Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: negative (BASF SE, 2006)

HPRT: negative (BASF SE, 2017)

MNT: positive in the absence of metabolic activation only (BASF SE, 2018)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his and trp gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S-9 Mix
Test concentrations with justification for top dose:
22 μg - 5 500 μg/plate (SPT)
11 μg - 2 750 μg/plate (PIT; TA 1535 and TA 100)
2 μg - 550 μg/plate (PIT; TA 1537 and TA 98)
22 μg - 5 500 μg/plate (PIT, E. coli WP2 uvrA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: Good solubility of the test substance in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
with S-9 Mix; strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanine (MNNG)
Remarks:
without S-9 Mix; strains TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine (NOPD)
Remarks:
without S-9 Mix; strains TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S-9 Mix; strains TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S-9 Mix; E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation


DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-72 hours in the dark


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test chemical 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
S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control
range under all experimental conditions in two experiments carried out independently of
each other.
Statistics:
not applicable
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
Remarks:
PIT: from about 275 μg/plate onward; SPT: from about 550 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
PIT: from about 275 μg/plate onward; SPT: from about 550 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 550 μg/plate onward.
In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 275 μg/plate onward.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
=> no E. coli or TA102 strain tested
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 or phenobarbital-induced S9
Test concentrations with justification for top dose:
30-1000 µg/plate
Vehicle / solvent:
Vehicle: DMSO (in 0.1 ml DMSO to top agar)
Justification: Good solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
TA1538, TA98 and TA100; S9 Aroclor-1254-induced
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamin
Remarks:
TA1538 and TA98; without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537; without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Glycidyl methacrylate
Remarks:
TA 1535 and TA 100; without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracen
Remarks:
TA 1535 and TA1537; with S9 (phenobarbital-induced)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 and TA100; without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);


- Exposure duration: after addition of the test substance in top agar, the plates were incubated for 48-72 hours.
- all test were performed on triplicate plates.
- Compunds were once tested with phenobarbital-induced S9, once with Aroclor-1254-induced S9, and twice without any additional metabolizing system.
- Plated were counted manually
- Controls: Solvent controls, positive controls and sterility controls for S9 mix were run with each experiment.
Evaluation criteria:
Increase of revertant rate was considered signifcant when their number is more than 2-fold compared to spontaneous revertant rate.
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: toxic in the highest concentration tested
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / micronucleus study
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 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
July 2016
Qualifier:
according to guideline
Guideline:
other: EU method B49; No L193: In vitro Mammalian Cell Micronucleus Test
Version / remarks:
May 2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
CELLS USED
- Cell cycle length, doubling time or proliferation index: 12 -14h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 containing an L-glutamine source supplemented with 10% FCS, 1% penicillin/streptomycin, 1% amphocericine B
- 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:
other: p53 proficient
Cytokinesis block (if used):
cytochalasin-B
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Without S9: 0.16 - 100µg/mL (different concentrations in different experiments)
With S9: 1.56 - 200µg/ml
The concentration was limited by cytotoxicity
Vehicle / solvent:
Due to the insufficient solubility of the test substance in water, dimethylsulfoxide (DMSO) was used as vehicle, which has been demonstrated to be suitable in the in vitro cytogenetic assay and for which historical control data are available.
Untreated negative controls:
yes
Remarks:
24h exposure only
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h (with and without S9), 24h (without S9)
- Expression time (cells in growth medium): 20 or 40h, none in the case of the 24h exposure
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 44h (with S9 only)

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin-B

STAIN (for cytogenetic assays): DAPI and PI

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION: method according to Fenech: 5x104 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides. At least two slides per replicate were prepared. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes.

NUMBER OF CELLS EVALUATED: 2000 binucleated cells per test group

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells were scored.

DETERMINATION OF CYTOTOXICITY
- Method: proliferation index: The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it was determined in at least 1000 cells per culture (at least 2000 cells per test group). This value indicates the average number of cell cycles per cell during the period of exposure to the actin polymerisation inhibitor cytochalasin B. The CBPI is used to calculate the % cytostasis (relative inhibition of cell growth compared to the respective vehicle control group) i.e. a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

OTHER EXAMINATIONS:
- pH
- Osmolarity
- Precipitation
Evaluation criteria:
A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data

A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data.
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PH and osmolarity were not influenced.
Precipitation was observed at 100µg/mL -S9 and at 800µg/mL +S9 in the first experiment.

