Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Negative Ames test performed with 3-PPB Negative mouse bone-marrow micronucleus test with benzyl alcohol Negative mouse bone-marrow micronucleus test with benzyl acetate Negative rat bone marrow chromosome aberration test with sodium benzoate Negative rat dominant lethal assay with sodium benzoate Negative sex-linked recessive lethal mutations in Drosophila melanogaster with benzyl acetate
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 December 2013 to 17 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to approved guidelines and in compliance with GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Copy of certificate of compliance from UK GLP Monitoring Authority included in report
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compound insoluble in water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period:
- Exposure duration: 72h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers
of at least twice (three times in the case of strains TA1535 and TA1537) that of the
concurrent vehicle controls, with some evidence of a positive concentration-response
relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test substance does not produce a reproducible increase in revertant colony
numbers, it is considered to show no evidence of mutagenic activity in this test system. No
statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response,
even after additional testing, the test data may be subjected to analysis to determine the
statistical significance of any increases in revertant colony numbers. The statistical
procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test
followed, if appropriate, by trend analysis. Biological importance will be considered along
with statistical significance. In general, treatment-associated increases in revertant colony
numbers below two or three times those of the vehicle controls (as described above) are not
considered biologically important. It should be noted that it is acceptable to conclude an
equivocal response if no clear results can be obtained
Statistics:
The mean number and standard deviation of revertant colonies were calculated for all groups.
The “fold-increases” relative to the vehicle controls were calculated in order to compare the
means for all treatment groups with those obtained for the vehicle control groups.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
It was concluded that 3-PPB showed no evidence of mutagenic activity in this bacterial
system under the test conditions employed.
Executive summary:

No evidence of toxicity was obtained following exposure to 3-PPB. No precipitate was observed on any plates containing 3-PPB. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to 3-PPB at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

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

Additional information

Additional information from genetic toxicity in vitro:

The mutagenic potential of 3-PPB was assessed in Ames testin a new GLP study in accordance with current OECD guideline No 471 (HLS report No OR0064, 2014). No evidence of toxicity was obtained following exposure to 3-PPB. No precipitate was observed on any plates containing 3-PPB. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to 3-PPB at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Genotoxicity studies in vivoperformed with the category members have been used to investigate the genotoxicity of 3 -PPB in mammalian cells.

Micronucleus tests with benzyl acetate or benzyl alcohol have been found to be negative for induction of bone marrow micronuclei in mice treated intraperitoneally with a single dose at 0, 50, 100, or 200 mg/kg benzyl alcohol or 0, 312.5, 625 or 1250 mg/kg benzyl acetate.

Sodium benzoate produced no detectable significant increase in the number of aberrations in bone marrow metaphase chromosomes of rats administered orally a single dose or a daily dose for five consecutive days at the dosage levels 50, 500 and 5000 mg/kg/day (Fabrizio, 1974).This compound was also considered to be non-mutagenic in rats in a Dominant Lethal Assay when administered at the same dose regimen.

No increase in sex-linked recessive lethal germ cell mutations occurred in male Drosophila melanogaster administered benzyl acetate in feed or by injection.

Based on the results of genotoxicity studies performed in vitro with 3-PPB and in vitro or in vivo with the benzyl derivatives category indicating that compounds in this category are non-mutagenic and non-clastogenic, it is considered likely that 3-PPB does not possess a genotoxic potential.



Justification for selection of genetic toxicity endpoint
One Ames test study performed with 3-PPB to GLP and current guidelines. The in vivo genotoxicity of 3-PPB has been assessed using the read across category approach.

Justification for classification or non-classification

Genotoxicity studies performed in vitro with 3-PPB and in vitro or in vivo with the benzyl derivatives category indicate that compounds are non-mutagenic and non-clastogenic.Taking into account the available data on the genotoxicity of the members of this category it is considered likely that 3-PPB is not genotoxic and therefore it is not classified as mutagenic under EU Regulation 1272/2008.