Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not mutagenic in a test with bacteria.

The analoge test substance Reaction products of benzeneamine, N-phenyl with nonene (branched) was tested for its genotoxic potential in the Ames Test (OECD 471, GLP), the HPRT test (OECD 476, GLP) and the micronucleus test in lymphocytes (OECD 487, GLP).

A chromosome aberration test is available for the constituent CAS 27177 -41 -9 ( ar-nonyldiphenylamine) showing absence of a clastogenic potential.

Literature in vitro and in vivo data (MLA, UDS and Dominant lethal assay) is available for the dioctylated diphenylamine (Brusick 1978). This data does not give rise of concern for genotoxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15.11-25.01.1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
non GLP (performed 1989), only 4 strains
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
His operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsomes and co-factors
Test concentrations with justification for top dose:
without activation: 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500 µg/ml and 19.5, 39.1, 78.1, 156, 313, 625, 1250 and 2500 µg/mL
with activation: 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL
Vehicle / solvent:
- Vehicle used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunorubicin-HCl
Remarks:
Strain TA 98 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
strain TA100 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
strain TA1535 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
strain TA1537 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2—aminoanthracene
Remarks:
strain TA98 with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
strain TA100 with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
strain TA1535 with activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
strain TA1537 with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: approx. 46 hours.
Evaluation criteria:
The cultures were shaken before each measurement and turbidity of the chamber content was measured automatically at the wave length of 546 nm at intervals of 20 min over a period of approx. 46 hours.
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at all concentrations

The test substance precipitated in the measurement medium in the experiments without and with microsomal activation at all concentrations. At the concentrations of 500 and 1000 µg/ml in the experiments with microsomal activation precipitates covered the bacterial growth.

Conclusions:
Interpretation of results (migrated information):
negative

In the experiments performed without and with microsomal activation, the various concentrations of TK 12340 did not induce histidine-prototrophic back-mutants.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
issued by Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Liqiude (viscous), yellowish to pink
Expiry date: 28 September 2018
Batch: 0016046440
Species / strain / cell type:
lymphocytes: human lymphocytes, primary culture
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication.
- Suitability of cells: yes
- Sex, age and number of blood donors if applicable: Blood was collected from a female donor (32 years old) for Experiment I and from a male donor (25 years old) for Experiment II.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Method of maintenace: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-naphthoflavone induced rat liver microsomal fraction S9 Mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
- Justification for choice of solvent/vehicle: 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
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours in Experiment I, 20 hours in Experiment II (without S9 Mix), 4 hours in Experiment II (with S9 Mix)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The harvested cells were spun down by gentle centrifugation, re-suspended in "saline G", spun down once again by centrifugation and resuspended in 5 mL KCl solution and incubated at 37 °C. Ice-cold fixative mixture of methanol and glacial acetic acid was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
- the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
- no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent contrl

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976)
- 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.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
Evaluation criteria:
The micronucleus assay will be considered acceptable if it meets the following criteria:

a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.

A test item can be classified as clastogenic and aneugenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.

Statistics:
Chi square test (α < 0.05)
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
tested up to phase separation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In both experiments in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.
In both experiments inthe absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item.

A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 11 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.

Table:: Summary of results of thein vitromicronucleus test in human lymphocytes

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

 

interval

concentration

index

in %*

cells

95% Ctrl limit

 

 

in µg/mL

CBPI

 

in %**

 

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

1.88

 

0.65

0.06 – 1.19

 

 

Positive control2

1.68

22.8

13.65S

3.92 – 25.34

 

 

11.3

1.93

n.c.

0.65

 

 

 

19.7

1.87

1.7

0.85

 

 

 

34.6PS

1.79

10.0

0.90

 

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.82

 

0.85

0.00 – 1.11

 

 

Positive control3

1.62

25.3

 4.75S

1.47 – 5.89

 

 

18.8

1.70

15.3

0.40

 

 

 

37.5

1.66

19.6

0.35

 

 

 

75.0PS

1.60

27.5

0.50

 

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1

1.77

 

0.50

0.08 – 1.38

 

 

Positive control4

1.70

9.6

 3.00S

0.70 – 10.20

 

 

19.7

1.71

8.9

0.75

 

 

 

34.6

1.72

6.8

0.30

 

 

 

60.5PS

1.79

n.c.

