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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Salmonella/microsome test (Ames test): positive with strains TA 98 and TA 100 (+ S9 mix) and negative with strains TA 102, TA 1535 and TA 1537 (+/- S9 mix)
Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
DEREK Nexus 6.1
2. MODEL (incl. version number)
DEREK Nexus 6.1
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
n/a
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: TOX 7.6.1. Genetic toxicity in vitro
- Unambiguous algorithm: logic of argumentation. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features) inactive
- Defined domain of applicability: The scopes of the structure-activity relationships describing the mutagenicity endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for mutagenicity it can be considered to be within the applicability domain. If a compound does not activate an alert or reasoning rule then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features. The applicability domain of each alert is defined by the alert developer on the basis of the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. For non-alerting compounds, users should determine the applicability of negative predictions by evaluating the information supplied by Derek (i.e. the presence or absence of misclassified and/or unclassified features).
- Appropriate measures of goodness-of-fit and robustness and predictivity: n/a
- Mechanistic interpretation: All alerts describing structure-activity relationships for the mutagenicity endpoint have a mechanistic basis wherever possible.
Mechanistic information is detailed in the comments associated with an alert and can include information on both the mechanism of action and biological target. The mechanistic basis of the model was developed a priori by examining the toxicological and mechanistic evidence before developing the structure-activity relationship.

5. APPLICABILITY DOMAIN

- Descriptor domain:
[1]Markush structures encoding activating and deactivating features (known as patterns in the Derek Nexus knowledge base)
[2]count of non-hydrogen atoms
[3]ClogP
[4]2D structural fragments
There is an a priori assumption that patterns and associated reasoning will be used to model toxicity within Derek Nexus. Further, experts identified that misclassified and unclassified features were useful descriptors for determining the reliability of negative predictions for non-alerting compounds.
- Similarity with analogues in the training set: Non-proprietary elements of the training set are available through the references, and illustrated by the examples, within Derek Nexus. The illustrative examples are not available, due to the proprietary nature of Derek Nexus.

6. ADEQUACY OF THE RESULT
Based on the common structure of the substance, the result is considered reliable.
Qualifier:
according to guideline
Guideline:
other: REACH Guidance on QSARs R.6
Version / remarks:
Version 3.1 July 2016
Principles of method if other than guideline:
- Software tool(s) used including version: DEREK Nexus 6.1
- Model(s) used: DEREK Nexus 6.1
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
bacteria, other: Predictions are made for the domain of bacteria and can be broken down into species (e.g. Salmonella typhimurium and Escherichia coli)
Additional strain / cell type characteristics:
other: The prediction is based on results from all tester strains recommended by the OECD Test Guideline
Evaluation criteria:
Two types of models were used to predict the mutagenic potential of the test item.
The DEREK Nexus model was used as a rule-based model which is based on the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. The second model used was the Leadscope Applier which is a statistical model using structural fragments to set an alert. If experimental data are available the prediction of the statistical model may be overruled.
Key result
Species / strain:
other: Not applicable for in silico study
Metabolic activation:
not applicable
Genotoxicity:
positive
Remarks:
The test item showed alerts for mutagenicity. Therefore Picolinamid-phenylether was considered to be mutagenic.
Cytotoxicity / choice of top concentrations:
other: not applicable
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
not applicable
Conclusions:
Based on the predictions performed with the statistical QSAR model Leadscope Applier and the rule-based model DEREK Nexus Picolinamid-phenylether is mutagenic in a bacterial reverse mutation assay.
Executive summary:

In a QSAR prediction using DEREK Nexus v6.1 the potential of Picolinamid-phenylether to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have been shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.


 


The query structure does match a structural alert or examples for (bacterial in vitro) mutagenicity in Derek.


Based on these results Picolinamid-phenylether is considered mutagenic as predicted by DEREK Nexus.


