iSafeRat®: HA-QSARs studies for REACH endpoints

ENDPOINTS:

  • OCTANOL/WATER PARTITION COEFFICIENT (LOG KOW)
  • WATER SOLUBILITY
  • Vapour pressure
  • Acute Fish toxicity
  • Acute daphnid toxicity
  • Algae toxicity
  • CHRONIC FISH TOXICITY
  • CHRONIC DAPHNID TOXICITY
  • Aquatic toxicity for mixtures
  • TOXICITY TO MICROORGANISMS
  • SKIN IRRITATION
  • EYE IRRITATION
  • SKIN SENSITISATION
  • DERMAL ABSORPTION
  • Endocrine Disruption potential ( ED SAR )
  • Endocrine Disruption Potential (SESAME-3D)
  • Third-party tools for endocrine disruption

OCTANOL/WATER PARTITION COEFFICIENT (LOG KOW)

iSafeRat® KOW study can test the hydrophobicity (as log KOW) of a substance

Study/studies replaced and value reported:
  • OECD 107 (Partition coefficient (n-octanol/water): Shake flask method)
  • OECD 117 (Partition coefficient (n-octanol/water): High Performance Liquid Chromatography (HPLC) method)
  • OECD 123 (Partition coefficient (n-octanol/water): Slow-stirring method
Domain:
  • organic chemicals with H, C, N, O, P, S, F, Cl, Br
  • log KOW between -2 and 10 and possibly beyond
Methodology:
  • From structure: Core-Centred Substitution Approach
  • Or from high quality water solubility and ecotoxicity data provided by the client: Holistic Approach
Accuracy:

As accurate as an OECD 107 study

Click for further information & price list

WATER SOLUBILITY

iSafeRat® SOL study can test the water solubility (in mg/L, at 25°C) of a substance

Study/studies replaced and value reported:
  • OECD 105 (Water solubility: Shake flask method)
  • modified OECD 105 (Water solubility: slow stirring method)
Domain:
  • organic chemicals with H, C, N, O, S, , F, Cl (and some with Br and I)
  • water solubility between 100 mol/L and 0.01 µmol/L and possibly beyond
Methodology:
  • From log KOW: a set of simple linear regressions between Water Solubility and log KOW
  • from high quality ecotoxicity data provided by the client (Holistic Approach)
Accuracy:

As accurate as a modified OECD 105 slow stirring study (Thomas & Burosse, 2012, see downloads)
95%-Confidence Intervals available
 

Click for further information & price list

Vapour pressure

iSafeRat® VAP study can test the vapour pressure (in Pa, at 25°C) of a substance

Study/studies replaced and value reported:
  • OECD 104 (Vapour Pressure)
Domain:
  • organic chemicals with H, C, N, O, S (and some halogenated compounds)
  • vapour pressure between 10-2 Pa to 105 Pa and possibly beyond
Methodology:
  • a set of simple linear regressions between Vapour Pressure and Boiling Point
Accuracy:

As accurate as an OECD 104 study
95%-Confidence Intervals available

 

Click for further information & price list

Acute Fish toxicity

iSafeRat® fishLC50 study can test 96h-acute toxicity on fish (in mg/L) of a substance

Study/studies replaced and value reported:
  • OECD 203 (Fish Acute Toxicity Test)
  • OECD 236 (Fish Embryo Acute Toxicity (FET) Test)
Domain:

Currently, the ecotoxicity module of the iSafeRat® HA-QSAR models can reliably predict the acute toxicity to fish 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 & 5.2)
  • 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)

with log KOW between 0 and ca. 5 (and possibly higher), i.e. the point at which acute toxicity is no longer found below the limit of solubility.

