR ErC50 and NOECr) of the test item was both predicted as greater than the water solubility value within the exposure period of the test.
95% confidence interval (α = 0.05) for 72h-ErC50: not applicable
95% confidence interval (α = 0.05) for 72h-NOECr: not applicable

Executive summary:

Two Quantitative Structure-Activity Relationship (QSAR) models were used to calculate the TOXICITY TO ALGAE (72-HOUR ErC50 and NOECr) of the test item. These QSAR models have been validated to be compliant with the OECD  recommendations for QSAR modeling (OECD, 2004) and predict the endpoint values which would be expected when testing the substance under experimental conditions in a laboratory following the Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (OECD, 2006), referenced as Method C.3 of Commission Regulation No. 440/2008 (European Commission, 2008). The criterions predicted were the Median Effective Concentration (ErC50), a statistically derived concentration which is expected to cause 50% inhibition of intrinsic rate of growth of the test system and the No Observed Effect Concentration (NOECr), a tested concentration which is expected to cause no effect on intrinsic rate of growth of the test system. Both criterions were determined for a period exposure of 72 hours.

 

The TOXICITY TO ALGAE (72-HOUR ErC50 and NOECr) was determined using iSafeRat® algEC50 and iSafeRat® algNOEC, two validated QSAR models for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). The QSAR models provide in silico predictions for the 72-hour ErC50 and NOECr values that can effectively be used in place of an experimentally derived results. The QSAR models are based on validated data for a training set of 40 chemicals derived from 72-hour ErC50 and 32 chemicals derived from 72-hour NOECr test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period.

 

The test item falls within the applicability domain of the models except for the descriptor domain. Therefore, both predicted TOXICITY TO ALGAE (72-HOUR ErC50) and TOXICITY TO ALGAE (72-HOUR NOECr) are considered as extrapolations. These results can be considered as reliable with restrictions (descriptor domain).

The TOXICITY TO ALGAE (72-HOUR ErC50 and NOECr) of the test item was both predicted as greater than the water solubility value within the exposure period of the test.
95% confidence interval (α = 0.05) for 72h-ErC50: not applicable
95% confidence interval (α = 0.05) for 72h-NOECr: not applicable

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling schedule:
Control : at 72 hours
Test concentrations : at 0 and 72 hours

Vehicle:

no

Details on test solutions:

A range finding test preceded the main test and provided information about the concentration which was used in the main test. In the main test, the algae were exposed to the test item added to dilution water at a limit concentration at the limit of water solubility under test conditions.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Strain No.: 86.81 SAG

Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).

Maintenance and Acclimatisation: Exponentially-growing stock cultures were maintained in the test facility under constant temperature conditions (21-24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 – 120 μE. x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to BRINGMANN & KÜHN, 1977) was renewed once a week. Cell density measurements were made using a microcell- counter, Sysmex F300, Digitana.

Preparation of pre-cultures: Pre-cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different nutrient medium

Test cultures: The algal inocula for a test were taken from an exponentially-growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Water hardness of the final nutrient medium: 1.3 °dH (22.5 mg/L CaCO3)

Test temperature:

21-24 °C

pH:

7.9 after 0 hour
9.0 after 72 hours

Dissolved oxygen:

No data

Nominal and measured concentrations:

100 mg/l test item corresponds to < 0.0176 HPLC-value (mg/l)

Details on test conditions:

Pre-TREATMENT OF THE TEST ITEM:
- 100.4 mg of the test item were added to 1 litre of dilution water, treated for 1 hour in an ultrasonic bath and afterwards stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter (pore size of 7-12 μm) and an aseptic filter Sartobran 150 Sterile Capsule (pore size 0.45 + 0.2 μm)
- pH was measured to be 7.7
- 100 mL of the solution were taken and 0.495 mL of the algal inoculum resulting in a final cell density of 5000 cells/mL was added to each replicate

