Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: negative (OECD 471, GLP, BASF 2012)
HPRT: negative (OECD 476, GLP, BASF 2012)

MNT in vitro: positive in the absence of S9, negative in the presence of S9 (OECD 487, GLP, BASF 2019)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 October 2018 to 20 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU method B.49 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
- Batch No.of test material: 180006P040
Species / strain / cell type:
lymphocytes: primary human
Details on mammalian cell type (if applicable):
CELLS USED
- Type: primary human lymphocytes

For lymphocytes:
- Sex, age and number of blood donors: in the 1st Experiment a 30 year old female donor and in the 2nd Experiment a male (32 years old) donor were used.
- Whether whole blood or separated lymphocytes were used: separated lymphocytes
- Whether blood from different donors were pooled or not: not pooled
- Mitogen used for lymphocytes: yes, phytohemagglutinin (PHA)

MEDIA USED
- Type and composition of media,
All media were supplemented with:
- 1% [v/v] penicillin/streptomycin (final concentration 100 μg/mL)
- 1% [v/v] HEPES buffer (1 M)

For the stimulation the medium was supplemented with:
- 0.5% [v/v] Phytohemagglutinin (PHA, stock solution 0.6 mg/mL, final concentration 3 μg/mL)
- 0.5% [v/v] sodium heparin (25000 IE)
For the Cytochalasin B treatment the medium was supplemented with:
- 30 μL Cytochalasin B (Cyt B, stock solution: 2 mg/mL in DMSO, final concentration: 6 μg/mL)

Culture medium
DMEM / Ham's F12 (1:1) (DMEM/F12) medium containing stable glutamine supplemented with 10% [v/v] fetal calf serum (FCS).

Treatment medium (4 hours treatment without and with S9 mix)
DMEM/F12 medium containing stable glutamine

All incubations were performed at 37°C with a relative humidity of ≥ 90% in a 5% [v/v] CO2 atmosphere.
Cytokinesis block (if used):
Cyt B
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The S9 fraction was prepared according to Ames et al. (1975) at BASF SE. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g) received 80 mg/kg b.w. phenobarbital i.p. and ß-naphthoflavone orally 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 dark 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 induced 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 (1 mL ≙ 1 g wet liver), then 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) was stored at -70°C to -80°C.
- method of preparation of S9 mix
The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawn at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors). The mixture of both components (S9 mix) was kept cool until use.

Following the concentrations of the cofactors:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer was prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.

- concentration or volume of S9 mix in the final culture medium : 6 mL (20% v/v)
- quality controls of S9 (metabolic capability): Metabolic activation of Cyclophosphamide
Test concentrations with justification for top dose:
1st experiment
Without S9 mix, 4 hours exposure
5.6, 10.1, 18.1, 32.7, 58.8, 105.8, 190.5, 342.9 μg/mL

With S9 mix, 4 hours exposure
32.7, 58.8, 105.8, 190.5, 342.9, 617.3, 1111.1, 2000 µg/mL

2nd experiment
without S9 mix, 4 hours exposure
0.5, 0.9, 1.6, 2.9, 5.1, 9.3, 16.7, 30.0 µg/mL

Test substance precipitation in culture medium occurred at 125.0 μg/mL and above 4 hours after start of treatment in the absence and at 1000 µg/mL and above in the presence of S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: used as vehicle, which has been demonstrated to be suitable in the in vitro cytogenetic assay and for which historical control data are available. The final concentration of the vehicle DMSO in culture medium was 1% (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Stimulating time: 48 hours
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment: recovery time 16 hours, harvest time 20 hours