Methodology:
  • simple linear regression between subcooled liquid water solubility and toxicity (Mackay et al., 2009; Thomas et al., 2015)
Accuracy:

As accurate as an OECD 203 study (in terms of finding true toxicity)
95%-Confidence Intervals

Click for further information & price list

Acute daphnid toxicity

iSafeRat® daphEC50 study can determine 48h-acute toxicity to daphnids (in mg/L) based on mobility for a substance

Study/studies replaced and value reported:
  • OECD 202 (Daphnia sp., Acute Immobilisation Test)
Domain:

Currently, the ecotoxicity module of the iSafeRat® HA-QSAR models can reliably predict the acute toxicity to daphnids 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 & 5.2)
  • 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)

 

with log KOW between 0 and ca. 5 (and possibly higher), i.e. the point at which acute toxicity is no longer found below the limit of solubility.

Methodology:
  • simple linear regression between subcooled liquid solubility and toxicity (Mackay et al. 2009, Thomas et al. 2015)
Accuracy:

As accurate as an OECD 202 study (in terms of finding true toxicity)
95%-Confidence Intervals

Click for further information & price list

Algae toxicity

iSafeRat® algEC50 and iSafeRat® algNOEC studies can test 72h-toxicity (as inhibition of growth) on algae (in mg/L) of a substance

Study/studies replaced and value reported:
  • OECD 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Domain:

Currently, the ecotoxicity module of the iSafeRat® HA-QSAR models can reliably predict the toxicity to algae 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 & 5.2)
  • 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)

with log KOW between 0 and ca. 5 (and possibly higher), i.e. the point at which toxicity is no longer found below the limit of solubility.

Note: iSafeRat® algNOEC is only available for compounds with MechoA 1.1 and 2.1

Methodology:
  • simple linear regression with subcooled liquid water solubility (Mackay et al. 2009, Thomas et al. 2015)
Accuracy:

As accurate as an OECD 201 study (in terms of finding true toxicity)
95%-Confidence Intervals

Note: In certain studies on algae the test substance may be lost due to metabolisation or adsorption by the algal cells. The iSafeRat® value will equate to the ErC50 (or NOEC) value obtained in a study where the substance was maintained over the whole study whether or not this can be achieved experimentally.

Click for further information & price list

CHRONIC FISH TOXICITY

iSafeRat® fishEC10 study can test 32d-chronic toxicity on fish (in mg/L) of a substance

Study/studies replaced and value reported:
  • OECD 210 (Fish, Early-life Stage Toxicity Test)
Domain:

Currently, the ecotoxicity module of the iSafeRat® HA-QSAR models can reliably predict the chronic toxicity to fish for chemicals with the following mechanisms of action of toxicity (MechoA):

  • non-polar narcosis (MechoA 1.1)
  • mono-/poly-esters whose hydrolysis products are narcotics (MechoA 2.1)

 

with log KOW between 0 and ca. 5.5 (and possibly higher), i.e. the point at which toxicity is no longer found below the limit of solubility.

Methodology:
  • simple linear regression with subcooled liquid water solubility (Mackay et al., 2009; Thomas et al., 2015)
Accuracy:

As accurate as an OECD 210 study (in terms of finding true toxicity)
95%-Confidence Intervals

Click for further information & price list

CHRONIC DAPHNID TOXICITY

iSafeRat® daphEC10 study can test 21d-chronic toxicity to daphnids (in mg/L) of a substance

Note: The iSafeRat® daphEC10 study will provide a calculated EC10 value (QSAR based on reproduction effects further to a 21-day study on daphnids using measured concentrations).

Study/studies replaced and value reported:
  • OECD 211 (Daphnia magna Reproduction Test)
Domain:

Currently, the ecotoxicity module of the iSafeRat® HA-QSAR models can reliably predict the chronic toxicity to daphnids for chemicals with the following mechanisms of action of toxicity (MechoA):

  • non-polar narcosis (MechoA 1.1)
  • mono-/poly-esters whose hydrolysis products are narcotics (MechoA 2.1)

 

with log KOW between 0 and ca. 6 (and possibly higher), i.e. the point at which toxicity is no longer found below the limit of solubility.

