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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Epoxide valerate is negative with and without metabolic activation in bacterial reverse mutation assays with the S. typhimurium strains TA 98, TA 100, TA1535, TA1537 and TA1538 using the direct plate incorporation procedure (Lang and Schmitt, 1991a) as well as the preincubation method (Lang and Schmitt, 1991b).


Additionally, a QSAR prediction was performed using DEREK Nexus v6.1. According to the prediction the test item is not mutagenic.

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:
May 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)shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are ynthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay unitsapplied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.

- 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 and the absence of any strucutral alert, 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
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:
negative
Remarks:
The test item showed no alerts for mutagenicity and has a negative Ames test in four strains. Therefore Epoxid-Valerate was considered to be non-mutagenic and was assigned to mutagenic impurity class 5.
Cytotoxicity / choice of top concentrations:
other: not applicable for in silico study
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 Epoxide-Valerate is nor mutagenic in a bacterial reverse mutation assay.
Executive summary:

In a QSAR prediction using DEREK Nexus v6.1 the potential of Epoxide-Valerate 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 beenshown 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 not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Although the query structure does  contain at least an unclassified feature and is consequently predicted to be indeterminate in the bacterial in vitro (Ames) mutagenicity test. However, experimental data are available clearly reporting a negative result.


Based on these results Epoxide-Valerate is considered non-mutagenic as predicted by DEREK Nexus.


This study is classified as acceptable for assessment based on methodolgy 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:
experimental study
Adequacy of study:
key study
Study period:
March 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
26 May 1983
Deviations:
no
Principles of method if other than guideline:
Direct plate incorporation procedure was performed. No E. coli WP2 or S. typhimurium TA102 strain tested; no preincubation test performed. These requirements were first formulated in the adoption of the guideline in 1997 and thus the study was conducted prior to implementation of these requirements. The current OECD TG 471 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system.
GLP compliance:
yes
Remarks:
- but a QA check was not performed
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene locus
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
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 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:
0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
cyclophosphamide
other: with metabolic activation: 2-AA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Between 13.3 and 24 xE+08 cells/mL
- Test substance added in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition


Evaluation criteria:
The plates were scored for the number of mutant colonies with an automated colony counter (Artek M 982B, Artek Systems Corporation, Farmingdale, NY, USA). The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of induced revertants compared to the number of spontaneous ones was reproducibly higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.
Species / strain:
S. typhimurium TA 102
Remarks:
or E.coli WP2 uvr A
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
Remarks:
The study was conducted prior to implementation of the fifth tester strain into OECD guideline 471. However, based on the additionally performed QSAR analysis, the substance showed no alert for mutagenicity. Please refer to the respective robust study sum
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
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
Remarks:
Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no data
- Data on osmolality: not data
- Precipitation and time of the determination: Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
0.05 ml of the solvents (0.1 M sodium phosphate buffer or DMSO) were plated as negative controls. In order to check the activity of the metabolizing system and the mutability of the bacteria at least two reference mutagens were tested for each strain. Their mutagenic effect occurred either directly {9-AA, 2-NF, NaN3} or after metabolic activation (2-AA, BP, CP). Sterility controls were performed additionally.
According to the evaluation criteria the vehicle controls and the positive control were valid.
Please refer to the tables under any other information on results incl. tables.


Ames test:
- Signs of toxicity: no
- Individual plate counts:
TA1535: 226, 259, 236
TA1537: 126, 127, 146
TA1538: 188, 167, 176
TA100: 193, 198, 178
TA98: 206, 242, 243
- Mean number of revertant colonies per plate and standard deviation:
Please refer to table 2 under any other information on results incl. tables.

None of the five tester strains showed increased reversion to prototrophy with the test item at the concentrations tested, either in the absence or presence of S9 mix.


 


Growth inhibition of the background lawn was not observed. Precipitates in the agar were found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with metabolic activation.


 


Negative controls and positive controls with known mutagens (9-acridinamine hydrochloride, anthracen-2-amine, benzo(a)pyrene, cyclophosphamide, 2-nitro-9H-fluorene, sodium azide) produced the expected numbers of revertant colonies.


Table 1: Negative and positive control data:



































































































































 



TA1535



TA100



TA1537



TA1538



TA98



 



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



DMSO



20±5



14±4



106±11



88±14



9±4



12±1



17±6



41±4



29±6



40±9



phosphate buffer



19±4



12±3



107±4



88±5



8±4



15±4



19±1



41±5



36±2



44±3



2-AA



16±2



102±19



131±13



802±84



10±1



234±13



19±4



1501±62



38±7



1610±19



CP



23±3



89±3



 



 



 



 



 



 



 



 



BaP



 



 



107±8



622±36



10±3



59±7



23±7



135±27



27±3



283±26



2-NF



 



 



 



 



 



 



1046±63



470±32



937±93



420±33



NaN3



630±57



88±9



699±45



145±11



 



 



 



 



 



 



9-AA



 



 



 



 



89±18



37±9



 



 



 



 



mean values ± SD


Table 2:Mean number of revertant colonies per plate and standard deviation



































































































































 