FOR MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: cytB, 6 µg/mL for 20 hours
- Number of cells spread and analysed per concentration: 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei .
- Criteria for scoring micronucleated cells: The analysis of micronuclei was carried out the following criteria of Countryman and Heddle.
> The diameter of the micronucleus was less than 1/3 of the main nucleus
> The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
> Only binucleated cells were scored.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The cells were transferred into tubes, centrifugated at 900 g for 5 min and washed with HBSS. After washing, the cells were centrifuged (900 g, 5 min) and suspended in 0.0375 M KCl (37°C), incubation for 20 minutes at 37°C. After the hypotonic treatment, the cells were fixed by adding of fixative (19 parts methanol and 1 part acetic acid). The cells were centrifuged (900 g, 5 min, 4°C) and suspended in fresh fixative and incubated for 20 min at 4°C. The fixation step will be repeated twice. After the last fixation step, the cells can be centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fresh fixative and spread on slides. The slides were dipped in deionized water, the cells were pipetted on the slide and fixed by passing through a flame. The cells were stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2, 10 min) and mounted. The slides were scored microscopically.
Evaluation criteria:
Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the evaluation of a sufficient number of analyzable cells in the control groups (vehicle/positive) and in at least three exposed test groups.
- Sufficient cell proliferation was demonstrated in the vehicle control.
- The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive controls both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.

Assessment criteria
A test substance is considered to be clearly positive if all 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 concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significantly 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 (95% control limit).
Statistics:
An appropriate statistical analysis was performed. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each
test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE). If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (s) were printed in the tables.
In addition, a statistical trend test (SAS procedure REG (15)) was performed to assess a possible dose-related increase of micronucleated cells. The used model is one of the proposed
models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the number of micronucleated cells and the independent variable
was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05. However, both, biological and statistical significance were considered together.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH was not influenced
- Data on osmolality: osmolality was not influenced
- Precipitation and time of the determination: in the absence of S9 mix, test substance precipitation in culture medium was observed at the end of treatment at 58.8 μg/mL and above in the 1st Experiment. In the 2nd Experiment precipitation was not observed up to the highest concentration applied (30.0 μg/mL). In the presence of S9 mix (1st Experiment) precipitation occurred at the end of treatment at the concentrations 342.9 μg/mL and above.

STUDY RESULTS
Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
Reduced proliferation was observed in the 1st Experiment in the absence of S9 mix at all concentrations evaluated for cytotoxicity. The cytostasis at 10.1 μg/mL and 18.1 μg/mL was 31.4% and 69.2%, respectively; higher concentrations were not evaluable for cytotoxicity. Thus, in this experimental part only two cultures (treated with 5.6 and 10.1 μg/mL) could have been evaluated for the occurance of micronuclei, therefore this experimental part did not fulfill the acceptance criteria of the current OECD guidline and was repeated in the 2nd Experiment. In the 2nd Experiment without S9 mix relevantly reduced cell proliferation was observed at 16.7 μg/mL (CBPI cytostasis: 46.7%); higher concentrations were not evaluable for cytotoxicity. In the presence of S9 mix a cytostasis of 20.7% was observed at 105.8 μg/mL. Treatment with higher concentrations induced too strong cytotoxic effects. The cytostasis at 190.5 μg/mL was 67.9% and cultures treated with higher concentrations were not scorable.

- Genotoxicity results
Please refer to table 1 - 3 in the section "any other information on results".

HISTORICAL CONTROL DATA
- Positive historical control data: please refer to table 5 in the section "any other information on results".
- Negative (solvent/vehicle) historical control data: please refer to table 4 in the section "any other information on results".

Table 1: Analysis of micronuclei – 1st Experiment; 4 hours exposure, without S9 mix

 

Test group

[µg/mL]

Culture

No. of

evaluated cells

Cells containing Micronuclei

[n]

[n]

[%]

Vehicle control*

A

1000

6

12

0.6

B

1000

6

5.6

A

n.d.

B

10.1

A

1000

19

33

1.7s

B

1000

14

18.1

A

n.s.

B

32.7

A

n.s.

B

58.8

A

n.s.

B

105.8

A

n.p.

B

190.5

A

n.p.

B

342.9

A

n.p.

B

MMC 0.31

A

n.d.

B

 

Table 2: Analysis of micronuclei – 1st Experiment; 4 hours exposure, with S9 mix

 

Test group

[µg/mL]

Culture

No. of

evaluated cells

Cells containing Micronuclei

[n]

[n]

[%]

Vehicle control*

A

1000

13

17

0.9

B

1000

4

32.7

A

1000

6

10

0.5

B

1000

4

58.8

A

1000

6

13

0.7

B

1000

7

105.8

A

1000

8

12

0.6

B

1000

4

190.5

A

n.s.

B

342.9

A

n.s.

B

617.3

A

n.p.

