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Toxicological information

Genetic toxicity: in vitro

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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:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N,N’-(isobutylidene)diurea
- Physical state: solid
- Analytical purity: 87.6 - 89.2 g/100 g (calculated); water content: 1.8 g/100 g (see analytical report, study code 15L00187)
- Lot/batch No.: 2015-03-24
- Expiration date of the lot/batch: 23 Mar 2020
- Stability under test conditions: Room temperature
- Storage condition of test material: The stability of the test substance under storage conditions is guaranteed until 23 Mar 2020 as indicated by the sponsor, and the sponsor holds this responsibility.

Method

Target gene:
HIS and TRP
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
1st Experiment
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
2nd Experiment
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [acetone]
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone 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
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD)
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. coliWP2 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 was 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 30 - 70%. 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.
The preparation date and protein content of the S9 fraction were given in Appendix 4.

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 (see Appendix 4).

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 PREPARATION
The test substance was grind in a mortar, was weighed and topped up with the chosen vehicle
to achieve the required concentration of the stock solution.
The test substance was suspended in acetone.

EXPERIMENTAL PROCEDURE
Choice of the vehicle
Due to the insolubility of the test substance in water, acetone 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 or vehicle (negative 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 be 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 hinders the counting
using the Image Analysis System.
To achieve homogeneity of the test substance in the vehicle, the test substance preparation
was treated with ultrasonic waves and was shaken thoroughly.
To keep the test substance homogeneously in the vehicle, the test substance preparation was
carefully pipetted before the removal.
The further concentrations were diluted according to the planned doses.
All test substance formulations were prepared immediately before administration.
• 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 or vehicle (negative 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 be 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 hinders 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).
0.1mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5mL 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 (Perseptive 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.
3.7.3.2. 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.

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
Precipitation of the test material was 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.
Evaluation criteria:
Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the
historical negative control data for each tester strain (see Appendix 5).
• The sterility controls revealed no indication of bacterial contamination (see Appendix 3).
• 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 (see Appendix 5).
• Fresh bacterial culture containing approximately 109 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli
WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and
TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix
or after adding a metabolizing system.
A test substance was 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.
Statistics:
N/A

Results and discussion

Test resultsopen allclose all
Key result
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
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
Positive controls validity:
valid
Additional information on results:
TOXICITY
A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was
occasionally observed in the standard plate test depending on the strain and test conditions
from 2500 μg/plate onward.
In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease
in the number of his+ or trp+ revertants) was not observed up to the highest required
concentration.

SOLUBILITY
Test substance precipitation was found in the standard plate test from 1000 μg/plate onward
and in the preincubation assay from 2500 μg/plate onward, both with and without S9 mix.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.  In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within or nearby the range of the historical positive control data.

Applicant's summary and conclusion