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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative (±S9 mix)

HPRT test: negative (±S9 mix)

MNT test: negative (± S9 mix)

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared from liver homogenates taken from Sprague-Dawley male rats aged 8 to 10 weeks, induced with Arochlor 1254
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
Ethanol was used as solvent
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium acetate, 2-nitrofluorene, 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS:
- Three per concentration and control

DETERMINATION OF CYTOTOXICITY
- Method: diminution of the background lawn was taken as an indication of bacteriotoxicity

Two independent experiments were performed.
Evaluation criteria:
Details on evaluation criteria are not given but should be in accordance with the publication by Ames et al. (1975), Mutation Research 32, 347-364.
Statistics:
The statistical difference between the mean number of revertants in the negative controls and the plates at each dosage level was tested using the Chi2 test (Mohn and Ellenberger 1977).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9: at 500 µg/plate; +S9: at 5000g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9: at 1500 µg/plate; +S9: at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9: at 500 mg/plate; +S9: at 5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9: at 500 µg/plate; +S9: at 5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9: at 500 g/plate; +S9: at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

see attached document

Conclusions:
The substance Neononyl acetate was not mutagenic to Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in an Ames test in the presence and absence of metabolic activation (S9-mix).
Executive summary:

The potential of the test substance Neononyl acetate to induce reverse mutations in Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 was tested in an Ames test under GLP in accordance with OECD TG 471. The test substance was dissolved in ethanol and tested in concentrations ranging from 5 to 5000 µg/plate in the presence and absence of a metabolic activation (S9-mix obtained from liver homogenates from Sprague-Dawley male rats aged 8 to 10 weeks). All strains were incubated at 37 °C for 48 to 72 hours in three replicates. In the presence of S9-mix the substance was cytotoxic to strains TA98, 100, 102 and 1535 at 500 µg/plate and to strain TA1537 at 1500 µg/plate. In the absence of S9-mix bacteriotoxicity to strain TA102 was seen at 1500 µg/plate and toxicity to strains TA98, 100, 1535 and 1537 occurred at 5000 µg/plate. Precipitation of the test compound on the plates was not observed. The test substance did not induce a significant or dose-related increase in the mutation frequency of all tested strains in the absence or presence of S9-mix. The number of spontaneous revertants observed in the controls was comparable to historical controls. The results of the positive control substances confirmed the known reversion properties and the specificity of the tested strains as well as the activity of the metabolising system.

It was concluded that the substance Neononyl acetate under the experimental conditions of an Ames test was not mutagenic to bacteria (Salmonella typhimurium) strains TA98, TA100, TA102, TA1535 and TA1537 in the absence and presence of metabolising system S9-mix.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2013-01-07 to 2013-03-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Deviations are considered to have no impact on the purpose or integrity of the study.
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Deviations are considered to have no impact on the purpose or integrity of the study.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
yes
Remarks:
Deviations are considered to have no impact on the purpose or integrity of the study.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal Essential Medium (MEM) supplemented with 10 % foetal bovine serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
4-hour exposure group (-S9 mix): 3.75, 7.5, 15, 30, 45, 60, 90, 120 µg/mL
4-hour exposure group (+S9 mix, 2 %): 7.5, 15, 30, 45, 60, 90, 120, 180 µg/mL
24-hour exposure group (-S9 mix) and 4-hour exposure group (+S9 mix, 1 %): 1.88, 3.75, 7.5, 15, 30, 60, 90 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (±S9 mix), 24 h (-S9 mix)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 6 or 7 days

SELECTION AGENT (mutation assays): 6-Thioguanine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2x10^5

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test item is classified as mutagenic if there is a reproducible dose-related increase in the mutation frequency where at least a threefold increase in the mutant frequency over the vehicle control value is observed. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
A test item producing neither a dose-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A single dose level that meets the minimum criterion for a positive response within a range of assayed concentrations is not sufficient to evaluate the test item as a mutagen.
Statistics:
If a test item gives a marked and dose-related increase in the mutant frequency over the vehicle controls it will be designated as mutagenic and statistical analysis will not be required. However, if weaker responses are observed then statistical analysis will be performed using the SPSS program or a suitable alternative.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 90 µg/mL and precipitate at and above 60 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 180 µg/mL and precipitate at and above 120 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 90 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: at and above 60 µg/mL (4-hour exposure, -S9 mix), at and above 120 µg/mL (4-hour exposure, +S9 mix 2 %)

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Remarks on result:
other: 4-hour exposure (Experiment 1)
Conclusions:
The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.
Executive summary:

The purpose of this study is to assess the potential mutagenicity of a test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. The test methods described are designed to be compatible with the procedures indicated by the OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No 440/2008 of 30 May 2008 and US EPA OPPTS 870.5300 Guideline.