B

1111.1

A

n.p.

B

2000.0

A

n.p.

B

CPA 2.50

A

1000

94

137

6.9s

B

1000

43

 

 

Table 3:Analysis of micronuclei – 2nd Experiment; 4 hours exposure, without S9 mix

 

Test group

[µg/mL]

Culture

No. of

evaluated cells

Cells containing Micronuclei

[n]

[n]

[%]

Vehicle control*

A

1000

3

9

0.5

B

1000

6

0.5

A

n.d.

B

0.9

A

n.d.

B

1.6

A

n.d.

B

2.9

A

n.d.

B

5.1

A

1000

6

14

0.7

B

1000

8

9.3

A

1000

11

29

1.5s

B

1000

18

16.7

A

1000

14

31

1.6s

B

1000

17

30.0

A

n.s.

B

MMC 0.31

A

1000

128

236

11.8s

B

1000

108

 

* DMSO 1% (v/v)

s = Frequency statistically significantly higher than corresponding control values

n.s. = not scorable due to strong cytotoxicity

n.p. = not scorable due to strong cytotoxicity

n.d. = not determined

 

Table 4

Historical negative control data

 

Summary – Without S9 Mix, All Vehicles*

Period: April 2018 – February 2019

Micronucleated cells [%]

Exposure period

4 hrs

Mean

0.6

Minimum

0.4

Maximum

1.0

Standard Deviation

0.15

95% Lower Control Limit

0.3

95% Upper Control Limit

0.9

No. of Experiments

23

*culture medium, DMSO 1%, Aceton 1%, Ethanol 1%

 

Summary – With S9 Mix, All Vehicles*

Period: April 2018 – February 2019

Micronucleated cells [%]

Exposure period

4 hrs

Mean

0.7

Minimum

0.3

Maximum

1.3

Standard Deviation

0.26

95% Lower Control Limit

0.2

95% Upper Control Limit

1.3

No. of Experiments

23

* culture medium, DMSO 1%, Aceton 1%, Ethanol 1%

 

Table 5 Historical positive control data

 

Summary – without S9 Mix, Mitomycin C and Colchicin

Period: April 2018 – February 2019

Micronucleated cells [%]

Exposure period

4 hrs

Substance and Concentration

MMC 0.31 µg/mL

Mean

8.1

Minimum

3.8

Maximum

12.4

Standard Deviation

2.91

No. of Experiments

21

 

Summary – with S9 Mix, Cychlophosphamid

Period: April 2018 – February 2019

Micronucleated cells [%]

Exposure period

4 hrs

Substance and Concentration

CPA 2.50 µg/mL

Mean

3.7

Minimum

1.9

Maximum

5.8

Standard Deviation

1.12

No. of Experiments

19

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 201 to 04 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch identification: 110007P040
Target gene:
his (salmonella)
trp (e.coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: The Salmonella strains TA 98, TA 100 and TA 1537 were obtained from KNOLL Aktiengesellschaft, Ludwigshafen, Germany. The Salmonella strain TA 1535 was obtained from Merck KGaA, Darmstadt, Germany.

The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (uvrB); ampicillin resistance (R factor plasmid).
Histidine auxotrophy is checked in each experiment via the spontaneous rate.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: The Escherichia coli strain was obtained from Merck KGaA, Darmstadt, Germany.
E. coli WP2 uvrA is checked for UV sensitivity.
Tryptophan auxotrophy is checked in each experiment via the spontaneous rate.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : 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 each on three consecutive days. During this time, the animals are housed in Makrolon cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a relative humidity of 30 - 70%. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours).
Standardized pelleted feed and tap water from bottles were available ad libitum. 24 hours after the last administration, the rats are sacrificed, and the livers are prepared using sterile solvents and glassware at a temperature of +4°C. The livers are weighed and washed in a weight-equivalent volume of a 150 mM KCl solution, then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9 000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (so-called S9 fraction) arestored at -70°C to -80°C.
- method of preparation of S9 mix: The S9 mix is prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient amount of S9 fraction is thawed at room temperature and 1 volume of S9 fraction is mixed with 9 volumes of S9 supplement (cofactors). This preparation, the so-called S9 mix, is kept on ice until used. The concentrations of the cofactors in the S9 mix are:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
15 mM phosphate buffer (pH 7.4)
The phosphate buffer ( 6) is prepared by mixing an Na2HPO4 solution with an NaH2PO4 solution in a ratio of about 4:1.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 mL
- quality controls of S9: 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:
1st experiment (standard plate test, no preincubation, with and without S9)
0, 33, 100, 333, 1000, 2500, 5000 µg/plate
2nd experiment (preincubation test, with and without S9)
0, 10, 33, 100, 333, 1000, 2500 µg/plate (TA strains)
0, 33, 100, 333, 1000, 2500, 5000 µg/plate (e.coli strains)
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate are generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose will be 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.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility 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.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with S9
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
TA 98, TA 100, TA 1535, TA1537, E.coli
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
TA 100, TA 1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD)
Remarks:
TA 98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9
Positive control substance:
9-aminoacridine
Remarks:
TA 1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
E.coli
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation) (first experiment); preincubation (second experiment)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min
- Exposure duration/duration of treatment: 48 to 72 hours

