N,N''-(isobutylidene)diurea

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data from two vitro Ames tests (BASF AG, 1988 and BASF SE, 2015), and in vitro HPRT-Test (BASF SE, 2010) and an in vivo micronucleus test (BG Chemie, 1999) shows that IBDU does not possess a genotoxic potential.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS and TRP

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.

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

Key result

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium, with different supplements
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

1st Experiment
without S9 mix (4-hour exposure period)
0; 10; 20; 40; 500; 1 000 μg/mL
with S9 mix (4-hour exposure period)
0; 10; 20; 40; 500; 1 000 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period)
0; 10; 20; 40; 500; 1 000 μg/mL
with S9 mix (4-hour exposure period)
0; 15; 30; 60; 500; 1 000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: Due to insolubility of the test substance in water, acetone and ethanol, dimethylsulfoxide
(DMSO) was the most suitable vehicle which led to a homogenous suspension at 1 000 μg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: EMS and MCA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: attachment period of 20 - 24 hours
- Exposure duration: treatment period of 4 hours
- Expression time (cells in growth medium): expression phase of about 6 - 8 days
- Selection time (if incubation with a selection agent): selection period of about 1 week


SELECTION AGENT (mutation assays): 6-thioguanine ("TG-medium")

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0 – 15 mutants per 106 clonable cells.
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data.
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

Due to clearly negative findings with mutation frequencies within the range of our laboratory's historical negative control data, a statistical evaluation was not carried out.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In both experiments in the absence and presence of S9 mix, no cytotoxicity indicated by reduced relative cloning efficiency of below 20% relative survival was observed up to the highest applicable concentration of 1 000 μg/mL under all test conditions.

In this study, no relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. In both experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.00 – 10.75 per 106 cells) were close to the respective vehicle control values (MFcorr.: 1.75 – 3.03 per 106 cells) and clearly within the range of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 15.95 per 106 cells; with S9 mix: MFcorr.: 0.00 – 12.62 per 106 cells).

The positive control substances EMS (without S9 mix; 300μg/mL) and MCA (with S9 mix; 20μg/mL) induced clearly increased mutant frequencies as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 137.16 – 486.06 per 106 cells; with S9 mix: MFcorr.: 75.37 – 76.23 per 106 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 48.83 – 999.46 per 106 cells; with S9 mix: MFcorr.: 26.29 –413.54 per 106 cells).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: not according to current but former guideline. Only 4 strains tested, (not tested with TA 102 or E.coli)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

0 ; 20 ; 100 ; 500 ; 2500 ; 5000 µg/plate

Vehicle / solvent:

- Solventused: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (dissolved in DMSO)

Remarks:

with S-9 mix for strains TA 98, TA 100, TA 1535 and TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO)

Remarks:

without S-9 mix for strains TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine (dissolved in DMSO)

Remarks:

without S-9 mix for the strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine chloride monohydrate (dissolved in DMSO)

Remarks:

without S-9 mix for the strain TA 1537

Details on test system and experimental conditions:

The standard plate test and the preincubation test were used

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

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:

other: not observed at the concentrations tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

No signs of precipitation of test substance.

Chemical    Dose         Tester 

    

         (µg/plate)      strain      -S9      +S9

Standard plate test
0 TA1535 15 20
20 19 20
100 17 23
500 14 19
2500 14 25
5000 17 18

0 TA100 151 156
20 145 168
100 130 165
500 119 144
2500 137 127
5000 130 138

0 TA1537 9 10
20 11 10
100 10 11
500 10 12
2500 8 14
5000 9 11

0 TA98 24 34
20 22 33
100 22 32
500 19 32
2500 17 29
5000 19 31 Preincubation test

0 TA1535 18 20
20 15 13
100 20 14
500 16 8
2500 14 10
5000 13 11

0 TA100 126 138
20 129 138
100 124 125
500 126 131
2500 127 136
5000 122 126

0 TA1537 8 10
20 15 13
100 11 13
500 13 11
2500 12 8
5000 12 11

0 TA98 24 37
20 27 35
100 31 34
500 27 40
2500 28 35
5000 25 38

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro data

The test substance N,N’-(isobutylidene)diurea was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay according to OECD 471 guideline and GLP (BASF, 2015).

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33μg - 5000μg/plate (SPT); 33μg - 5000μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: Precipitation of the test substance was found from 1000μg/plate onward depending on the test conditions.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from 2500μg/plate onward.

MUTAGENICITY:

A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

CONCLUSION:

Thus, under the experimental conditions of this study, the test substance N,N’-(isobutylidene)diurea is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

IBDU (purity 92 %) was not mutagenic in the standard and pre-incubation Ames test (OECD guideline 471) with and without metabolic activation (tested up to 5000μg/plate in Salmonella typhimurium TA1535, 1537, 98, 100; metabolic activation: liver S-9 mix from Aroclor 1254-induced rats). Cytotoxicity was not observed, positive controls were functional (BASF, 1988). As this test was performed according to the older guideline version TA 102 and E. coli were not used for testing. These strains were introduced later to cover oxidizing mutagens, cross-linking agents and hydrazines. However, since IBDU does not have these properties, the study is comparable to a current guideline study.

 

IBDU (purity 94 %) did not lead to a relevant increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other (BASF SE, 2010) in a study performed in accordance with OECD 476 guideline and GLP.

The mutant frequencies at any concentration were close to the range of the concurrent vehicle control values and within the range of the historical negative control data.

The mutation frequencies of the vehicle control groups were within the historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study

The increase in the frequencies of mutant colonies induced by the positive control substances EMS and MCA clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

 

In vivo data

IBDU (purity 90.1 %) did not induce micronuclei in bone marrow cells of male NMRI mice dosed by gavage with up to 2000 mg/kg bw, given twice within 24 hours in a study performed in line with OECD guideline 474 (BG Chemie, 1999). The bone marrow cells were evaluated at 24 hours after the last dosing. The ratio between polychromatic and normochromatic erythrocytes were not substantially increased as compared to the corresponding vehicle controls, indicating that the test substance had no substantial cytotoxic effects on bone marrow cells.

Justification for classification or non-classification

No classification and labeling for mutagenic effects is necessary as IBDU was not mutagenic in two Ames Tests as well as HPRT-Test and did not induce micronuclei in bone marrow cells of male NMRI mice.