This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
Leadscope model applier (v3.1.0)

2. MODEL (incl. version number)
Leadscope model applier (v3.1.0)
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS: 244462-37-9; Chemical name: Picolinamid-phenylether; SMILES: CNC(=O)C1=NC=CC(=C1)OC2=CC=C(C=C2)N

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: QMRF 4.10. Mutagenicity OECD 471 Bacterial Reverse Mutation Test

- Unambiguous algorithm:
A new ICH M7 compliant expert alert system to predict the mutagenic potential of impurities (white paper) http://www.leadscope.com/white_papers/ICHM7-WhitePaper-0314.pdf
The logic for matching alerts is detailed in "A new ICH M7 compliant expert alert system to predict the mutagenic potential of impurities" (white paper): http://www.leadscope.com/white_papers/ICHM7-WhitePaper-0314.pdf

- Defined domain of applicability: The applicability domain is defined as having at least one chemical in a reference set with at least 30% global similarity to the test structure (using the Leadscope 27,000 chemical fragments as descriptors and the Tanimoto similarity score).

- Appropriate measures of goodness-of-fit and robustness and predictivity: Chemicals/descriptor ratio: 241 alerts for 11,528 reference chemicals (ratio = 48); Alerts are run within the Leadscope model applier that provides the capability to specify one or more compounds (using SMILES, Mol files, SD files, or copying from the clipboard), select and run the alerts, assess the applicability domain, and view the results including an explanation for any prediction (such as a full description of any matched alerts). The performance was assessed using the Hansen dataset comprised of 3,700 chemicals (47% positive).
Concordance = 83%, Sensitivity = 92%, Specificity = 70%, Positive
Predictivity = 81%, Negative Predictivity = 86% , coverage = 95% were
obtained.

- Mechanistic interpretation: Accompanying any positive prediction, any alert(s) that match the test compounds are described including a description of the mechanistic basis from the literature reference that cites the alert.

5. APPLICABILITY DOMAIN

- Descriptor domain: The applicability domain is defined as having at least one chemical in a reference set with at least 30% global similarity to the test structure (using the Leadscope 27,000 chemical fragments as descriptors and the Tanimoto similarity score).

- Structural domain: Leadscope Predictive Data Miner is a software program for systematic sub‐structural analysis of a chemical using predefined structural features stored in a template library, training set‐dependent generated structural features (scaffolds) and calculated molecular descriptors. The feature library contains approximately 27,000 pre‐defined structural features and the structural features chosen for the library are motivated by those typically found in small molecules: aromatics, heterocycles, spacer groups, simple substituents. Leadscope allows for the generation of training set‐dependent structural features (scaffold generation), and these features can be added to the pre‐defined structural features from the library and be included in the descriptor selection process.

- Mechanistic domain: The global model identifies structural features and molecular descriptors which in the model development was found to be statistically significant associated with effect. Many predictions may indicate modes of action that are obvious for persons with expert knowledge for the endpoint

- Similarity with analogues in the training set: The original data set from Kazius et al. (2005) consisted of 4337 molecular structures with corresponding Ames test data.
The structural similarity of the test compound with respect to the training set compounds was analysed and quantified in terms of Tanimoto distance, which provides a quantitative measure of structural relatedness between the test compound and each training set compound. The 25 training set compounds found to be mostly similar to the test compound.

