N-benzylprop-2-enamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance (in DMSO) was tested in the Ames test in Salmonella typhimurium strains TA 1535, TA1537, TA98 and TA100 and in E.coli strain WP2uvrA at concentrations of 3 to 5000 ug/plate with and without metabolic activation.

No cytotoxicity or precipitate was observed up to 5000 ug/plate. The number of revertants was not increased in any of the evaluated concentrations (100 -5000 ug/plate). Vehicle and positive controls included gave results within the historical control ranges. An independent repeat with the same evaluated concentrations gave a similar result. It is therefore concluded that the substance is not mutagenic in bacteria with and without metabolic activation.

In a chromosome aberration assay the substance was tested in 2 independent assays with and without metabolic activation.

In the first assay human peripheral lymphocytes were exposed during 3 hours (24 fixation time with colchicine added 2.5 -3 h before fixation) in absence and presence of metabolic activation at concentrations of 333, 1000 and 1612 ug/mL (highest dose is 0.01 M). No increase of the number of aberrations was seen at any of the concentrations tested. Vehicle (DMSO) and positive controls were within the expected ranges.

In the second assay human peripheral lymphocytes were exposed during 24 hours (24 h fixation time with colchicine added 2.5 -3 h before fixation) in absence of metabolic activation at concentrations of 100, 200, 400, 600, 800,1000, 1200, 1400 and 1612 ug/mL Based on mitotic index the concentrations evaluated were 100, 200 and 600 ug/mL. No increase of the number of aberrations was seen at any of the concentrations tested. Vehicle (DMSO) and positive controls were within the expected ranges.

In an addtional assay human peripheral lymphocytes were exposed during 48 hours (48 h fixation time with colchicine added 2.5 -3 h before fixation) in absence of metabolic activation at concentrations of 33, 100, 300, 400, 500, 600, 700, 800, 900 and 1000 ug/mL. Based on mitotic index the concentrations evaluated were 100, 200 and 600 ug/mL. No increase of the number of aberrations was seen at any of the concentrations tested. Vehicle (DMSO) and positive controls were within the expected ranges.

In another assay human peripheral lymphocytes were exposed during 3 hours (48 fixation time with colchicine added 2.5 -3 h before fixation) with metabolic activation at concentrations of 333, 1000 and 1612 ug/mL (highest dose is 0.01 M). No increase of the number of aberrations was seen at any of the concentrations tested. Vehicle (DMSO) and positive controls were within the expected ranges.

It can be concluded that the substance is not clastogenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2007 to 19 February 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His+ (S. typhimurium), Trp+ (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver of phenobarbital/beta-naphthoflavone (80/100 mg/kg bw) treated rats
- method of preparation of S9 mix (10 mL): A mixture of 30 mg NADP and 15.2 mg glucose-6-phosphate dissolved in 5.0-5.5 mL Milli-Q water, 2 mL 0.5 M sodium phosphate buffer, 1 mL 0.08 M MgCl2 and 1 mL 0.33 M KCl was filter (0.22 um) sterilized. To this 0.5 to 1 mL S9 homogenate was added
- concentration or volume of S9 mix and S9 in the final culture medium : experiment 1 5%, experiment 2 10%

Test concentrations with justification for top dose:

experiment 1: 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate with and without metabolic activation (evaluated concentrations 100-5000 ug/plate)
experiment 2: 100, 333, 1000, 3330 and 5000 ug/plate with and without metabolic activation

Maximum concentration based on the absence of toxicity (bacterial background lawn, size of micro colonies and number of revertants) and precipitate in a dose range finding study in TA100 and TA98 at 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate with and without metabolic activation

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplcate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density: 1.0E09 cells/mL
- Test substance in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; size of micro-colonies and number of revertants

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
TA100 number of revertants >2 times vehicle control
TA98, TA1535, TA1537 and Wp2uvrA number of revertants >3 times vehicle control
Negative response confirmed in separate experiment

Evaluation criteria:

TA100 number of revertants >2 times vehicle control
TA98, TA1535, TA1537 and Wp2uvrA number of revertants >3 times vehicle control
Negative response confirmed in separate experiment

Statistics:

NA

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

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within historical control ranges

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

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within historical control ranges

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

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within historical control ranges

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

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within historical control ranges

Species / strain:

E. coli WP2 uvr A

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

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within historical control ranges

see attached document

Conclusions:

The substance is not mutagenic in bacteria with and without metabolic activation

Executive summary:

The substance (in DMSO) was tested in the Ames test in Salmonella typhimurium strains TA 1535, TA1537, TA98 and TA100 and in E.coli strain WP2uvrA at concentrations of 3 to 5000 ug/plate with and without metabolic activation.

No cytotoxicity or precipitate was observed up to 5000 ug/plate. The number of revertants was not increased in any of the evaluated concentrations (100 -5000 ug/plate). Vehicle and positive controls included gave results within the historical control ranges. An independent repeat with the same evaluated concentrations gave a similar result.