FOR GENE MUTATION:
- Selective agent: Salmonella: 0.5 mM histidine + 0.5 mM biotin, E.coli: 0.5 mM tryptophan

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition (titer determination)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Individual plate counts, the mean number of revertant colonies per plate and the standard deviation were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.
Evaluation criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

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

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
standard plate test: at 5000 µg/plate; pre-incubation test: at 333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Remarks:
standard plate test: at 5000 µg/plate; pre-incubation test: at 1000 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
standard plate test: no cytotoxicity; pre-incubation test: at 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Remarks:
standard plate test: at 2500 µg and above; pre-incubation test: at 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Remarks:
standard plate test: no cytotoxicity; pre-incubation test: at 2500 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation found

STUDY RESULTS
- Concurrent vehicle negative and positive control data : please refer to table 1-10.

Ames test:
- Signs of toxicity : yes
- Individual plate counts : please refer to table 1-10.
- Mean number of revertant colonies per plate and standard deviation : please refer to table 1-10.

HISTORICAL CONTROL DATA
Please refer to table 11-15 (negative control data) and table 16 to 20 (positive control data).

Standard plate test

 

Table 1: TA 1535

TA 1535

Without S9 mix

With S9 mix

 

DOSE/PLATE

REV

M

SD

FAC

REV

M

SD

FAC

TITRE

DMSO

16

14

3

1.0

15

15

2

11.0

44

10

 

 

 

14

 

 

 

37

15

 

 

 

17

 

 

 

29

33 µg

14

12

2

0.9

15

16

3

1.0

 

13

 

 

 

19

 

 

 

 

10

 

 

 

14

 

 

 

 

100 µg

10

12

3

0.9

17

15

3

1.0

 

15

 

 

 

11

 

 

 

 

11

 

 

 

16

 

 

 

 

333 µg

12

12

3

0.9

17

15

3

1.0

 

14

 

 

 

12

 

 

 

 

9

 

 

 

15

 

 

 

 

1000 µg

10

12

2

0.9

16

15

1

1.0

 

14

 

 

 

14

 

 

 

 

12

 

 

 

16

 

 

 

 

2500 µg

14

12

2

0.9

19

15

4

1.0

26

11

 

 

 

12

 

 

 

32

11

 

 

 

14

 

 

 

24

5000 µg

7

6

1

0.5

11

10

1

0.7

27

6

 

 

 

10

 

 

 

29

6

 

 

 

9

 

 

 

28

MNNG 5.0 µg

1126

1152

57

84.3

 

 

 

 

 

1217

 

 

 

 

 

 

 

 

1113

 

 

 

 

 

 

 

 

2-AA 2.5 µg

 

 

 

 

169

180

9

11.7

 

 

 

 

 

184

 

 

 

 

 

 

 

 

186

 

 

 

 

(REV=revertants/plate, M=mean, SD=standard deviation, FAC=factor)

 

Table 2: TA 100

TA 100

Without S9 mix

With S9 mix

 

DOSE/PLATE

REV

M

SD

FAC

REV

M

SD

FAC

TITRE

DMSO

75

76

3

1.0

99

99

12

1.0

25

79

 

 

 

111

 