0.45

 

*      For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

PS     Phase separation occurred at the end of treatment

S       The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c.  Not calculated as the CBPI is equal or higher than the solvent control value

1        DMSO                 1.0 % (v/v)
2
        MMC                    0.8 µg/mL
3
        Demecolcine         50 ng/mL
4
        CPA                    17.5 µg/mL

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Conclusions:
The substance did not cause genotoxicity in the in-vitro micronucleus test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
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)
Version / remarks:
2016
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
Type of assay:
other: Gene Mutation Assay in Mammalian Cells in vitro (HPRT)
Specific details on test material used for the study:
Batch 0016046440
Content: sum of all peaks: 99.2 area-% (HPLC, 210 nm)
sum of all peaks: 99.5 area-% (HPLC, 290 nm)
Liquid, viscous, yellowish to pink, clear

The stability of the test substance under storage conditions throughout the study period was guaranteed until 07 Jan 2020
Storage conditions: Room temperature (avoid temperatures < 10°C)
Target gene:
hprt
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Suitability of cells: The CHO cell line is a permanent cell line derived from the Chinese hamster and has a high proliferation rate, high plating efficiency (about 90 %) and karyotype with a modal number of 20 chromosomes.
- Cell cycle length, doubling time or proliferation index: 12 - 16 hours
- Number of passages if applicable: At least 2 passages were performed before cells were taken for the experiment. A further passage was also necessary in order to prepare test cultures.
- Methods for maintenance in cell culture if applicable: For cell cultivation, deep-frozen cell suspensions were thawed at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm² plastic flasks containing about 5 mL Ham's F12 medium including 10 % (v/v) FCS. Cells were grown with 5 % (v/v) CO2 at 37 °C and ≥ 90 % relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 12 - 16 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10 % (v/v) fetal calf serum (FCS), 1 % (v/v) penicillin / streptomycin (stock solution: 10000 IU / 10000 µg/mL) and 1 % (v/v) amphotericine B (stock solution: 250 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and ß-naphthoflavone induced rat liver S9 fraction
Test concentrations with justification for top dose:
without S9
0, 20.00; 30.00; 40.00; 80.00 μg/mL

with S9
0, 5.00; 10.00; 20.00; 30.00 μg/mL

Precipitation of the test substance in the vehicle dimethyl sulfoxide (DMSO) was not observed in the stock solution (Test group: 5000 μg/mL). However, in the dose groups 2500.0 μg/mL,
1250.0 μg/mL and 625.0 μg/mL the dilutions were homogenous dispersions. In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 39.1 μg/mL and above in the absence and presence of S9 mix. After 4 hours treatment in the absence and presence of S9 mix, cytotoxicity was observed as indicated by a reduced RS of about or below 20% of control at 1250.0 μg/mL and 2500.0 μg/mL. At the highest applied test substance concentration of 5000.0 μg/mL no relevant cytotoxicity was observed
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 20 x 10^6 cells in 40 mL

DURATION
- Preincubation period: about 20 - 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 16 days

SELECTION AGENT (mutation assays): 6-thioguanine

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF CELLS EVALUATED: 200 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative survival after treatment; mutant frequency
Evaluation criteria:
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative controls should not be less than 50 % (with and without S9 mix).
- The background mutant frequency in the negative controls should be within the historical negative control data range (95 % control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not "under control".
- The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range.
A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation Frequencies exceeds both the concurrent negative / vehicle control value and the range of the laboratory's historical negative control data (95 % control limit).
Isolated increases of mutant frequencies abouve the historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of the laboratory's historical negative control data (95 % control limit).
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
If the results of these tests were statistically significant compared with the respective vehicle control, labels are printed in the tables.
However, both, biological and statistical significance are considered together.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: yes

RANGE-FINDING/SCREENING STUDIES:
In the pre-test the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.
Precipitation of the test substance in the vehicle dimethyl sulfoxide (DMSO) was not observed in the stock solution (Test group: 5000 μg/mL). However, in the dose groups 2500.0 μg/mL,
1250.0 μg/mL and 625.0 μg/mL the dilutions were homogenous dispersions. In culture medium, test substance precipitation occurred by the end of treatment at concentrations of
39.1 μg/mL and above in the absence and presence of S9 mix. After 4 hours treatment in the absence and presence of S9 mix, cytotoxicity was observed as indicated by a reduced RS of about or below 20% of control at 1250.0 μg/mL and 2500.0 μg/mL. At the highest applied test substance concentration of 5000.0 μg/mL no relevant cytotoxicity was observed