6. ADEQUACY OF THE RESULT
As can be seen from Annex A and B of the QPRF the result is considered adequate due to the presence of almost all structural features of the parent compound which can also be found in the training/validation dataset. Furthermore the prediction substantiate the experimental result for the substance of interest.
Qualifier:
according to guideline
Principles of method if other than guideline:
- Software tool(s) used including version: Leadscope model applier (v3.1.0)
- Model(s) used: Leadscope model applier (v3.1.0)
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Combination of results from the S. typhimurium histidine reversion gene mutation test using tester strains TA97, TA97a, TA1537, TA98, TA100, TA1535, TA102, E.coli (any variant)
Additional strain / cell type characteristics:
other: The QSAR prediction is based on results from all tester strains recommended by the OECD Test guideline
Evaluation criteria:
The model used was the Leadscope Applier which is a statistical model using structural fragments to set an alert. Only descrete organic compounds can be predicted. The model searches for structural fragments and combines them with eight molecular descriptors. Thus, a probability of either a negative or positive result is calculated. If experimental data are available the prediction of the statistical model may be overruled.
Key result
Species / strain:
other: not applicable for in silico study
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
The test item showed alerts for mutagenicity. Therefore the test item was considered to be mutagenic.
Cytotoxicity / choice of top concentrations:
other: not applicable
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
not applicable
Conclusions:
Based on the predictions performed with the statistical QSAR model Leadscope Applier Picolinamid-phenylether is mutagenic in a bacterial reverse mutation assay.
Executive summary:

In a QSAR prediction using Leadscope Model Applier (v3.1.0) the potential of Picolinamid-phenylether to induce mutagenicity was assessed. Leadscope uses two parameters to guide the applicability of model domain: 1) having at least one structural feature defined in the model in addition to all the property descriptors; 2) having at least one chemical in a training neighbourhood with at least 30% global similarity to the test structure. In this case the prediction is within the applicability domain, since 28 training compounds were identified in the model training set being structurally similar to the test compound.


 


The query structure does match some structural alerts or examples for (bacterial in vitro) mutagenicity in Leadscope.


Based on these results Picolinamid-phenylether is considered mutagenic as predicted by Leadscope.


 


This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13/07/2021 to 31/10/2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Rat S9-Liver-mix
- source of S9: GLP-Prüfeinrichtung Early Development Bayer, Genetic Toxicology Wuppertal
- method of preparation of S9 mix: Liver homogenates (S9: 9000 x g fraction) were isolated in house from the livers of Aroclor 1254-induced male Sprague-Dawley rats. The used S9 fraction was derived from preparation dated 26 Nov 2019, color code green (protein content 23.8 mg/mL).
For use, frozen aliquots of the S9 fraction were slowly thawed and mixed with a cofactor
solution (2+3 parts). The S9 mix contained 40 % S9 fraction to result in a final concentration
of 2 % S9 in cultures and was kept in refrigerator and used on the same day.
Vehicle / solvent:
- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: Generally, the test item was dissolved in a suitable solvent which was selected based on the solubility of the test item or according to the information given by the sponsor. Based on a solubility test, DMSO was selected as solvent. In this solvent the test item was soluble at least up to 200 mg/mL.

- Justification for percentage of solvent in the final culture medium: A 1 % (v/v) dilution of DMSO in the treatment medium was used as solvent control.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
vinblastine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: sextuplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): For the experiment about 2500 cells per well and 6 wells per concentration were seeded in 100 µL medium per well of a 96-well plate
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 and 24 h
- Harvest time after the end of treatment (sampling/recovery times): 24 h after treatment

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Approximately 24 hours after the start of treatment cells were harvested and then stained with EMA (Dye A). In addition, RNase and counting beads were added according to the pertinent instruction manual (version no. 171207) of the Micronucleus Analysis Kit (Litron).
Thereafter, cells were lysed and simultaneously the nuclei were stained with SYTOX (Dye B)
green. After this, samples were submitted to flow cytometric analysis.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The target sample size per well of approximately 3,000 nuclei for the determination of the frequency of micronuclei was met in most wells, if not limited by excessive toxicity. The percentage of micronuclei per nucleated events (%MN), indicative of clastogenic effects,
or hypodiploid nuclei per nucleated events (%HD), indicative of aneugenic effects, was
determined. In parallel, the proportion of nuclei stemming from apoptotic or necrotic cells
was detected (%A/N).
- %A/N = (A/N / Total Events) x100
- %MN = (MN / Nucleated) x100
- %HD = (HD / Nucleated) x100




METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative Increase in Nuclei Count (RINC)
Additionally, the number of nuclei originating from viable cells was related to an internal
standard (Cell Sorting Set-up Beads) as a measure of relative increase in nuclei count. For this relative increase in nuclei count, nuclei in cultures of up to 30 parallel wells were counted at the start of treatment time to determine start values.
The percentage was calculated as follows:
Mean (Nuclei/Beads) well 1-n Test Item - Mean (Nuclei/Beads) well 1-n Start
%RINC = ---------------------------------------------------------------------------------------------------------------------------- x 100
Mean (Nuclei/Beads) well 1-n SC- Mean (Nuclei/Beads) well 1-n Start

- Any supplementary information relevant to cytotoxicity:
Determination of Relative Cytotoxicity
Relative cytotoxic effects of the test item were assessed using the relative increase in nuclei
count (RINC) in the presence and absence of S9 mix. The results of the solvent controls were
set 100 % and compared to the test substance treated cultures. A change of the RINC relative
to the corresponding solvent control was calculated as follows:
Relative Cytotoxicity % = 100% - RINC %

Evaluation criteria:
Assessment Criteria
Providing that all acceptability criteria were fulfilled (see Chapter 5.3), the test item was considered to be positive if:
- the test item induced a micronucleus frequency in one of the test item concentrations
that is two-fold higher compared to the micronucleus frequency of concurrent solvent control
- at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical negative control data
Statistics:
Please refer to 'Any other information on material and methods incl. tables'
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No biologically relevant increases in the numbers of apoptotic/necrotic nuclei were detected; no biologically relevant increases in the numbers of hypodiploid nuclei were detected. both after 4 and 24 h
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
from 600µg/mL, biologically relevant and statistically significant increase in the numbers of micronuclei was detected. Moreover, a concentration-related trend in the micronucleus frequency across the increasing concentration levels with S9 was detected.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 600 µg/mL onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
After 24 hours treatment no biologically relevant or statistically significant increase of the micronucleus frequency was observed.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 140 µg/mL onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
after 4h at 24.7 µg/mL, no dose dependent increase
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 222.2 µg/mL onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Concentrations of the test item of up to 2000 µg/mL did not change the pH in the medium (phenol red-containing medium).
- Data on osmolality: The osmolality in the medium was not changed by concentrations of up to 2000 µg/mL test item (highest concentration tested), analyzed with an Osmometer (Gonotec).
- Precipitation and time of the determination: No


STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes: please refer to 'Any other information on results incl. tables'


Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:

o When cytokinesis block is not used: RICC, RPD or PD, as well as the number of cells treated and of cells harvested for each culture


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data): Please refer to 'any other information on results incl. tables'


































































































































































































































































































































Summary of the Results (4 Hours Treatment –S9 Mix)



 



Conc. µg/mL



%A/N



% MN



% HD



% Rel. Cytotoxicity



Precipitation (P)



Solvent control



0.0



0.5



0.9



0.1



--



no



Positive control MMC



0.1



3.0



18.5*



0.2



41.7



no



test item



0.91



0.6



1.0



0.1



11.4



no



 



2.74



0.6



1.1



0.1



1.7



no



 



8.23



0.7



1.1



0.1



2.4



no



 



24.7



0.9



1.8



0.1



7.3



no



 



74.1



0.6



1.0



0.1



6.8



no



 



222.2



0.5



1.2



0.0



22.4 a



no



 



666.7



0.6



1.2



0.0



59.5 a



no



 



2000



88.3



DIV/0!



DIV/0!