 

 

27

74

 

 

 

87

 

 

 

27

33 µg

77

81

6

1.1

109

86

11

1.0

 

88

 

 

 

92

 

 

 

 

78

 

 

 

88

 

 

 

 

100 µg

81

77

4

1.0

77

88

10

0.9

 

77

 

 

 

91

 

 

 

 

74

 

 

 

96

 

 

 

 

333 µg

66

72

5

0.9

70

87

17

0.9

 

74

 

 

 

103

 

 

 

 

76

 

 

 

88

 

 

 

 

1000 µg

74

77

3

1.0

72

77

5

0.8

 

79

 

 

 

82

 

 

 

 

77

 

 

 

77

 

 

 

 

2500 µg

93

82

10

1.1

60

67

6

0.7

27

76

 

 

 

70

 

 

 

25

76

 

 

 

71

 

 

 

21

5000 µg

62

51

10

0.7

37

39

2

0.4

17

45

 

 

 

41

 

 

 

28

46

 

 

 

40

 

 

 

12

MNNG 5.0 µg

913

1038

112

13.7

 

 

 

 

 

1129

 

 

 

 

 

 

 

 

1072

 

 

 

 

 

 

 

 

2-AA 2.5 µg

 

 

 

 

745

724

57

7.3

 

 

 

 

 

660

 

 

 

 

 

 

 

 

767

 

 

 

 

(REV=revertants/plate, M=mean, SD=standard deviation, FAC=factor)

 

Table 3: TA 1537

TA 1537

Without S9 mix

With S9 mix

 

DOSE/PLATE

REV

M

SD

FAC

REV

M

SD

FAC

TITRE

DMSO

4

5

1

1.0

5

6

1

1.0

29

6

 

 

 

7

 

 

 

29

6

 

 

 

6

 

 

 

35

33 µg

6

5

1

0.9

6

6

2

1.1

 

4

 

 

 

8

 

 

 

 

5

 

 

 

5

 

 

 

 

100 µg

7

5

2

0.9

5

6

1

0.9

 

3

 

 

 

7

 

 

 

 

5

 

 

 

5

 

 

 

 

333 µg

5

6

1

1.1

7

7

1

1.2

 

5

 

 

 

8

 

 

 

 

7

 

 

 

6

 

 

 

 

1000 µg

4

6

2

1.1

7

6

1

1.0

 

6

 

 

 

5

 

 

 

 

7

 

 

 

6

 

 

 

 

2500 µg

4

4

1

0.7

2

3

1

0.6

17

3

 

 

 

4

 

 

 

21

4

 

 

 

4

 

 

 

24

5000 µg

3

3

1

0.5

4

2

2

0.4

10

2

 

 

 

1

 

 

 

18

3

 

 

 

2

 

 

 

14

AAC 100 µg

419

443

25

83.1

 

 

 

 

 

441

 

 

 

 

 

 

 

 

469

 

 

 

 

 

 

 

 

2-AA 2.5 µg

 

 

 

 

139

154

17

25.7

 

 

 

 

 

150

 

 

 

 

 

 

 

 

173

 

 

 

 

(REV=revertants/plate, M=mean, SD=standard deviation, FAC=factor)

 

Table 4: TA 98

TA 98

Without S9 mix

With S9 mix

 

DOSE/PLATE

REV

M

SD

FAC

REV

M

SD

FAC

TITRE

DMSO

20

18

4

1.0

30

26

5

1.0

26

21

 

 

 

20

 

 

 

37

14

 

 

 

27

 

 

 

22

33 µg

18

20

2

1.1

24

23

1

0.9

 

22

 

 

 

24

 

 

 

 

19

 

 

 

22

 

 

 

 

100 µg

14

18

4

1.0

27

25

3

1.0

 

20

 

 

 

21

 

 

 

 

21

 

 

 

26

 

 

 

 

333 µg

16

18

2

1.0

17

21

5

0.8

 

17

 

 

 

26

 

 

 

 

20

 

 

 

19

 

 

 

 

1000 µg

18

18

1

1.0

21

24

3

0.9

 

19

 

 

 

24

 

 

 

 

18

 

 

 

27

 

 

 

 

2500 µg

12

13

2

0.7

22

18

4

0.7

 