Cytotoxicity
Cell numbers were not reduced to below 50% at any concentration tested. But proliferation was strongly reduced at and above 2.5µg/mL without S9 and at and above 12.5µg/mL with S9.
Remarks on result:
other: positive results obtained only after 24h of treatment, not after 4h treatment

The following experiments were invalid due to the indicated reasons. The results from these experiments are not reported, yet will be archived in the raw data of this study:

Experiment I in the presence and absence of S9 mix: Due to strong cytotoxicity in both experimental parts not enough scorable dose groups were provided. Thus, a guideline-conform scoring of three dose groups per experimental part was not given.

Experiment III in the presence of S9 mix: Due to a faulty application of CytB the cultures were abandoned.

Results without S9 mix

Experiment Exposure / Preparation Interval Group Micronucleated cells [%] Citotoxicity (CBPI) [%]
2 4/24 hrs neg. control 1.4 0
0.63 1 19.5
1.25 1.1 22.9
2.5 1.1 47.3
5 n.s. 80.4
pos. control 6.1* 29.6
3 24/24 hrs neg. control 1.1 0
0.63 1.2 17.2
1.25 1.3 19.1
2.5 3.2* 44.2
5 n.s. n.s.
pos. control 2.4* 28.4
5 24/24 hrs neg. control 0.6 0
0.63 0.8 16.3
1.25 1 10.1
2.5 2.6* 28.9
3.75 n.s. n.s.
pos. control 2.3* 15.8

* significant differences

n.s.: not scorable due to strong cytotoxicity

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)
Version / remarks:
July 16
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell transformation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 12-16h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Ham's F12 supplemented with 10% FCS except during treatment. All media were supplemented with 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine B. CO2 concentration was 5%
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
A pretest was performed up to 2200µg/ml (app. 10.7mM). PH was not influenced. Precipitation occured at and above 275µg/ml in the absence at and above 68.75µg/ml in the presence of S9 mix.
After 4 hours of treatment without S9, relative survival of the cells was reduced to below 20% at 8.59µg/ml. In the presence of S9, relative survival was reduced at and above 68.75µg/ml.
Based on these results the following doses were selected for the main study:
-S9, 1st exp. +S9, 1st exp.
0.10 μg/mL 1.56 μg/mL
0.20 μg/mL 3.13 μg/mL
0.39 μg/mL 6.25 μg/mL
0.78 μg/mL 12.50 μg/mL
1.56 μg/mL 25.00 μg/mL
3.13 μg/mL 50.00 μg/mL
6.25 μg/mL 100.00 μg/mL
12.50 μg/mL 200.00 μg/mL

-S9, 2nd exp. +S9, 2nd exp.
0.29 μg/mL 2.34 μg/mL
0.59 μg/mL 4.69 μg/mL
1.17 μg/mL 9.36 μg/mL
2.34 μg/mL 18.75 μg/mL
4.69 μg/mL 37.50 μg/mL
9.36 μg/mL 75.00 μg/mL
18.75 μg/mL 150.00 μg/mL
Vehicle / solvent:
DMSO
Selected, due to the insolubility of the test substance in aqueous media
Negative solvent / vehicle controls:
yes
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: 20x10^6 cells /40mL

DURATION
- Preincubation period: 1 day
- Exposure duration: 4h
- 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): 16

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if:
• 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 be within our historical negative control data range (95% control limit).
• The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range

Assessment criteria
A test substance is considered to be clearly positive if:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
And
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit)
Isolated increases of mutant frequencies above our 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:
• 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 our laboratory’s historical negative control data (95% control limit)
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Cytotoxic effects, as indicated by clearly reduced relative survival of about or below 20% of the respective negative control values were observed in both experiments in the absence and presence of S9 mix at least at the highest applied concentrations. In the absence of S9 mix, there was a strong decrease in the number of colonies at 12.50 μg/mL (RS: 0.0%) in the 1st Experiment and from 9.36 μg/mL (RS: 3.4%) onward in the 2nd Experiment after an exposure period of 4 hours. In addition, in the presence of S9 mix, there was a severe decrease in the number of colonies from 100.00 μg/mL (RS: 1.0%) onward in the 1st Experiment and from 75.00 μg/mL (RS: 1.3%) onward in the 2nd Experiment.