108.3 bc



no



a relevant cytotoxicity


b excessive cytotoxicity, (above limit = 55 ± 5%)


c excluded from assessment


* statistically significant increase in micronucleated events (P = < 0.05)



Summary of the Results (4 Hours Treatment +S9 Mix)



 



Conc. µg/mL



%A/N



% MN



% HD



% Rel. Cytotoxicity



Precipitation (P)



Solvent control



0.0



0.9



2.0



0.1



--



no



Positive control CP



2



3.2



18.5*



0.3



32.5 a



no



test item



200



0.8



1.9



0.1



0.0



no



 



300



0.8



1.8



0.1



1.1



no



 



400



0.9



2.0



0.1



0.0



no



 



470



1.0



2.1



0.1



1.6



no



 



530



0.8



2.1



0.1



8.4



no



 



600



0.8



2.4*



0.1



34.9 a



no



 



700



0.9



3.0*



0.2



39.3 a



no



 



800



1.0



3.1*



0.1



59.0 a



no



a relevant cytotoxicity


* statistically significant increase in micronucleated events (P = < 0.05)



 



Summary of the Results (24 Hours Treatment –S9 Mix)



 



Conc. µg/mL



%A/N



% MN



% HD



% Rel. Cytotoxicity



Precipitation (P)



Solvent control



0.0



0.6



1.0



0.1



--



no



Positive control VSS



0.0018



4.7



26.7*



7.8#



82.3 b



no



test item



10



0.6



1.0



0.1



11.6



no



 



30



0.6



1.0



0.1



0.0



no



 



90



0.6



0.8



0.1



1.2



no



 



110



0.6



0.8



0.1



10.0



no



 



140



0.8



0.7



0.1



27.3 a



no



 



170



0.9



1.0



0.1



48.3 a



no



 



200



1.3



0.8



0.1



67.3 bd



no



 



250



2.0



0.7



0.1



77.2 bc



no



a relevant cytotoxicity


b excessive cytotoxicity, (above limit = 55 ± 5%)


c excluded from assessment


d statistically analyzed despite excessive cytotoxicity


* statistically significant increase in micronucleated events (P = < 0.05)


# biologically relevant increase in hypodiploid events



 


Historical Controls


9000 – 18000 nuclei per study on flow cytometer MACSQuant 10 or Accuri C6 were evaluated.



































































































































Historical Controls 2018-2020, 4 Hours Treatment, 24 Hours Harvest Time



Solvent or


Substance



S9


Mix



Conc.



Number


of


Studies



Micronuclei in %



Mean



SD



Min



Max



Water



-



1% v/v



11



1.1



0.6



0.4



2.5



DMSO



-



1% v/v



150



1.0



0.4



0.4



2.0



Mitomycin C



-



0.1 µg/mL



168



15.8



3.7



7.0



27.4



Water



+



1% v/v



11



1.4



0.5



0.7



2.4



DMSO



+



1% v/v



162



1.2



0.4



0.5



2.2



CP



+



2 µg/mL



173



16.0



4.3



5.9



29.2



 



Historical Controls 2018 - 2020, 24 Hours Treatment, 24 Hours Harvest Time



Solvent or


Substance



S9


Mix



Conc.



Number


of


Studies



Micronuclei in %



Mean



SD



Min



Max



Water



-



1% v/v



12



1.2



0.8



0.4



2.9



DMSO



-



1% v/v



158



1.2



0.5



0.4



2.5



Vinblastine



-



0,0018 µg/mL



177



18.1



5.6



8.5



42.6



 


 


 


 


 


 


 

Conclusions:
The present study was conducted according to OECD guideline 487 (2016) Chinese hamster lung fibroblasts (V79) were exposed to Picolinamid phenylether at concentrations of 0, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, 666.7 and 2000 µg/mL without S9 mix and of 0, 200, 300, 400, 470, 530, 600, 700 and 800 µg/mL with S9 mix for 4h and with 10, 30, 90, 110, 140, 170, 200 and 250 µg/mL without S9 mix for 24 h. The frequency of micronuclei, apoptotic/necrotic nuclei and of hypodiploid nuclei were determined. A statistically significant and biologically relevant increase in the frequencies of micronuclei was seen after 4 hour treatment in the presence of S9 mix. In addition, a positive trend was demonstrated after 4 hours treatment with S9 mix. After 24 hours treatment without S9 mix with the test item no biologically relevant or statistically significant increase in the micronucleus frequency was observed. No biologically relevant increase in hypodiploid or apoptotic/necrotic nuclei was observed. Evaluation of the data indicates that the test item is genotoxic in the micronucleus test in vitro, when tested up to cytotoxic concentrations.
Executive summary:

In a mammalian cell micronucleus assay according to OECD guideline 487 (2016), V79 cells cultured in vitro were exposed to Picolinamid phenyletherin DMSO at concentrations of 0, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, 666.7 and 2000 µg/mL without S9 mix and of 0, 200, 300, 400, 470, 530, 600, 700 and 800 µg/mL with S9 mix for 4h and with  10, 30, 90, 110, 140, 170, 200 and 250 µg/mL without S9 mix for 24 h.


Picolinamid phenylether was tested up to cytotoxic concentrations (i.e., 140 µg/mL (24 h) and 222.2 µg/mL (4 h, without S9 mix) and 600 µg/mL (4h, with S9 mix). After 4 h treatment in the absence of metabolic activation a statistically significant increase in the frequency of micronuclei (up to two fold over solvent control) was observed. All percentages of micronucleated cells were within the range of the historical control data. Moreover, a concentration-dependent increase in the frequency of micronuclei was lacking. Therefore, the induction of micronuclei following treatment with the test item in the absence of S9 mix was considered as borderline. A statistically significant and biologically relevant increase in the frequencies of micronuclei was seen after 4 h treatment in the presence of S9 mix. In addition, a positive trend was demonstrated after 4 hours treatment with S9 mix. After 24 h treatment without S9 mix with the test item no biologically relevant or statistically significant increase in the micronucleus frequency was observed.


 


The positive controls did induce the appropriate response. There was a concentration related positive response of induced micronuclei over background.


 


This study is classified as acceptable.  This study satisfies the requirement for Test Guideline 487 for in vitro mammalian cell micronucleus data.


 


Based on the described results Picolinamid phenylether is considered genotoxic in the micronucleus test in vitro, when tested up to cytotoxic concentrations in Chinese hamster lung fibroblasts (V79).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct to Nov 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
yes
Remarks:
only the plate incorporation test was used
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene locus
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male rat liver S9 mix
Test concentrations with justification for top dose:
0, 50, 158, 500, 1581, 5000 µg/plate (first test, +/-S9 mix, all strains)
0, 156, 312, 624, 1248, 2496, 4992 µg/plate (repeat test, +S9 mix, TA 98 and TA 100 only)







Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
No solvent control was used since sufficient evidence was available in the literature and from testing laboratory experience, indicating that the solvents used had no influence on the spontaneous mutant counts of the used strains.
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
mitomycin C
other: nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), 2-aminoanthracene (all strains).
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E+06 cells/mL
- Test substance added in medium; in agar (plate incorporation)
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537 at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
+S9-mix: from 1581 µg to 5000 µg and 2nd experiment: from 624 µg to 4992 µg
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
+S9-mix: from 500 µg to 5000 µg and 2nd experiment: from 624 µg to 4992 µg
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Summary of results from the Salmonella mutagenicity assay (first test) with Picolinamid-Phenylether (mean values of revertants per plate)

 Dose (µg per plate)

Without metabolic activation

 

TA 1535

 TA 100

 TA 1537

 TA 98

 TA 102

0

8

135

8

38

267

50

10

135

7

36

256

158

9

158

6

29

262

500

9

153

5

39

245

1581

9

158

5

37

218

5000

11

145

6

39

192

 Positive control

758*

317*

88*

150*

572* 

 Dose (µg per plate )

With metabolic activation (liver S9 mix)