It is therefore concluded that the substance is not mutagenic in bacteria with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 October 2007 to 19 December 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

NA

Species / strain / cell type:

lymphocytes: primary lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human peripheral lymphocytes
- Suitability of cells: standard according to the guideline

For lymphocytes:
- Sex, age and number of blood donors:
range finding: male 37 years, AGT 14.5 h
1st exp: male 30 years, AGT 13.5 h
2nd exp: male 40 years, AGT 13.9 h
- Whether whole blood or separated lymphocytes were used: whole blood treated with heparine
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED : RPMI1640 medium supplemented with 20% v/v foetal calf serum, L-glutamine, penicillin/streptomycin and 30U/mL heparin
CO2 concentration 5 mg/L, humidity level 66-91%, temperature 34.8-37.4 ˚C (deviations were evaluated as of no influence on the study result).

Cytokinesis block (if used):

colchicine

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: liver of phenobarbital/beta-naphthoflavone (80/100 mg/kg bw) treated rats
- method of preparation of S9 mix: A mixture in 0.5 M sodium phosphate buffer of 3.4 mg/mL NADP, 1.7 mg/mL glucose-6-phosphate, 1.63 mg/mL MgCl2, 2.46 mg/mL KCl and 4 umol HEPES was filter (0.22 um) sterilized. To this 0.5 to 1 mL S9 homogenate was added
- concentration or volume of S9 mix and S9 in the exposure medium: 1.8 %v/v

Test concentrations with justification for top dose:

exp 1: with and without S9: 333, 1000 and 1612 ug/mL (highest dose is 0.01 M)
exp 2: with S9: 333, 1000 and 1612 ug/mL (highest dose is 0.01 M)
without S9 24 exposure: 100, 200, 400, 600, 800,1000, 1200, 1400 and 1612 ug/mL (based on mitotic index evaluated concentrations selected were 100, 200 and 600 ug/mL)
without S9 48 exposure: 33, 100, 300, 400, 500, 600, 700, 800, 900 and 1000 ug/mL (based on mitotic index evaluated concentrations selected were 100, 500 and 600 ug/mL)

Vehicle / solvent:

DMSO (HBBS for positive controls)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preculture for 48 hours
- Exposure duration/duration of treatment: exp 1: 3 h (24 h fixation time)
exp 2: with S9 3 h (48 h fixation time); without S9 24 h (24 h fixation time), 48 h (48 h fixation time)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colchicine (0.5 ug/mL medium) at 2.5-3 hours before fixation
- Methods of slide preparation and staining technique: cultures were centrifuged (1300 rpm) and the supernatant was discarded, The cells were swollen during 5 min with potassium chloride and fixed with 3 changes of methanol. Fixed cells were dropped on slides (2/culture) and stained with Giemsa during 10-30 min.
- Number of cells spread and analysed per concentration: 100 metaphases per culture were analysed for chromosomal aberrations by light microscopy (1000 metaphases were counted for assessment of mitotic index)
- Determination of polyploidy: included, but none found
- Determination of endoreplication: included, but none found

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, mitotic index (MI)

Evaluation criteria:

positive: if it induced a dose-related statistically significant increase in the number of chromosomal aberration compared with the concurrent negative control; if a statistically significant and biologically relevant increase in the number of chromosomal aberrations is observed without dose-response relationship

Species / strain:

lymphocytes: huma

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

in all 3 h experiments

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

24 h exposure cytotoxicity at 800 ug/mL, 48 h exposure cytotoxicity at 600 ug/mL

Positive controls validity:

valid

Additional information on results:

details see attached tables

Conclusions:

The substance did not induce chromosomal aberrations in presence and absence of metabolic activation

Executive summary:

In a chromosome aberration assay the substance was tested in 2 independent assays with and without metabolic activation.

In the first assay human peripheral lymphocytes were exposed during 3 hours (24 fixation time with colchicine added 2.5 -3 h before fixation) in absence and presence of metabolic activation at concentrations of 333, 1000 and 1612 ug/mL (highest dose is 0.01 M). No increase of the number of aberrations was seen at any of the concentrations tested. Vehicle (DMSO) and positive controls were within the expected ranges.

In the second assay human peripheral lymphocytes were exposed during 24 hours (24 h fixation time with colchicine added 2.5 -3 h before fixation) in absence of metabolic activation at concentrations of 100, 200, 400, 600, 800,1000, 1200, 1400 and 1612 ug/mL Based on mitotic index the concentrations evaluated were 100, 200 and 600 ug/mL. No increase of the number of aberrations was seen at any of the concentrations tested. Vehicle (DMSO) and positive controls were within the expected ranges.

In an addtional assay human peripheral lymphocytes were exposed during 48 hours (48 h fixation time with colchicine added 2.5 -3 h before fixation) in absence of metabolic activation at concentrations of 33, 100, 300, 400, 500, 600, 700, 800, 900 and 1000 ug/mL. Based on mitotic index the concentrations evaluated were 100, 200 and 600 ug/mL. No increase of the number of aberrations was seen at any of the concentrations tested. Vehicle (DMSO) and positive controls were within the expected ranges.

In another assay human peripheral lymphocytes were exposed during 3 hours (48 fixation time with colchicine added 2.5 -3 h before fixation) with metabolic activation at concentrations of 333, 1000 and 1612 ug/mL (highest dose is 0.01 M). No increase of the number of aberrations was seen at any of the concentrations tested. Vehicle (DMSO) and positive controls were within the expected ranges.

It can be concluded that the substance is not clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information the substance does not need to be classified for mutagenicity according to EU Regulation 1272/2008 (CLP).