In this study, in the absence of S9 mix, after 4 hours treatment the morphology and attachment of the cells was adversely influenced (grade > 2) from 6.25 μg/mL onward in the 1st Experiment and at 4.69 μg/mL and above in the 2nd Experiment. In addition, in the presence of S9 mix, after 4 hours treatment with 50.00 μg/mL and above in the 1st Experiment and with 75.00 μg/mL and above in the 2nd Experiment, the morphology and attachment of the cells was adversely influenced (grade > 2).

Osmolality and pH values were not influenced by test substance treatment. In this study, in the absence of S9 mix precipitation was not observed up to the highest concentration applied in both experiments. In the presence of S9 mix, test substance precipitation was observed macroscopically in culture medium at the end of treatment at the highest applied concentrations in the 1st and the 2nd Experiment (200.00 or 150.00 μg/mL respectively).

Experiments without S9

Experiment Test Groups Mutant frequency (per 10^6 cells) Relative survival Cloning efficiency2
1 Neg. Control 1.04 100.0 100.0
0.10 2.05 124.0 101.4
0.20 0.70 129.0 98.6
0.39 1.45 110.3 95.2
0.78 4.35 124.2 95.5
1.56 3.17 102.3 87.2
3.13 2.88 110.5 84.1
6.25 2.51 62.4 68.9
12.50 n.c. 0.0 n.c.
  Pos. Control 110.18S 155.9 78.2
2 Neg. Control 4.50 100.0 100.0
0.29 2.21 117.0 81.4
0.59 3.79 117.3 95.2
1.17 3.69 106.1 89.5
2.34 10.54S 98.9 94.0
4.69 6.53 74.4 87.4
9.36 n.c. 3.4 n.c.
18.75 n.c. 0.0 n.c.
Pos. Control 109.29S 94.2 80.8

Experiments with S9

Experiment Test Groups Mutant frequency (per 10^6 cells) Relative survival Cloning efficiency2
1 Neg. Control 1.27 100.0 100.0
1.56 1.75 122.8 108.2
3.13 2.90 96.7 87.3
6.25 2.41 99.4 78.8
12.5 2.73 99.5 81.0
25 4.61 76.9 68.7
50 1.67 24.6 75.6
100 n.c. 1.0 n.c.
200 n.c. 0.0 n.c.
  Pos. Control 99.02S 69.3 64.9
2 Neg. Control 2.92 100.0 100.0
2.34 3.51 95.7 99.7
4.69 3.31 112.1 96.8
9.36 2.86 137.3 91.8
18.75 1.67 104.7 87.5
37.5 3.09 65.0 94.5
75 n.c. 1.3 n.c.
150 n.c. 0.0 n.c.
Pos. Control 101.49S 86.4 78.1

n.c.: not continued due to strong cytotoxicity

S: significantly different compared to control

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

Genetic toxicity in vivo

Description of key information

MNT in vivo: negative (BASF 2018)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
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 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks
- Assigned to test groups randomly: yes
- Housing: Makrolon cages
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Limited solubility of the test substance in water
Duration of treatment / exposure:
24 (all treatments), 48 h (control and high dose)
Frequency of treatment:
once
Remarks:
Females: 1500, 750, 375mg/kg
Remarks:
Males: 750, 375, 187.5mg/kg
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide: 20 mg/kg
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION
The slides were stained in eosin and methylene blue (modified May-Gruenwald solution or Wrights solution) for about 5 minutes.
After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
Subsequently, the slides were stained in Giemsa solution (15 ml Giemsa, 185 ml purified water) for about 15 minutes.
After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Gorbit-Balsam.
Evaluation criteria:
Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. >= 2000 PCEs and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs.
- The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.


Assessment criteria
A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.

A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
mortality at 1500mg/kg
Vehicle controls validity:
valid
Positive controls validity:
valid
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Since mortality occured in 1500mg/kg males, this dose could not be scored. An additional experiment was performed, adding a new low dose of 187.5mg/kg as well as the 48h evaluation for the new high dose of 750mg/kg for males. Concurrent controls were included in this experiment and valid.
The slight increase observed in the 48h control samples (females) and in the low dose (females) had no relation to dosing and was not confirmed when twice the number of PCEs were recounted.
There was no significant difference in the percentage of PCEs per erythrocytes between groups.