TA1535



TA100



TA1537



TA1538



TA98



 



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



DMSO



20±5



14±4



106±11



88±14



9±4



12±1



17±6



41±4



29±6



40±9



phosphate buffer



19±4



12±3



107±4



88±5



8±4



15±4



19±1



41±5



36±2



44±3



0.10



17±4



14±5



106±11



89±3



7±4



15±2



18±6



32±6



33±11



38±8



0.25



21±4



13±1



102±7



87±3



9±4



15±6



16±1



35±11



31±4



45±12



0.50



23±4



15±3



99±7



105±10



8±1



12±1



18±7



37±5



33±4



44±4



1.00



24±2



16±5



123±14



92±8



10±2



11±9



18±4



47±5



30±3



39±5



2.50



19±4



12±1



116±9



112±5



8±3



14±4



19±2



42±12



34±12



47±3



5.00



23±12



14±2



121±8



112±4



6±2



13±3



23±4



36±4



32±6



40±5



 


 


 


 

Conclusions:
The test item was tested for mutagenic activity effects in five histidine-dependent strains of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538 ) using the direct plate incorporation procedure equivalent to OECD TG 471. The study was performed with and without metabolic activation, employed a range of the test item concentrations from 0.1 to 5.0 mg per plate. No increased reversion to prototrophy was seen neither without nor with metabolic activation. Growth inhibition of the background lawn was not observed. Precipitation was found starting at 1 mg/plate without S9 mix and at 2.5 mg/plate with metabolic activation. Therefore, the test item was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 26 May 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Epoxide-Valerate in DMSO at concentrations of 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.


The test item was tested limit concentration 5000 µg/plate. None of the five tester strains showed increased reversion to prototrophy at any of the concentrations tested between 100 and 5000 µg/plate, either in the absence or presence of S9 mix. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was not observed. Precipitates in the agar were found starting at 1000 µg/plate without S9 mix and at 2500 µg/plate with metabolic activation.


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


The test material is considered non-mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
26 May 1983
Deviations:
no
Principles of method if other than guideline:
Preincubation procedure was performed. no E. coli WP2 or S. typhimurium TA102 strain tested; no direct plate incorporation test performed. These requirements were first formulated in the adoption of the guideline in 1997 and thus the study was conducted prior to implementation of these requirements. The current OECD TG 471 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system.
GLP compliance:
yes
Remarks:
- but a QA check was not performed
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene locus
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
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 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:
0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
cyclophosphamide
other: with metabolic activation: 2-AA, DMNA.
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Between 14.6 and 30.2 xE+08 cells /mL
- Test substance added in preincubation method

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:
The plates were scored for the number of mutant colonies with an automated colony counter (Artek M 9828, Artek Systems Corporation, Farmingdale, NY, USA). The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of induced revertants compared to the number of spontaneous ones was reproducibly higher than 2-fold. Also a dose-dependent increase in the number of revertants was considered to indicate a mutagenic effect. A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicroscopically.
Species / strain:
S. typhimurium TA 102
Remarks:
or E.coli WP2uvr A
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
Remarks:
The study was conducted prior to implementation of the fifth tester strain into OECD guideline 471. However, based on the additionally performed QSAR analysis, the substance showed no alert for mutagenicity. Please refer to the respective robust study sum
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no data
- Data on osmolality: not data
- Precipitation and time of the determination: Precipitates in the agar were found starting at 0.5 mg/plate without S 9 mix and generally at 2.5 mg/plate with activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
0.025 ml of the solvents were plated as negative controls. In order to check the activity of the metabolizing system and the mutability of the bacteria at least two reference mutagens were tested for each strain. Their mutagenie effeet oceurred either directly (9-M, 2-NF, NaN3) or after metabolic activation (2-AA, BP, CP, DMNA). Sterility controls were performed additionally.
According to the evaluation criteria the vehicle controls and the positive control were valid.
Please refer to the tables under any other information on results incl. tables.


Ames test:
- Signs of toxicity: no
- Individual plate counts:
TA1535: 286, 295, 324
TA1537: 177, 165, 170
TA1538: 125, 153, 160
TA100: 276, 254, 300
TA98: 261, 241, 263
- Mean number of revertant colonies per plate and standard deviation:
Please refer to table 2 under any other information on results incl. tables.

None of the five tester strains showed increased reversion to prototrophy with the test item at the concentrations tested, either in the absence or presence of S9 mix.


 


Growth inhibition of the background lawn was not observed. Precipitates in the agar were found starting at 0.5 mg/plate without S9 mix and generally at 2.5 mg/plate with metabolic activation.


 


Negative controls and positive controls with known mutagens (9-acridinamine hydrochloride, anthracen-2-amine, benzo(a)pyrene, cyclophosphamide, 2-nitro-9H-fluorene, sodium azide, N-nitrosodimethylamine) produced the expected numbers of revertant colonies.