Methodology:
  • simple linear regression with subcooled liquid water solubility (Mackay et al. 2009, Thomas et al. 2015)
Accuracy:

As accurate as an OECD 211 study (in terms of finding true toxicity)
95%-Confidence Intervals

Click for further information & price list

Aquatic toxicity for mixtures

As opposed to the other High Accuracy-QSAR models this is a multistep calculation method requiring knowledge of the toxicity properties of each constituent. These can initially be calculated using one of the above ecotoxicity acute modules. Currently KREATiS can accurately predict mixture toxicity (e.g. Natural Complex Substances) for up to 29 constituents. Even if your mixture contains more, it may be possible to get a high quality prediction based on the major constituents and those expected to have the highest toxicity.

iSafeRat® fishLL50 WAF, daphEL50 WAF or algErL50 WAF study can test the acute toxicity on fish, daphnid or agae (in mg/L) of the Water Accommodated Fraction (WAF) of a substance (expressed as the lethal or effective loading rate, L/EL50, in mg/L)

Note. The calculation method is also available to get the chronic result (based on the loading rate once again).

Study/studies replaced and value reported:
  • OECD 203 (Fish Acute Toxicity Test)
  • OECD 210 (Fish, Early-life Stage Toxicity Test)
  • OECD 236 (Fish Embryo Acute Toxicity (FET) Test)
  • OECD 202 (Daphnia sp., Acute Immobilisation Test)
  • OECD 211 (Daphnia magna Reproduction Test)
  • OECD 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

using WAF method (following OECD Guidance No. 23 Guidance on Aquatic testing of difficult substances and mixtures)

Domain:
  • mixture consituents should share the same Mechanism of Action (MechoA), ideally MechoA 1.1 (non-polar narcotics)
  • with log KOW between 0 and ca. 5 (and possibly higher), i.e. the point at which toxicity is no longer found below the limit of solubility
Methodology:

The toxicity of mixtuures to aquatic organisms was determined using a calculation method based on toxic additivity principle. That means the toxic parts of each constituent are added up. Therefore the constituents considered within the mixture should act with a similar MechoA. To maximise accuracy of the prediction, the concentration of each constituent in the mixture should be known and is used as inputs into the model.

  1. First a thermodynamic approach is used to calculate the influence of each constituent on the solubility of the others allowing the concentration of each constituent within the mixture to be determined (i.e. providing the “loading rate”).
  2. In the second step, the non-bioavailable fraction is removed to determine the true concentration of each constituent exerting toxicity.
  3. Then, the bioavailable concentrations of each constituent is converted into chemical activity prior to be summed.
  4. Finally, the loading rate is adjusted until the value of the sum of activities of the bioavailable fractions is equal to the fraction-weighted average of toxic activity of each constituent. This can be seen as a mechanistic description of the process which occurs in an experimental WAF study.

This method has been validated internally on a dozen of Natural Complex Substances for acute exposure with non-polar narcosis constituents. The methodology will soon be available in literature (Bicherel and Thomas, in prep).

Accuracy:

As accurate as a WAF experimental study following the specific guideline
(no confidence limits available)

Click for further information & price list

TOXICITY TO MICROORGANISMS

iSafeRat® asritEC50 study can test the toxicity to microorganisms of activated sludge (as EC50) of a substance

Study/studies replaced and value reported:
  • OECD 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Domain:

Currently, the ecotoxicity module of the iSafeRat® HA-QSAR models can reliably predict the toxicity to microorganisms for chemicals with the following mechanisms of action of toxicity (MechoA):

  • non-polar narcosis (MechoA 1.1)
  • mono-/poly-esters whose hydrolysis products are narcotics (MechoA 2.1)

with log KOW between 0 and ca. 3 (and possibly higher), i.e. the point at which toxicity is no longer found below the limit of solubility

Methodology:
  • simple linear regression with subcooled liquid water solubility (Mackay et al., 2009; Thomas et al., 2015)
Accuracy:

As accurate as an OECD 209 study (in terms of finding true toxicity)
95%-Confidence Intervals

Click for further information & price list

SKIN IRRITATION

iSafeRat® iSafeRabbit Skin study can predict the irritant potential to the skin of a substance

Study/studies replaced and value reported:
  • OECD 404 (Acute Dermal Irritation/Corrosion)
  • OECD 431 & 439 (In vitro )
The reported output is the GHS/CLP classification for skin irritation/corrosion:

  • Corrosive to skin, Category 1
  • Irritant to skin, Category 2
  • Non-irritant to skin, No category
Domain:
Currently, the iSafeRat Skin Irritation/Corrosion model can reliably predict the acute skin irritation/corrosion for chemicals within 7 local models grouping the following structural domain:

Alcohols, Aldehydes, Esters, Ethers, Ketones, Organic Acids and Bases
Methodology:
The iSafeRat Skin Irritation/Corrosion prediction model determines whether the applied dose of a chemical substance causes cytotoxicity, thereby responsible for erythema and/or oedema as both lesions are driving the classification according to CLP and GHS. The dose applied and the physico-chemical properties of the test substance are the input of a series of algorithms to determine: the substance concentration in the viable epidermis to establish if this concentration reaches a cytotoxic concentration for the keratinocytes. These concentrations are plotted against the logarithm of the subcooled water solubility of the substances (Log SCLS). The plot is subdivided into corrosive, irritant and non-irritant zones. The test item will fall into one of these zones, thereby allowing a classification.

Goodness-of-fit & predictive performance:
Accuracy: 80% - 100%

Predictive capacity for:
  • Corrosives (Cat. 1): 100%
  • Irritants (Cat. 2.): 67%
  • No category (NC) : 78%
Click for further information & price list

EYE IRRITATION

iSafeRat® iSafeRabbit Eye study can predict the irritant potential to the eyes of a substance

Study/studies replaced and value reported:
  • OECD 405 (Acute Eye Irritation/Corrosion)
The reported output is the GHS/CLP classification for skin irritation/corrosion:

  • Corrosive to eye, Category 1
  • Irritant to eye, Category 2
  • Non-irritant to eye, No category
Domain:
Currently, the eye irritation/corrosion iSafeRat model can reliably predict the acute eye irritation/corrosion for chemicals within 12 local models grouping the following structural domain:

Alcohols, Aldehydes, Esters, Ethers, Ketones, Simple linear and cyclic substituted hydrocarbons (H, C, O, Br, Cl) and Organic Acids and Bases.
Methodology:
The iSafeRat Eye Irritation/Corrosion prediction model determines whether the applied dose of a chemical substance causes cytotoxicity, thereby responsible for corneal opacity and/or conjunctival redness as both lesions are driving the classification according to CLP and GHS. The dose applied and the physico-chemical properties of the test substance are the input of a series of algorithms to determine: the substance concentration in the viable epidermis to establish if this concentration reaches a cytotoxic concentration in the eye tissue. These concentrations are plotted against the logarithm of the subcooled water solubility of the substances (Log SCLS). The plot is subdivided into corrosive, irritant and non-irritant zones. The test item will fall into one of these zones, thereby allowing a classification.

Goodness-of-fit & predictive performance:
Accuracy: 74% - 100%

Predictive capacity for:
  • Corrosive (Cat. 1): 71 – 100%
  • Irritant (Cat. 2.): 75 -100%
  • No category (NC) : 75 – 100%
Click for further information & price list

SKIN SENSITISATION

iSafeRat® Skin Sensitisation model can predict the molecular initiating event (MIE) of the skin sensitization AOP of a substance.