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Corrected mutant frequency: mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

"Without S9 mix, 400 µg/mL ethyl methanesulfonate (EMS)"
Corrected Mutant Frequency
Exposure period 4 hrs
Mean 158.01
Minimum 42.47
Maximum 419.90
Standard Deviation 80.87
95% Lower Control Limit 0.00
95% Upper Control Limit 320.94
No. of Experiments 63 (March 2016 – December 2017)

"With S9 mix, 1.25 µg/mL 7,12-Dimethylbenz[a]anthracene (DMBA)"
Corrected Mutant Frequency
Exposure period 4 hrs
Mean 125.89
Minimum 21.52
Maximum 270.48
Standard Deviation 56.16
95% Lower Control Limit 13.02
95% Upper Control Limit 238.77
No. of Experiments 69 (March 2016 – December 2017)

Without S9 mix - all vehicles With S9 mix - all vehicles
Corrected Mutant Frequency
Exposure period 4 hrs 4 hrs
Mean 2.86 2.93
Minimum 0.00 0.00
Maximum 7.09 9.93
Standard Deviation 1.81 2.24
95% Lower Control Limit 0.00 0.00
95% Upper Control Limit 6.49 7.43
No. of Experiments 69 72 (March 2016 – December 2017)

Table 1:  Summary of results
Exp. Exposure period
[h]
Test groups [µg/mL] S9
mix
Prec.* Genotoxicity**
MFcorr.[per 106 cells]
Cytotoxicity***
RS
[%]
CE2
[%]
1 4 Vehicle control (DMSO 1% v/v) - n.d. 2.11 100.0 100.0
1.25 - - n.c.1 112.0 n.c.1
2.50 - - n.c.1 90.9 n.c.1
5.00 - - n.c.1 126.4 n.c.1
10.00 - - n.c.1 160.2 n.c.1
20.00 - - 0.68 124.6 104.2
30.00 - - 0.00 173.8 87.7
40.00 - - 2.44 138.9 101.1
80.00 - + 1.30 158.2 81.3

EMS 400 µg/mL

- n.d 321.86S 97.8 64.4
1 4 Vehicle control (DMSO 1% v/v) + n.d. 2.27 100.0 100.0
1.25 + - n.c.1 73.2 n.c.1
2.50 + - n.c.1 82.4 n.c.1
5.00 + - 1.09 90.8 88.7
10.00 + - 0.32 84.8 100.3
20.00 + - 0.00 85.1 88.0
30.00 + + 2.48 65.3 91.3
40.00 + + n.c.2 n.c.2 n.c.2
80.00 + + n.c.2 n.c.2 n.c.2
   

DMBA 1.25 µg/mL

+ n.d. 143.06S 76.2 69.9

*: Macroscopically visible precipitation in culture medium at the end of exposure period

**: Mutant frequency MFcorr.: mutant colonies per 106cells corrected with the CE2value

***: Cloning efficiency related to the respective vehicle control

s: Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

n.c.1: Culture was not continued since a minimum of only four analyzable concentrations is required

n.c.2: Culture was not continued since only one concentration beyond the solubility limit is required

n.d.: Not determined

Conclusions:
The substance did not cause gene mutations in mammalian cells in vitro.
Executive summary:

The substance Reaction products of benzeneamine, N-phenyl with nonene (branched) was assessed for its potential to induce gene mutations at the hypoxanthine-guanine

phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. One experiment was carried out, with and without the addition of liver S9 mix from phenobarbitaland

β-naphthoflavone induced rats (exogenous metabolic activation).

Due to strong precipitation (from 39.1 μg/mL onward) of the test substance in culture medium in an initial range-finding cytotoxicity test, the maximum concentration to be used in

this study was determined to be 80.0 μg/mL. The following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 1.25; 2.50; 5.00; 10.00; 20.00; 30.00; 40.00; 80.00 μg/mL

with S9 mix

0; 1.25; 2.50; 5.00; 10.00; 20.00; 30.00; 40.00; 80.00 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were

cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and

counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-

anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

The highest tested concentrations in the main experiment showed clear test substance precipitates in culture medium macroscopically at the end of exposure period. No cytotoxicity

was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a

metabolizing system.