Males
Interval Exp. Micronuclei Range PCEs relative to control
Vehicle control 24 2 1.1 ‰ 2-6 100%
Positive control 24 2 16.9 ‰** 51-86 104%
187.5mg/kg 24 2 1.5 ‰ 2-10 104%
Vehicle control 24 1 1.9 ‰ 2-10 100%
Positive control 24 1 13.5 ‰** 34-71 130%
375mg/kg 24 1 1.7 ‰ 5-8 115%
750mg/kg 24 1 1.9 ‰ 3-12 125%
Vehicle control 48 2 1.1 ‰ 3-7 100%
750mg/kg 48 2 1.3 ‰ 1-9 97%
Females
Interval sample size Micronuclei Range PCEs relative to control
Vehicle control 24 4000 1.7 ‰ 3-7 100%
Vehicle control 24 8000 1.5 ‰ 10-14 100%
Positive control 24 4000 12.8 ‰** 28-79 94%
375mg/kg 24 4000 2.5 ‰ 5-16 85%
375mg/kg 24 8000 1.7 ‰ 8-21 89%
750mg/kg 24 4000 1.3 ‰ 3-7 97%
1500mg/kg 24 4000 1.5 ‰ 4-8 79%
Vehicle control 48 4000 2.4 ‰ 6-14 100%
1500mg/kg 48 4000 1.8 ‰ 2-11 94%

** significantly different from control

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

Additional information

The substance Laromer BDDA 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. The test strains are TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The test concentrations are 22 µg - 5500 µg/plate (SPT), 11 µg - 2750 µg/plate (PIT; TA 1535 and TA 100), 2 µg - 550 µg/plate (PIT; TA 1537 and TA 98) and 22 - 5500 µg/plate (PIT, E.coli WP2 uvrA). No precipitation of the test substance was found. A bacteriotoxic effect was observed depending on the strain and test conditions from about 275 μg/plate onward. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance Laromer BDDA is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

This result is supported by a publicised plate incorporation Ames Test. Exposure of Salmonelly strains TA 1535, 1537, 100, 98 to up to 1000µg/plate with or without the addition of a metabolizing system led to bacteriotoxicity, but not to an increase in mutant frequencies.

In an HPRT assay in mammalian cells, up to 6.25µg/ml without metabolic activation and up to 50µg/ml with metabolic activation of BDDA were tested. The concentration was limited by the strong cytotoxic effects of the test substance. 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. A single statistically significant mutant frequency observed with the test substance in the 2nd Experiment in the absence of metabolic activation was considered biologically irrelevant. 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 in two experiments performed independently of each other.

The substance Laromer BDDA was assessed for its potential to induce micronuclei in TK6 cells in vitro (clastogenic or aneugenic activity). Five independent experiments were carried out, with and/or without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Of these experiment I in the presence and absence of S9 mix was considered invalid due to strong cytotoxicity in both experimental parts, which led to an insufficient number of scorable dose groups. The cultures of experiment III in the presence of S9 mix were abandoned due to a faulty application of CytB. Of the remaining experiments, the highest concentrations that could be scored were 2.5µg/ml in the absence of S9 either for 4 or 24h treatment, and 25µg/mL in the presence of S9. Since no increase in the number of micronuclei was observed after treatment for 4h, an additional experiment was performed with addition of S9 with continous treatment for 24h. A significant increase in the number of micronuclei was observed at 2.5µg/mL in two independent experiments in the presence of strong cytotoxicity. A slightly higher concentration of 3.75µg/mL was not scorable due to excessive cytotoxicity. Consequently, BDDA was judged to be clastogenic in vitro at cytotoxic concentrations in the absence of metabolic activation.

To check the relevance of the results obtained in vitro, an in vivo MNT was subsequently performed.

Five male and female NMRI mice were orally exposed to doses of up to 1500mg/kg of BDDA in corn oil in a study according to OECD guideline 474. The highest dose led to mortality in male mice, so that the maximum evaluable dose in this sex is 750mg/kg. Treatment with BDDA did not lead to an increase in the number of micronuclei, while positive and negative (vehicle) controls gave the expected results. Two marginally (not significantly) increased values, one in the low dose in female animals, which was not confirmed after a higher number of cells were evaluated, and the other in the control group were considered as incidental. Consequently, it can be concluded that BDDA does not possess clastogenic properties.

Justification for classification or non-classification

BDDA did not cause point mutations either in bacteria or mammalian cells. In an MNT in vitro, an increase in the number of micronuclei was observed only without metabolic activation after 24h, and only at cytotoxic concentrations. This result could not be confirmed in an in vivo study. Consequently, there is no need to classify BDDA for mutagenicity according to Regulation (EC) No 1272/2008 (CLP).