15

 

 

 

15

 

 

 

 

11

 

 

 

17

 

 

 

 

5000 µg

9

9

1

0.5

16

13

2

0.5

22

8

 

 

 

12

 

 

 

24

10

 

 

 

12

 

 

 

26

NOPD 10 µg

772

779

37

42.5

 

 

 

 

 

745

 

 

 

 

 

 

 

 

819

 

 

 

 

 

 

 

 

2-AA 2.5 µg

 

 

 

 

860

803

49

31.3

 

 

 

 

 

773

 

 

 

 

 

 

 

 

776

 

 

 

 

(REV=revertants/plate, M=mean, SD=standard deviation, FAC=factor)

 

Table 5: E.coli WP2 uvrA

E.coli

Without S9 mix

With S9 mix

 

DOSE/PLATE

REV

M

SD

FAC

REV

M

SD

FAC

TITRE

DMSO

41

40

4

1.0

45

40

6

1.0

44

36

 

 

 

34

 

 

 

38

43

 

 

 

41

 

 

 

46

33 µg

30

36

6

0.9

39

41

10

1.0

 

40

 

 

 

52

 

 

 

 

39

 

 

 

32

 

 

 

 

100 µg

44

38

6

0.9

39

43

4

1.1

 

37

 

 

 

44

 

 

 

 

32

 

 

 

46

 

 

 

 

333 µg

39

39

5

1.0

32

39

8

1.0

 

44

 

 

 

38

 

 

 

 

35

 

 

 

48

 

 

 

 

1000 µg

32

33

1

0.8

45

36

8

0.9

 

34

 

 

 

30

 

 

 

 

34

 

 

 

33

 

 

 

 

2500 µg

37

36

2

0.9

37

37

5

0.9

36

34

 

 

 

32

 

 

 

44

38

 

 

 

41

 

 

 

35

5000 µg

36

39

3

1.0

35

37

3

0.9

33

40

 

 

 

41

 

 

 

32

41

 

 

 

36

 

 

 

41

4-NQO 5.0 µg

775

731

44

18.3

 

 

 

 

 

687

 

 

 

 

 

 

 

 

732

 

 

 

 

 

 

 

 

2-AA 60 µg

 

 

 

 

287

256

31

6.4

 

 

 

 

 

254

 

 

 

 

 

 

 

 

226

 

 

 

 

(REV=revertants/plate, M=mean, SD=standard deviation, FAC=factor)

 

Preincubation Test

 

Table 6: TA 1535

TA 1535

Without S9 mix

With S9 mix

 

DOSE/PLATE

REV

M

SD

FAC

REV

M

SD

FAC

TITRE

DMSO

15

15

3

1.0

16

15

1

1.0

28

17

 

 

 

15

 

 

 

44

12

 

 

 

15

 

 

 

34

10 µg

18

16

2

1.1

18

16

3

1.0

 

15

 

 

 

13

 

 

 

 

16

 

 

 

17

 

 

 

 

33 µg

12

15

3

1.0

16

14

2

0.9

 

11

 

 

 

15

 

 

 

 

21

 

 

 

12

 

 

 

 

100 µg

14

15

3

1.0

21

18

3

1.2

 

18

 

 

 

18

 

 

 

 

13

 

 

 

15

 

 

 

 

333 µg

15

16

1

1.1

10

14

4

0.9

 

17

 

 

 

17

 

 

 

 

16

 

 

 

14

 

 

 

 

1000 µg

12

12

5

0.8

11

10

2

0.6

30

17

 

 

 

10

 

 

 

26

8

 

 

 

8

 

 

 

34

2500 µg

11

8

3

0.5

11

8

3

0.5

14

6

 

 

 

5

 

 

 

11

6

 

 

 

7

 

 

 

19

MNNG 5.0 µg

887

831

83

56.7

 

 

 

 

 

736

 

 

 

 

 

 

 

 

871

 

 

 

 

 

 

 

 

2-AA 2.5 µg

 

 

 

 

136

145

10

9.5

 

 

 

 

 

155

 

 

 

 

 

 

 

 

145

 

 

 

 

(REV=revertants/plate, M=mean, SD=standard deviation, FAC=factor)

 