Reaction products of benzeneamine, N-phenyl with nonene (branched) is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(21 Jul 1997)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Name of test substance: Reaction products of benzeneamine, N-phenyl with nonene (branched)

Content: 100 g/100 g (UVCB substance)
water content: 0.03 g/100 g

The test material identity was confirmed analytically.
Target gene:
his+ / trp+
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
1st Experiment (Standard plate test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
2nd Experiment (Preincubation test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer


OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Positive controls:

With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA
Evaluation criteria:
Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
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
Remarks:
A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 1000 μg/plate onward with and without
S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the standard plate test only using tester strain TA 1535 with S9 mix at a concentration of 5000 μg/plate. In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants) was observed using tester strain TA 1535 from about 2500 μg/plate onwards and using tester strain TA 1537 from about 1000 μg/plate onwards both without metabolic activation.

Preincubation test, Without metabolic activation
Strain Test group Dose (µg/plate) mean counts Standard deviation Factor Individual revertant colony
TA 1535 DMSO - 13.3 1.2 - 12, 14, 14
Test item 33 9.0 3.0 0.7 6, 9, 12
100 11.7 5.5 0.9 18, 9, 8
333 8.3 1.2 0.6 9, 9, 7
1000 13.0 8.2 1.0 20 P, 15 P, 4 P
2500 8.0 2.6 0.6 6 P, 7 P, 11 P
5000 5.0 3.0 0.4 5 P, 8 P, 2 P
  MNNG 5.0 2417.3 309.5 181.3 2493, 2077, 2682
TA 100 DMSO - 115.0 11.4 - 102, 123, 120
Test item 33 102.3 9.9 0.9 109, 107, 91
100 111.3 4.5 1.0 111, 116, 107
333 114.3 9.6 1.0 123, 104, 116
1000 99.7 11.0 0.9 91 P, 112 P, 96 P
2500 98.7 2.1 0.9 101 P, 98 P, 97 P
5000 106.0 11.3 0.9 112 P, 113 P, 93 P
  MNNG 5.0 3483.3 252.7 30.3 3772, 3376, 3302
TA 1537 DMSO - 11.0 1.7 - 12, 12, 9
Test item 33 8.3 5.0 0.8 13, 9, 3
100 7.7 2.1 0.7 7, 6, 10
333 10.3 1.2 0.9 11, 11, 9
1000 5.7 1.5 0.5 7 P, 6 P, 4 P
2500 7.0 2.6 0.6 9 P, 8 P, 4 P
5000 5.0 2.0 0.5 3 P, 5 P, 7 P
  AAC 100 807.7 123.6 73.4 950, 727, 746
TA 98 DMSO - 16.3 0.6 - 17, 16, 16
Test item 33 18.3 2.1 1.1 16, 19, 20
100 14.3 4.0 0.9 12, 19, 12
333 16.3 1.5 1.0 15, 16, 18
1000 12.3 1.5 0.8 11 P, 14 P, 12 P
2500 14.3 3.1 0.9 11 P, 17 P, 15 P
5000 13.7 3.8 0.8 18 P, 11 P, 12 P
  NOPD 10 1234.0 113.2 75.6 1287, 1311, 1104
E. coli DMSO - 19.3 3.8 - 15, 22, 21
Test item 33 23.0 6.6 1.2 24, 16, 29
100 24.0 4.6 1.2 20, 29, 23
333 23.0 8.9 1.2 13, 30, 26
1000 22.7 7.2 1.2 18 P, 19 P, 31 P
2500 21.7 2.5 1.1 22 P, 19 P, 24 P
5000 23.0 4.6 1.2 27 P, 24 P, 18 P
  4-NQO 5 526.3 73.0 27.2 569, 568, 442

