Reaction products of N-Phenyl-1-Naphthylamine with Nonene, branched

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Constituent C25H31N | Nonyl (branched) substituted - N-Phenyl-1-naphthylamine

Molecular formula:

C25H31N

IUPAC Name:

Constituent C25H31N | Nonyl (branched) substituted - N-Phenyl-1-naphthylamine

Constituent 2

Reference substance name:

Constituents C34H49N | Di-nonyl (branched)- substituted N-Phenyl-1-naphthylamine

Molecular formula:

C34H49N

IUPAC Name:

Constituents C34H49N | Di-nonyl (branched)- substituted N-Phenyl-1-naphthylamine

Test material form:

liquid

Details on test material:

Name of test item: XPDL 958
Test item No.: 20/0205-1
Batch identification: 0021851006
CAS No.: 63451-49-0
Purity: 100% UVCB *; additionally, an analytical characterization was conducted (No. 20L00034).
Homogeneity: The test item was homogeneous by visual inspection.
Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Expiry date: November 18, 2021
Storage conditions: Room temperature
Physical state / color: Liquid, viscous/ brown to red

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction
The S9 fraction was prepared according to Ames et al. (1, 2) at BASF SE in an AAALACapproved
laboratory in accordance with the German Animal Welfare Act and the effective
European Council Directive.
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany
GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied
by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days.
During this time, the animals were housed in polycarbonate cages: central air conditioning with
a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The
day/night rhythm was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am.
Standardized pelleted feed and drinking water from bottles were available ad libitum.
24 hours after the last administration, the rats were sacrificed, and the livers were prepared
using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and
washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three
volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at
+4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

S9 mix
The S9 mix was prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient
amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with
9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on
ice until used. The concentrations of the cofactors in the S9 mix were:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer (6) is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution
in a ratio of about 4:1.
To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with
benzo(a)pyrene.

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (top dose as requested by the guideline)

Vehicle / solvent:

dimethyl sulfoxide (DMSO)
Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-aminoanthracene (2-AA) with S9, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 4-nitroquinoline-N-oxide (4-NQO) without S9

Details on test system and experimental conditions:

TEST SYSTEM
For testing, deep-frozen (-70°C to -80°C) bacterial cultures (Salmonella typhimurium TA 1535,
TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) were thawed at room temperature, and 0.1 mL
of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth
+ 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The
optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were
grown up to late exponential or early stationary phase of growth (approximately 109 cells per
mL). These cultures grown overnight were kept in iced water from the beginning of the
experiment until the end in order to prevent further growth.
The use of the strains mentioned was in accordance with the current scientific
recommendations for the conduct of this assay.
The Salmonella strains TA 1535, TA 100, TA 1537 and the Escherichia coli strain were
obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014. The
Salmonella strain TA 98 was obtained from Moltox Molecular Toxicology on 07 Jan 2015.

Salmonella typhimurium
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy
(his-) to histidine prototrophy (his+) is determined (2, 3, 4). The tester strains TA 1535, TA 1537,
TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium
LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision
repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and
this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all
strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an
increase in permeability to lipophilic substances.
The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by
the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and
TA 98 are strains for the detection of frameshift mutagens. These strains carry different
frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type
his D 3052 in the case of TA 98.
The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 (4) and, in addition to having
genes resistant to antibiotics, they have a modified postreplication DNA repair system, which
increases the mutation rate by inducing a defective repair in the DNA; this again leads to a
considerable increase in sensitivity.

Escherichia coli
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a
derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which
induce base pair substitutions (5). The rate of induced back mutations from tryptophan
auxotrophy (trp-) to tryptophan independence (trp+) is determined.

Checking the tester strains
The Salmonella strains were checked for the following characteristics at regular intervals:
deep rough character (rfa); UV sensitivity (Δ uvrB); ampicillin resistance (R factor plasmid).
E. coli WP2 uvrA was checked for UV sensitivity.
Histidine and tryptophan auxotrophy were checked in each experiment via the spontaneous
rate.