 

TA 1535

 TA 100

 TA 1537

 TA 98

TA 102

0

11

178

11

54

269

50

12

177

8

55

249

158

11

211

9

70

305

500

11

233*

8

76

296

1581

8

243*

10

120*

276

5000

10

294*

14

183*

244

 Positive control

131*

1661*

281*

1411*

690*

 

* = mutagenic effect

 

Table 2: Summary of results from the Salmonella mutagenicity assay (repeat test with strains TA 98 and TA 100 and metabolic activation) with Picolinamid-Phenylether (mean values of revertants per plate)

 

Dose (µg per plate)

With metabolic activation (liver S9 mix)

 

TA 1535

 TA 100

 TA 1537

 TA 98

 TA 102

0

-

153

-

41

-

156

-

201

-

60

-

312

-

190 

-

65

-

624

-

221*

-

87*

-

1248

-

241*

-

101*

-

2496

-

240*

-

140*

-

4992

-

260*

-

203*

-

 Positive control

-

1592* 

1305* 

 

* = mutagenic effect

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed.

Evidence of mutagenic activity of Picolinamid-Phenylether was seen. On Salmonella typhimurium TA 100 and TA 98, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. The lowest effective dose was 500 µg per plate for TA 100 and 624 µg per plate for TA 98. The Salmonella/microsome test thus showed Picolinamid-Phenylether to have a mutagenic effect.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

.
Conclusions:
The mutagenic potential of Picolinamid-Phenylether was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Evidence of mutagenic activity of Picolinamid-Phenylether was seen. On Salmonella typhimurium strains TA 100 and TA 98, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. The lowest effective dose was 500 µg per plate for strain TA 100 and 624 µg per plate for strain TA 98. The Salmonella/microsome test thus showed Picolinamid-Phenylether has a mutagenic effect.



Executive summary:

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 98, TA 100, TA 102, TA 1535, and TA 1537 of S. typhimurium were exposed to Picolinamid-phenylether in DMSO at concentrations of 0, 50, 158, 250, 500, 1581, and 5000 µg/plate in the first experiment in the presence and absence of mammalian metabolic activation using the plate incorporation method and 0, 156, 312, 624, 1248, 2496, 4992 µg/plate in the repeat test, +S9 mix, TA 98 and TA 100 only.


 


The test item was tested up to the limit concentration of 5000 µg/plate. Three (TA 102, TA 1535, and TA 1537) of the five tester strains showed no increased reversion to prototrophy at different concentrations tested between 50 and 5000 µg/plate, either in the absence or presence of S9 mix. The tester strains TA 98 and TA 100 showed an increased reversion to prototrophy from 1581 and 500 µg per plate onwards in the first experiment and from 624 µg per plate onwards in the second experiment with metabolic activation. There was no increase without metabolic activation in the first experiment. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was not observed up to the highest concentrations tested (5000 µg/plate) in all strains with or without metabolic acitvation. There were no precipitates in the agar found at all concentrations in all strains used in the tests without and with S9 mix.


 


This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.


 


Evaluation of the data does indicate a mutagenic potential of the test substance in the tester strains S. typhimurium TA 98 and TA 100 with metabolic activation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 98, TA 100, TA 102, TA 1535, and TA 1537 of S. typhimurium were exposed to Picolinamid-phenylether in DMSO at concentrations of 0, 50, 158, 250, 500, 1581, and 5000 µg/plate in the first experiment in the presence and absence of mammalian metabolic activation using the plate incorporation method and 0, 156, 312, 624, 1248, 2496, 4992 µg/plate in the repeat test, +S9 mix, TA 98 and TA 100 only.