Study/studies replaced and value reported:
  • OECD 429 (Skin Sensitisation: Local Lymph Node Assay)
The reported output is:
  • Alert for potential skin sensitizer via protein binding mechanism (parent and/or metabolite)
  • Negative for skin sensitization via protein binding (parent nor metabolite)
Domain:
Currently, the iSafeRat Skin Sensitisation model can reliably predict the skin sensitization potential for chemicals within the 26 Mechanisms of action of toxicity (MechoA).
Methodology:
Decision tree: iSafeRat MechoA scheme model is used to predict the MIE for the skin sensitization endpoint:
Positive predictions for skin sensitization via protein binding: Chemicals triggering MechoA 3 and/or 4.3 and/or 4.4
Negative predictions, i.e., non-protein binders: Chemicals triggering other MechoA classes than 3, 4.3 & 4.4.

Goodness-of-fit & predictive performance:
Positive predictivity (Chemicals triggering MechoA 3 and/or 4.3 and/or 4.4):
  • 62% relative to human clinical data
  • 57% relative to LLNA Click for further information & price list
Negative predictivity (Chemicals triggering other MechoA classes than 3, 4.3 & 4.4):
  • 83% relative to human clinical data
  • 78% relative to LLNA

Use of iSafeRat® MechoA scheme model as part of an in silico battery approach to strengthen in silico weight of evidence strategies for skin sensitisation, can help in the decision making of the in vitro testing strategy. Our team is currently working on combining MechoA with a dermal absorption model to improve its performance for skin sensitisation.

Click for further information & price list

DERMAL ABSORPTION

iSafeRat® Dermal Absorption model can estimate the amount of a substance that may be absorbed via a dermal exposure.

Study/studies replaced and value reported:
  • OECD 428 (In vitro Dermal absorption)
The reported output is:
  • % permeated substance (stratum corneum)
  • % evaporated substance
  • % Absorbed substance (systemic)
  • Kp (cm/h)
Methodology:
Physics-based model simulating in vitro TG 428 procedure
Click for further information & price list

Endocrine Disruption potential ( ED SAR )

- 2D SARs for EATS and positive and negative alerts

iSafeRat® ED SAR

Study/studies replaced and value reported:
The studies replaced by this 2D SAR include:
  • Reporter gene assay (RA)
  • Enzyme activity assay (inhibition)
Domain:
Currently, this is the list of endpoints that are covered by this model:
  • Estrogen Receptor α (ESR1) agonism and antagonism
  • Estrogen Receptor β (ESR2) agonism and antagonism
  • Androgen Receptor (AR) agonism and antagonism
  • Thyroid Hormone Receptor ? and ? (THRA and THRB) agonism and antagonism
  • Thyroperoxidase (TPO) inhibition
  • Iodothyronine Deiodinase type I, II and III (DIO1, DIO2 and DIO3) inhibition
  • Sodium-Iodide Symporter (SLC5A5) inhibition
  • Thyroid Stimulating Hormone Receptor (TSHR) agonism and antagonism
  • Progesterone Receptor (PGR) agonism and antagonism
  • Follicle-Stimulating Hormone Receptor (FSHR) antagonism
  • Aromatase (CYP19A1) inhibition
The structural domain is dependent on each endpoint and is constantly increasing.
Methodology:
  • Curation and thorough validation of in vitro assays (receptor binding, receptor (in)activation, enzyme inhibition) data from various sources, covering more than 8000 substances.
  • Grouping of chemicals with common structural patterns and assay outcomes to create structural alerts.
Accuracy:
Each structural alert is evaluated independently for its accuracy. All alerts obtained at least 85% accuracy, however most (95%) alerts reached accuracy >99%, on the training data set. External validation is in progress.
Click for further information & price list

Endocrine Disruption Potential (SESAME-3D)