Table 1: Negative and positive control data:





























































































































































 



TA1535



TA100



TA1537



TA1538



TA98



 



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



DMSO



31±1



24±4



123±12



141±12



7±2



11±2



21±6



29±6



44±6



49±5



phosphate buffer



29±11



19±2



123±7



133±13



8±4



12±4



19±1



27±5



34±2



46±3



2-AA



26±23



159±11



142±6



786±72



0±0



138±11



16±4



627±14



27±8



684±14



DMNA



 



 



131±6



1084±0



 



 



 



 



 



 



CP



39±10



556±36



 



 



 



 



 



 



 



 



BaP



 



 



 



 



9±2



97±24



18±8



442±33



34±4



445±86



acridinamine



 



 



 



 



19±0



160±17



 



 



 



 



2-NF



 



 



 



 



 



 



1103±53



180±13



865±7



273±33



NaN3



212±14



19±8



318±4



149±7



 



 



 



 



 



 



9-AA



 



 



 



 



 



 



 



 



 



 



 


Table 2: Mean number of revertant colonies per plate and standard deviation



































































































































 



TA1535



TA100



TA1537



TA1538



TA98



 



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



DMSO



31±1



24±4



123±12



141±12



7±2



11±2



21±6



29±6



44±6



49±5



phosphate buffer



29±11



19±2



123±7



133±13



8±4



12±4



19±1



27±5



34±2



46±3



0.10



25±7



20±8



119±8



133±15



8±3



11±2



22±5



28±3



46±12



53±8



0.25



23±10



19±3



127±11



144±21



7±1



11±5



18±5



24±2



39±6



51±7



0.50



19±8



18±5



140±9



144±4



6±5



12±1



20±4



32±4



38±5



55±15



1.00



21±6



19±2



127±4



133±16



9±4



9±2



20±3



30±7



42±10



52±4



2.50



26±2



17±1



144±4



142±4



7±2



5±3



20±2



28±4



47±3



51±8



5.00



21±4



23±5



144±8



135±5



8±3



7±217±2



17±2



29±2



36±6



42±5



 


 


 

Conclusions:
The test item was tested for mutagenic activity effects in five histidine-dependent strains of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) using the preincubation procedure equivalent to OECD TG 471. The study was performed with and without metabolic activation, employed a range of the test item concentrations from 100 to 5000 µg per plate. No increased reversion to prototrophy was seen neither without nor with metabolic activation. Growth inhibition of the background lawn was not observed. Precipitation was found starting at 500 µg/plate without S9 mix and generally at 2500 µg/plate with metabolic activation. Therefore, the test item was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 26 May 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Epoxide-Valerate in DMSO at concentrations of 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method.


The test item was tested limit concentration 5000 µg/plate. None of the five tester strains showed increased reversion to prototrophy at any of the concentrations tested between 100 and 5000 µg/plate, either in the absence or presence of S9 mix. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was not observed. Precipitates in the agar were found starting at 500 µg/plate without S9 mix and at 2500 µg/plate with metabolic activation.


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


The test material is considered non-mutagenic under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

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 26 May 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Epoxide-Valerate in DMSO at concentrations of 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.


 


The test item was tested limit concentration 5000 µg/plate. None of the five tester strains showed increased reversion to prototrophy at any of the concentrations tested between 100 and 5000 µg/plate, either in the absence or presence of S9 mix. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was not observed. Precipitates in the agar were found starting at 1000 µg/plate without S9 mix and at 2500 µg/plate with metabolic activation.


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


The test material is considered non-mutagenic under the conditions of the test.


 


In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 26 May 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Epoxide-Valerate in DMSO at concentrations of 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method.


The test item was tested limit concentration 5000 µg/plate. None of the five tester strains showed increased reversion to prototrophy at any of the concentrations tested between 100 and 5000 µg/plate, either in the absence or presence of S9 mix. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was not observed. Precipitates in the agar were found starting at 500 µg/plate without S9 mix and at 2500 µg/plate with metabolic activation.


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


The test material is considered non-mutagenic under the conditions of the test.


Both bacterial reverse mutation assays were performed prior to implementation of the additonal fifth tester strain (i.e. S. typhimurium TA102 or E. coli WP2 uvr A) to detect certain oxidizing mutagens, cross-linking agents and hydrazines.


In general the query substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, testing without the additional tester strains is basically considered sufficient to evaluate the mutagenic activity in a bacterial test system. However, additionally a QSAR preditcion was performed to substantiate the aforementioned results.


In a QSAR prediction using DEREK Nexus v6.1 the potential of Epoxide-Valerate 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 not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Although the query structure does  contain at least an unclassified feature and is consequently predicted to be indeterminate in the bacterial in vitro (Ames) mutagenicity test. However, experimental data are available clearly reporting a negative result.


Based on these results Epoxide-Valerate is considered non-mutagenic as predicted by DEREK Nexus.


This study is classified as acceptable for assessment based on methodolgy and documentation. The data is part of an overall assessment.


In summary, Epoxide-Valerate is not mutagenic under the conditions of the described test systems nor in the QSAR prediction. The results are considered to be sufficient for non-classification according to Regulation (EU) No. 1272/2008 (CLP) with respect to mutagenic activity in a bacterial test system.


 

Justification for classification or non-classification

Based on the study results a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.