The test item was tested up to the limit concentration of 5000 µg/plate. Three (TA 102, TA 1535, and TA 1537) of the five tester strains showed no increased reversion to prototrophy at different concentrations tested between 50 and 5000 µg/plate, either in the absence or presence of S9 mix. The tester strains TA 98 and TA 100 showed an increased reversion to prototrophy from 1581 and 500 µg per plate onwards in the first experiment and from 624 µg per plate onwards in the second experiment with metabolic activation. There was no increase without metabolic activation in the first experiment. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was not observed up to the highest concentrations tested (5000 µg/plate) in all strains with or without metabolic acitvation. There were no precipitates in the agar found at all concentrations in all strains used in the tests without and with S9 mix.


This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.


Evaluation of the data does indicate a mutagenic potential of the test substance in the tester strains S. typhimurium TA 98 and TA 100 with metabolic activation.


 


 


In a mammalian cell micronucleus assay according to OECD guideline 487 (2016), V79 cells cultured in vitro were exposed to Picolinamid phenyletherin DMSO at concentrations of 0, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, 666.7 and 2000 µg/mL without S9 mix and of 0, 200, 300, 400, 470, 530, 600, 700 and 800 µg/mL with S9 mix for 4h and with  10, 30, 90, 110, 140, 170, 200 and 250 µg/mL without S9 mix for 24 h.


Picolinamid phenylether was tested up to cytotoxic concentrations (i.e., 140 µg/mL (24 h) and 222.2 µg/mL (4 h, without S9 mix) and 600 µg/mL (4h, with S9 mix). After 4 h treatment in the absence of metabolic activation a statistically significant increase in the frequency of micronuclei (up to two fold over solvent control) was observed. All percentages of micronucleated cells were within the range of the historical control data. Moreover, a concentration-dependent increase in the frequency of micronuclei was lacking. Therefore, the induction of micronuclei following treatment with the test item in the absence of S9 mix was considered as borderline. A statistically significant and biologically relevant increase in the frequencies of micronuclei was seen after 4 h treatment in the presence of S9 mix. In addition, a positive trend was demonstrated after 4 hours treatment with S9 mix. After 24 h treatment without S9 mix with the test item no biologically relevant or statistically significant increase in the micronucleus frequency was observed.


The positive controls did induce the appropriate response. There was a concentration related positive response of induced micronuclei over background.


This study is classified as acceptable.  This study satisfies the requirement for Test Guideline 487 for in vitro mammalian cell micronucleus data.


Based on the described results Picolinamid phenylether is considered genotoxic in the micronucleus test in vitro, when tested up to cytotoxic concentrations in Chinese hamster lung fibroblasts (V79).


 


 


In a QSAR prediction using Leadscope Model Applier (v3.1.0) the potential of Picolinamid-phenylether to induce mutagenicity was assessed. Leadscope uses two parameters to guide the applicability of model domain: 1) having at least one structural feature defined in the model in addition to all the property descriptors; 2) having at least one chemical in a training neighbourhood with at least 30% global similarity to the test structure. In this case the prediction is within the applicability domain, since 28 training compounds were identified in the model training set being structurally similar to the test compound.


The query structure does match some structural alerts or examples for (bacterial in vitro) mutagenicity in Leadscope.


Based on these results Picolinamid-phenylether is considered mutagenic as predicted by Leadscope.


This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.


 


 


In a QSAR prediction using DEREK Nexus v6.1 the potential of Picolinamid-phenylether to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have been shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.


The query structure does match a structural alert or examples for (bacterial in vitro) mutagenicity in Derek.


Based on these results Picolinamid-phenylether is considered mutagenic as predicted by DEREK Nexus.


This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.


 


Based on the available experimental and in silico data that exhibited a mutagenic potential for Picolinamid-phenylether and gave strong alerts for a mutagenic effect, respectively, Picolinamid-phenylether is classified according to Regulation (EU) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) as mutagenic Category 2, 'Suspected of causing
genetic defects'.

Justification for classification or non-classification

Based on the study results a classification as mutagenic Categogy 2 according to Regulation (EC) No. 1272/2008 (CLP) is warranted.