EXPOSURE CONDITIONS:
- Test vessels: 300 mL Erlenmeyer flasks with cotton/cellulose stoppers, test volume: 100 mL
- Culturing apparatus: Light chamber in which a temperature in the range 21°C to 24°C was maintained at +/- 2°C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm
- Light intensity: At the average of the test solutions, a light intensity in the range 60 to 120 μE. x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was used. Light intensity was checked before start of the study
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask for measurements, which were not replaced
- Experimental design: 1 test concentration plus 1 control, 6 replicates per concentration, 6 replicates per control, Initial cell density in the test cultures approximately 5000 cells per millilitre
- Test item concentration/s: 100 mg/L,
- Method of administration: direct weighing
- Duration of exposure: 72 hours
- Criteria of effects: The criteria of adverse effects were the item-induced inhibition of yield [y] and growth rate [r], respectively, of the algal population

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The results are expressed in terms of Nominal concentrations. After 0 and 72 hours all values were below the quantification limit of the HPLC analysis (0.0176 mg/L).

Validity of the test

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16.

With the current test it was found to be 134.0. The test fulfils this validity criterion.

The mean of the replicate coefficients of variation in the section-by-section growth rate was: 15.8 %.

According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35 %. The test fulfils this validity criterion.

The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 0.8 %.

According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7 %. The test fulfils this validity criterion.

The following validity criteria of the test were met:

The cell density in the control cultures increased by a factor of at least 16 within 72 hours.

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35 %.

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 %.

Validity criteria fulfilled:

yes

Remarks:

(Cell density in control cultures increased by a factor of at least 16 within 72 hours; specific growth rates in the control cultures did not exceed 35%.)

Conclusions:

In a limit test Desmodesmus subspicatus was exposed under static conditions to a concentration of 100 mg/l (pH 7.9-9.0). After 72 h no toxic effects against algae were observed at a limit test concentration of 100 mg/L, corresponding to a measured concentration of < 0.0176 mg/L, which reflects the maximum water solubility under exposure conditions.

Executive summary:

A study was performed to assess the adverse effects of the test item on the yield (= biomass at time t minus initial biomass) and the growth rate (= rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Algae and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test' (2006). Exponentially growing algal cells were exposed for a period of 72 hours to a concentration, nominally of 100 mg/l. The cell densities were measured in 24 hrs intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to Williams Multiple Sequential t-Test Procedure. After 72 h no toxic effects against algae were observed at a limit test concentration of 100 mg/L, corresponding to a measured concentration of < 0.0176 mg/l, which reflects the maximum water solubility under exposure conditions. This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute Algae study.

Description of key information

72h-ErC10 and ErC50 (Desmodesmus subspicatus) > solubility limit; EU Method C.3; Currenta (2012).

72h-ErC10 and ErC50 (algae) > solubility limit; iSafeRat® High-Accuracy-Quantitative Structure-Activity Relationship; KREATiS (2022).

Additional information

The experimental result is supported by one HA-QSAR prediction (KREATiS, 2022) showing no toxicity up to the solubility limit of the registered substance. Also, experimental algae studies are available on other substances of the category: Solvent Violet 36 (CAS No 82-16-6) and Solvent Green 28 (CAS No 4851-50-7), with a similar result. See the category approach justification document, attached in section 13, for further details.

Global overview of the category approach for the endpoint:

	Solvent Violet 36	Solvent Green 3	Reinblau RLW	Reinblau BLW	Solvent Blue 104^#	Solvent Green 28
Toxicity to aquatic algae	72h-ErC10 and ErC50 (D.subspicatus) > 100 mg/L (nominal) or > 0.16 mg/L (measured)^$   72h-NOECr and ErC50 (algae) > solubility limit*	72h-NOECr and ErC50 (algae) > solubility limit*   Other Lead Registrant data available but not considered key	72h-ErC10 and ErC50 (D.subspicatus) > 100 mg/L (nominal)^$   72h-NOECr and ErC50 (algae) > solubility limit*	Study planned (OECD TG 201)   72h-NOECr and ErC50 (algae) > solubility limit*	72h-NOECr and ErC50 (algae) > solubility limit*	72h-ErC10 and ErC50 (D.subspicatus) > 100 mg/L (nominal)^$

^# not registered by LANXESS

* iSafeRat® High Accuracy QSAR predictions (KREATiS, 2022)

^$ experimental studies