EXOGENOUS METABOLIC ACTIVATION
S9 fraction
The S9 fraction was prepared according to Ames et al. (1, 2) at BASF SE in an AAALACapproved
laboratory in accordance with the German Animal Welfare Act and the effective
European Council Directive.
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany
GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied
by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days.
During this time, the animals were housed in polycarbonate cages: central air conditioning with
a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The
day/night rhythm was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am.
Standardized pelleted feed and drinking water from bottles were available ad libitum.
24 hours after the last administration, the rats were sacrificed, and the livers were prepared
using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and
washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three
volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at
+4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

S9 mix
The S9 mix was prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient
amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with
9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on
ice until used. The concentrations of the cofactors in the S9 mix were:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer (6) is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution
in a ratio of about 4:1.
To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with
benzo(a)pyrene.

DOSES
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were
generally selected as maximum test dose at least in the 1st Experiment. However, this
maximum dose was tested even in the case of relatively insoluble test compounds to detect
possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be
tested in repeat experiments for further clarification/substantiation.

TEST SUBSTANCE PREPARATIONS
The test substance was weighed and topped up with the chosen vehicle to achieve the required
concentration of the stock solution.
The test substance was dissolved dimethyl sulfoxide (DMSO).
To achieve a clear solution of the test substance in the vehicle, the test substance preparation
was treated with ultrasonic waves and was shaken thoroughly.
The further concentrations were diluted from the stock solution according to the planned doses.

ANALYSIS OF TEST SUBSTANCE PREPARATION
The stability of the test substance in the vehicle DMSO was not determined analytically,
because the test substance was administered immediately after preparation and is usually
stable.

EXPERIMENTAL PROCEDURE
Choice of the vehicle
Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had
been demonstrated to be suitable in bacterial reverse mutation tests and for which historical
control data are available (9).

Mutagenicity tests
Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based
on the method of Ames et al. (1, 2).
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL
agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino
acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept
in a water bath at about 42 - 45°C, and the remaining components were added in the
following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants)
were counted. The colonies were counted using the Sorcerer Image Analysis System with
the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK).
Colonies were counted manually, if precipitation of the test substance hindered the counting
using the Image Analysis System.
All test substance formulations were prepared immediately before use.
• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL
agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino
acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath
at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants)
were counted. The colonies were counted using the Sorcerer Image Analysis System with
the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK).
Colonies were counted manually, if precipitation of the test substance hindered the counting
using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. (7) and
Matsushima et al. (8).
0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix
(with metabolic activation) or phosphate buffer (without metabolic activation) were
incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of
soft agar was added and, after mixing, the samples were poured onto the agar plates within
approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The
colonies were counted using the Sorcerer Image Analysis System with the software program
Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted
manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Controls
Negative controls / Vehicle controls
Each experiment included negative controls in order to check for possible contaminants
(sterility control) and to determine the spontaneous mutation rate (vehicle control).
• Sterility control
Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance
but without the addition of tester strains (see Appendix 3).
• Vehicle control
The vehicle control with and without S9 mix only contains the vehicle used for the test
substance at the same concentration and volume for all tester strains.

Positive controls
The following positive controls were used to check the mutability of the bacteria and the activity
of the S9 mix:
With S9 mix
• 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Fluka; 97%)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD) (Sigma-Aldrich; 98%)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC) (Sigma-Aldrich; 98%)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich; 98%)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA
The stability of the selected positive controls was well-defined under the selected culture
conditions, since they were well-established reference mutagens.

Scope of tests and test conditions
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control
2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

Rationale for test conditions:

as specified by the guideline

Evaluation criteria:

EVALUATION
Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard
deviations were given for all dose groups as well as for the positive and negative (vehicle)
controls in all experiments. In general, six doses of the test substance were tested with a
maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the
1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat
studies or further experiments were based on the findings of the 1st Experiment.

Toxicity
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated
in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose
groups.

Solubility
If precipitation of the test material was observed, it would be recorded and indicated in the
tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was
generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at
least in the 1st Experiment even in the case of relatively insoluble test compounds to detect
possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat
experiments for further clarification/substantiation.

Statistics:

N/A

Results and discussion

Test results

Test results 1

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:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was observed in
the standard plate and preincubation test depending on the strain and test conditions at and
above 333 μg/plate.

Any other information on results incl. tables

The test substance XPDL 958 was tested for mutagenicity in the Salmonella typhimurium /

Escherichia coli reverse mutation assay both in the standard plate test and in the preincubation

test with and without the addition of a metabolizing system (S9 mix) obtained from rat liver

using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli

WP2 uvrA.

SOLUBILITY

Test substance precipitation was observed at and above 1000 μg/plate both with and without S9 mix.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that XPDL 958 is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.