P =Precipitation

Preincubation test, With metabolic activation
Strain Test group Dose (µg/plate) mean counts Standard deviation Factor Individual revertant colony
TA 1535 DMSO - 7.0 1.0 - 8, 6, 7
Test item 33 11.0 0.0 1.6 11, 11, 11
100 8.0 4.6 1.1 7, 4, 13
333 10.7 1.5 1.5 9, 12, 11
1000 11.3 3.2 1.6 15 P, 9 P, 10 P
2500 6.3 1.5 0.9 5 P, 8 P, 6 P
5000 5.0 1.7 0.7 7 P, 4 P, 4 P
  2-AA 2.5 206.0 17.8 29.4 200, 192, 226
TA 100 DMSO - 102.3 16.5 - 84, 107, 116
Test item 33 116.3 8.0 1.1 117, 124, 108
100 116.7 12.5 1.1 129, 117, 104
333 103.0 10.5 1.0 92, 104, 113
1000 102.3 11.0 1.0 97 P, 115 P, 95 P
2500 90.3 29.2 0.9 124 P, 73 P, 74 P
5000 84.7 9.6 0.8 95 P, 76 P, 83 P
  2-AA 2.5 2141.0 37.2 20.9 2170, 2099, 2154
TA 1537 DMSO - 9.3 3.2 - 8, 7, 13
Test item 33 8.0 0.0 0.9 8, 8, 8
100 8.3 4.5 0.9 4, 8, 13
333 6.7 1.5 0.7 7, 5, 8
1000 9.0 3.0 1.0 9 P, 6 P, 12 P
2500 8.3 0.6 0.9 8 P, 9 P, 8 P
5000 6.3 1.2 0.7 7 P, 5 P, 7 P
  2-AA 2.5 127.3 14.2 13.6 111, 137, 134
TA 98 DMSO - 20.0 1.0 - 21, 20, 19
Test item 33 18.3 3.8 0.9 20, 21, 14
100 23.3 3.8 1.2 26, 19, 25
333 22.7 4.2 1.1 18, 26, 24
1000 17.3 8.4 0.9 27 P, 13 P, 12 P
2500 19.0 2.6 1.0 17 P, 22 P, 18 P
5000 17.7 5.1 0.9 12 P, 22 P, 19 P
  2-AA 2.5 1494.3 318.9 74.7 1234, 1399, 1850
E. coli DMSO - 24.3 2.1 - 26, 25, 22
Test item 33 20.0 4.0 0.8 20, 24, 16
100 26.7 8.1 1.1 36, 21, 23
333 23.3 2.1 1.0 25, 21, 24
1000 20.0 1.0 0.8 19 P, 21 P, 20 P
2500 25.0 1.0 1.0 24 P, 26 P, 25 P
5000 25.7 2.5 1.1 28 P, 23 P, 26 P
  2-AA 60 127.3 16.1 5.2 134, 139, 109

P=Precipitation

Conclusions:
The substance is not mutagenic in the Ames test (OECD 471, GLP).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The substance Reaction products of benzeneamine, N-phenyl with nonene (branched) was assessed for its potential to inducegene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. One experiment was carried out, with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats (exogenous metabolic activation). Due to strong precipitation (from 39.1 μg/mL onward) of the test substance in culture medium in an initial range-finding cytotoxicity test, the maximum concentration to be used in this study was determined to be 80.0 μg/mL. The following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 1.25; 2.50; 5.00; 10.00;20.00; 30.00; 40.00; 80.00μg/mL

with S9 mix

0; 1.25; 2.50;5.00; 10.00; 20.00; 30.00; 40.00; 80.00 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. The highest tested concentrations in the main experiment showed clear test substance precipitates in culture medium macroscopically at the end of exposure period. No cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system. Reaction products of benzeneamine, N-phenyl with nonene (branched) is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

The test substance Reaction products of benzeneamine, N-phenyl with nonene (branched) was tested for its mutagenic potential based on the ability to induce mutations in selected loci of several bacterial strains, i.e.Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (OECD 71, GLP).The standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) were used.

Tested bacteria strains were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA.

The dose range was 33 μg - 5000 μg/plate (SPT) and 33 μg - 5000 μg/plate (PIT).

Precipitation of the test substance was found from about 1000 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 1000 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

The test item Reaction products of benzeneamine, N-phenyl with nonene (branched), dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments (OECD 487, GLP). In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (5000 μg/mL of the test item) was chosen with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation. In both experiments in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In this study in the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Reaction products of benzeneamine, N-phenyl with nonene (branched) is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.

The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours was verified analytically.

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 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and the in vitro chromosome aberration assay (OECD 473, GLP) for the analogue substance. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.