- Docking tools

Study/studies replaced and value reported:
The studies replaced by the workflow include:
  • Protein binding (Binder/Non Binder)
  • Relative binding affinity (RBA, percentage of test item binding to a receptor relative to its natural ligand)
We report a predicted affinity constant for the test molecule along with its related standard deviation. The calculated RBA in respect to the native hormone is also provided along with its error boundaries. Other RBA in respect to a restricted set of pharmacological actives can be provided on request.
Domain:
Currently, this is the list of endpoints that are covered by this model:
  • Thyroid Receptor α (THRA) binding
  • Thyroid Receptor β (THRB) binding
  • Estrogen Receptor α (ESR1) binding
  • Estrogen Receptor β (ESR2) binding
  • Progesterone Receptor (PGR) binding
  • Androgen Receptor (AR) binding
The KREATiS offer of protein targets for molecular docking will expand in the future, as the number of investigated conformations of each receptor in the biological domain. Nevertheless, extra biological targets can be included in a study on request. This last option, however, is subject to a limitation imposed by the availability within external sources of experimentally derived structural models for the requested proteins.

The chemical domain supported for the test molecule covers any type of organic chemicals, and is applicable to all of the listed biological endpoints.
Methodology:
  • To date SESAME-3D provides a molecular docking capability for the estimation of the possibility of existence of molecular complexes. In the future a technique called simulation of molecular dynamics will be included as an option for more thorough investigation.
  • The molecular docking workflow uses as docking targets only experimentally derived models of the ligand binding domains of the proteins of interest. Thus, the ensemble of experimental models available (from external sources) is curated and clustered into conformational classes, a single representative of which is used as self-standing protein target in a docking experiment.
  • Machine learning algorithms are further trained to calculate predicted affinities for the tested and the reference molecules.
  • The likelihood of a tested molecule to be a binder is derived from a comparison with the binding profiles of computed molecular complexes for known actives docked within a related biological target.
Accuracy:
The confidence level for each prediction is provided through the report and the interpretation of the results of various statistical tests implemented in the SESAME-3D workflow.

The overall accuracy of SESAME-3D is currently under investigation as this workflow is quite a new tool but is anticipated to predict the affinity of native hormones-receptor complexes listed above as studied biological domain, within ±10 nM from the AC50 (concentration providing 50% of maximum effect) reported from in vitro experiments in a consortium database.)
Click for further information & price list

Third-party tools for endocrine disruption

Currently, we use dozens of selected models that have been chosen to make sure we get the most reliable results to predict endocrine disruption potential of chemicals via Estrogenic, Androgenic, Thyroidal (EAT) and non-EATS modalities. We don’t just apply the models to the molecule and report the results. Each model is systematically challenged to make sure it is fit for purpose and a consensus approach is used to arrive at a meaningful conclusion.

Study/studies replaced and value reported:
The studies replaced by third party tools include:
  • Protein binding (Binder/Non Binder)
  • Relative binding affinity (RBA, percentage of test item binding to a receptor relative to its natural ligand)
  • Transactivation assays (RA)

Domain:
For the following list of endpoints:
  • Estrogen Receptor α and β (ESR1 and ESR2) binding, agonism and antagonism
  • Androgen Receptor (AR) agonism and antagonism
  • Thyroid Hormone Receptor ? (THRA) binding
  • Thyroid Hormone Receptor ? (THRB) binding
  • Pregnane X Receptor (NR1I2) binding

The applicability domain of each model is different and therefore it is assessed on a case-by-case basis. In general, it covers organic chemicals.

Methodology:
  • The in silico models available within the following software packages are currently used to predict endpoints related to ED potential: OECD QSAR Toolbox, Danish QSAR Database, VEGA and T.E.S.T. from US EPA.
  • Available third-party models are used to generate the predictions for each test item, followed by a manual reliability check of each prediction, i.e., relative to the prediction based on the models’ goodness-of-fit and/or performance and relative to the test item’s structure (when possible). This step allows to “turn OFF” low reliability alerts and therefore, to reach a conclusion based on only reliable predictions.

Goodness-of-fit & Performance:
Depending on each model used (Reliability cut-off was set at 70% for sensitivity or specificity).
Click for further information & price list