Chinese hamster (V79) cells were treated with the test item at up to seven dose levels, in duplicate, together with vehicle (solvent) and positive controls in the presence and absence of an S9 metabolic activation system. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2 % final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1 % final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose range of the test item was selected based on the results of a preliminary cytotoxicity test and were as follows:

Exposure Group

Final concentration of test item (µg/mL)

4-hour

(-S9mix)

3.75

7.5

15

30

45

60

90

120

4-hour

(+S9mix, 2 %)

7.5

15

30

45

60

90

120

180

24-hour

(-S9mix)

1.88

3.75

7.5

15

30

60

90

-

4-hour

(+S9mix, 1%)

1.88

3.75

7.5

15

30

60

90

-

The vehicle (solvent) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system. The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-06-06 to 2013-02-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 487 "In vitro Mammalian Cell Micronucleus Test"
Deviations:
yes
Remarks:
The expression phase and harvest time were slightly modified compared to the OECD TG 487
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/Ham's F12 medium, mixture 1:1) supplemented with 200 mM GlutaMax
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 mix
Test concentrations with justification for top dose:
Experiment I:
40 h / 4 h: 6.9-1863.0 µg/mL (-S9 mix)
40 h / 4 h: 6.9-1863.0 µg/mL (+S9 mix)

Experiment IIA:
40 h / 20 h: 12.1-1863.0 µg/mL (-S9 mix)
40 h / 4 h: 37.1-1863.0 µg/mL (+S9 mix)

Experiment IIB:
40 h / 4 h: 50.0-1200.0 µg/mL (+S9 mix)
Vehicle / solvent:
- Vehicle/solvent used: ethanol; final concentration of ethanol in the culture medium was 0.5 % v/v
- Justification for choice of solvent: the solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
mitomycin C
other: Demecolcin
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 h with and without S9 mix (experiment I), 4 h with S9 mix and 20 h without S9 mix (experiment IIA) and 4 h with S9 mix (experiment IIB)
- Expression time (cells in growth medium): cells exposed for 4 h have 16 h recovery period before fixation, no recovery period for 20 h exposure cells
- Selection time (if incubation with a selection agent): 20 h with Cytochalasin B (4 µg/mL)
- Cells were prepared 40 hrs after start of the exposure

SPINDLE INHIBITOR: Cytochalasin B (4 µg/mL)
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
cytotoxic effect the CBPI: ca 500 cells per culture and cytotoxicity is expressed as % cytostasis
micronuclei effects: at least 1000 binucleate cells per culture. The frequency of micronucleated cells was reported as % micronucleated cells

DETERMINATION OF CYTOTOXICITY
- percentages of reduction in the CBPI (cytokinesis-block proliferating index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate
- Exposure time 4 hrs (with and without S9 mix), cells were prepared 40 hrs after start of the exposure
Evaluation criteria:
cytotoxic effect: percentages of reduction in the CBPI (cytokinesis-block proliferating index) in comparison with the controls (% cytostasis)

micronuclei effects: 1000 binucleate cells per culture. The frequency of micronucleated cells was reported as % micronucleated cells

criteria for the evaluation of micronuclei:
The micronuclei were counted in cells showing a clearly visible cytoplasm area. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.


A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.
Statistics:
Statistical significance was confirmed by means of the Chi square test.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: Phase separation was observed in Experiment I at 347.6 Og/mL and above in the absence of S9 mix and at 198.6 Og/mL in the presence of S9 mix. In Experiment IIA phase separation was observed at 608.3 Og/mL in the absence of S9 mix and at 347.6 Og/mL and above in the presence of S9 mix. In Experiment IIB no phase separation was observed.
- Precipitation: no
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Conclusions:
Under the experimental conditions reported, the test item Neo Nonyl Acetate did not induce micronuclei in human lymphocytes in vitro, when tested up to cytotoxic, the highest required or evaluable concentration.
Executive summary:

The test item Neo Nonyl Acetate, dissolved in ethanol, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Three independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment IIA, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. In Experiment IIB, the exposure period was 4 hours with S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis. The highest treatment concentration in this study, 1863.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. No precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity or pH value was observed. Phase separation was observed in Experiment I at 347.6 µg/mL and above in the absence of S9 mix and at 198.6 µg/mL in the presence of S9 mix. In Experiment IIA phase separation was observed at 608.3 µg/mL in the absence of S9 mix and at 347.6 µg/mL and above in the presence of S9 mix. In Experiment IIB no phase separation was observed. In Experiment I in the absence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA in the absence of S9 mix cytotoxicity (48.1 %) was observed at the highest evaluated concentration. In Experiment I and IIA in the presence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment IIB in the presence of S9 mix cytotoxicity (48.4 %) was observed at the highest evaluated concentration. In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.00 - 0.95 % micronucleated cells) did not exceed the range of the solvent control values (0.05 - 1.00 % micronucleated cells) and were within the range of the laboratory historical control data. However, in Experiment IIA in the presence of S9 mix one single statistically significant increase in micronucleated cells (0.40 %) was observed after treatment with 347.6 µg/mL. The value is clearly within the range of the historical control data (0.20 – 1.70 % micronucleated cells) and therefore biologically irrelevant. Either Demecolcin (75.0 ng/mL), MMC (2.0 µg/mL) or CPA (15.0 or 20.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Under the experimental conditions reported, the test item Neo Nonyl Acetate did not induce micronuclei in human lymphocytes in vitro, when tested up to cytotoxic, the highest required or evaluable concentration.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Three testsin vitro(Ames, HPRT and MNT) according to data requirements are negative.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.