1,8-bis[(4-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
Cc1ccc(Nc2cccc3C(=O)c4cccc(Nc5ccc(C)cc5)c4C(=O)c23)cc1

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 (< -6.243 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 (< -6.243 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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The stability ot test concentration during exposure was examined by chemical analysis at 0, 24, 48 and 72 hours; the control was examined after 72 hours
Aliquots of the samples from the biological test were thawed at room temperature, homogenised and diluted (see above). Subsequently the samples were analysed by HPLC/MS/MS.

Vehicle:

no

Details on test solutions:

The test item was treated for 1 hour in an ultrasonic bath and stirred for 24 hours on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter (pore size 7-12 µm)

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Strain: Strain No. 86.81 SAG
- Source: 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 to 120 µE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter); the growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week; cell density measurements were made using a Particle and Count Size Analyzer, Z2 (Beckman Coulter)
- 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 growth medium
- Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth 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:

1.3 °dH (22.5 mg/L CaCO3)

Test temperature:

21-24 °C

pH:

7.9 - 8.1

Dissolved oxygen:

n.a.

Salinity:

n.a.

Conductivity:

no data

Nominal and measured concentrations:

Nominal concentration: 100 mg/L; After 72 hours the values were below or at the limit of quantification (0.003 mg/L); Effective concentrations ranged from 0.41 to 0.46 % of nominal values at 0 hours, corresponds 0.15 % of nominal values at 24 hours, from 0.12 % to 0.15 % of nominal values at 48 hours and from 0.07 % to 0.19 % of nominal values at 72 hours.

Details on test conditions:

EXPOSURE CONDITIONS:
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers, test volume 100 mL
- Culturing apparatus: Light chamber in which a temperature in the range 21 °C to 25 °C was maintained, and continuous uniform illumination was provided in the spectral range 400 to 700 nm
- Light intensity: A light intensity in the range 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lx, was measured; the light intensity was checked before the start of the study
- Cell density measurements: Cell densities were measured in a Particle and Size Analyzer (Z2, Beckman Coulter) by taking small aliquots from each test flask, 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, Additionally highest test concentration without algae
- Test item concentration: 100 mg/L
- Method of administration: direct weighing
- Criteria of effects: The criteria of adverse effects used in this study 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:

EC50

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/L (nominal)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/L (nominal)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/L (nominal)

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/L (nominal)

Details on results:

The results are expressed in terms of nominal concentrations and geometric mean measured values. Effective concentrations ranged from 0.41 to 0.46 % of nominal values at 0 hours, corresponds 0.15 % of nominal values at 24 hours, from 0.12 % to 0.15 % of nominal values at 48 hours and from 0.07 % to 0.19 % of nominal values at 72 hours.

Reported statistics and error estimates:

After 72 h, the coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) exceeded the validity criterion of 35%.

Validity criteria fulfilled:

yes

Remarks:

Factor of the biomass parameter (241), measured in the control between 0 and 72 h, was at least 16; the mean coefficient of variation (23.6%) was not higher than 35%; the coefficient of variation of the mean specific growth rate (2.7%) did not exceed 7%

Conclusions:

No toxic effect at against algae at the limit of water solubility under exposure conditions. The results are expressed in terms of nominal concentrations and geometric mean measured values. Effective concentrations ranged from 0.41 to 0.46 % of nominal values at 0 hours, corresponds 0.15 % of nominal values at 24 hours, from 0.12 % to 0.15 % of nominal values at 48 hours and from 0.07 % to 0.19 % of nominal values at 72 hours.

Executive summary:

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test' (2006). The purpose of this method was to determine those concentrations which caused a 10 % and a 50 % adverse effect (EC 10, EC 50) or, if conducted as a limit test, to determine the adverse effects at a maximum test concentration of 100 mg/L or at the limit of water solubility. Effect data are expressed on the basis of growth rate [r] (and yield [y]). A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test. The following nominal concentrations of the test item were tested in the range finding test: 0.1, 1.0, 10 and 100 mg/L (nominal). The test item was treated for 1 hour in an ultrasonic bath and stirred for 24 hours on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7 -12 µm. 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 for a period of 72 hours. At this concentration no inhibition of algal growth rate and yield was observed at the end of the 72 hour study period. Cell densities were recorded at 24 hour intervals. All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000. During the test a temperature range of 21 -24 °C was maintained in the test vessels. The pH was measured at the beginning of the test and after 72 hours of exposure. No toxic effect at against algae at the limit of water solubility under exposure conditions. The results are expressed in terms of nominal concentrations and geometric mean measured values. Effective concentrations ranged from 0.41 to 0.46 % of nominal values at 0 hours, corresponds 0.15 % of nominal values at 24 hours, from 0.12 % to 0.15 % of nominal values at 48 hours and from 0.07 % to 0.19 % of nominal values at 72 hours. This toxicity study is classified as acceptable and satisfies the guideline requirements for the Algae study.

Description of key information

72h-ErC10 and ErC50 (Desmodesmus subspicatus) > solubility limit; OECD TG 201; Spoo-kloeppel (2018).

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

Additional information

The experimental study 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: Reinblau RLW (CAS No 41611-76-1) 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