Table 7: TA 100

TA 100

Without S9 mix

With S9 mix

 

DOSE/PLATE

REV

M

SD

FAC

REV

M

SD

FAC

TITRE

DMSO

78

80

2

1.0

98

96

9

1.0

45

82

 

 

 

104

 

 

 

35

81

 

 

 

87

 

 

 

33

10 µg

86

84

9

1.0

111

97

13

1.0

 

91

 

 

 

96

 

 

 

 

74

 

 

 

85

 

 

 

 

33 µg

79

83

5

1.0

96

99

5

1.0

 

88

 

 

 

97

 

 

 

 

83

 

 

 

105

 

 

 

 

100 µg

69

78

10

1.0

80

85

13

0.9

 

76

 

 

 

75

 

 

 

 

88

 

 

 

99

 

 

 

 

333 µg

79

85

6

1.1

114

93

18

1.0

 

90

 

 

 

85

 

 

 

 

87

 

 

 

80

 

 

 

 

1000 µg

74

66

7

0.8

70

71

6

0.7

28

64

 

 

 

65

 

 

 

21

60

 

 

 

77

 

 

 

34

2500 µg

55

43

11

0.5

50

48

6

0.5

22

41

 

 

 

41

 

 

 

17

34

 

 

 

52

 

 

 

21

MNNG 5.0 µg

777

820

42

10.2

 

 

 

 

 

861

 

 

 

 

 

 

 

 

821

 

 

 

 

 

 

 

 

2-AA 2.5 µg

 

 

 

 

886

857

28

8.9

 

 

 

 

 

831

 

 

 

 

 

 

 

 

855

 

 

 

 

(REV=revertants/plate, M=mean, SD=standard deviation, FAC=factor)

 

Table 8: TA 1537

Ta 1537

Without S9 mix

With S9 mix

 

DOSE/PLATE

REV

M

SD

FAC

REV

M

SD

FAC

TITRE

DMSO

8

8

1

1.0

10

8

3

1.0

25

8

 

 

 

9

 

 

 

27

7

 

 

 

5

 

 

 

22

10 µg

6

8

2

1.0

9

9

3

1.1

 

9

 

 

 

6

 

 

 

 

8

 

 

 

11

 

 

 

 

33 µg

5

7

3

1.0

7

7

1

0.8

 

7

 

 

 

7

 

 

 

 

10

 

 

 

6

 

 

 

 

100 µg

6

7

1

0.9

9

8

3

1.0

 

8

 

 

 

10

 

 

 

 

6

 

 

 

5

 

 

 

 

333 µg

9

8

1

1.0

8

7

1

0.8

 

7

 

 

 

6

 

 

 

 

8

 

 

 

6

 

 

 

 

1000 µg

5

4

1

0.6

7

6

1

0.8

26

5

 

 

 

6

 

 

 

29

3

 

 

 

5

 

 

 

25

2500 µg

4

3

1

0.3

3

3

2

0.3

12

2

 

 

 

1

 

 

 

18

2

 

 

 

4

 

 

 

13

AAC 100 µg

448

435

11

56.8

 

 

 

 

 

430

 

 

 

 

 

 

 

 

428

 

 

 

 

 

 

 

 

2-AA 2.5 µg

 

 

 

 

111

129

21

16.1

 

 

 

 

 

124

 

 

 

 

 

 

 

 

152

 

 

 

 

(REV=revertants/plate, M=mean, SD=standard deviation, FAC=factor)

 

Table 9: TA 98

TA 98

Without S9 mix

With S9 mix

 

DOSE/PLATE

REV

M

SD

FAC

REV

M

SD

FAC

TITRE

DMSO

24

21

3

1.0

27

29

2

1.0

24

18

 

 

 

29

 

 

 

28

21

 

 

 

31

 

 

 

35

10 µg

28

26

7

1.3

28

31

8

1.1

 

19

 

 

 

40

 

 

 

 

32

 

 

 

26

 

 

 

 

33 µg

14

22

7

1.1

35

34

1

1.2

 

28

 

 

 

35

 

 

 

 

25

 

 

 

33

 

 

 

 

100 µg

23

23

6

1.1

29

31

3

1.1

 

28

 

 

 

29

 

 

 

 

17

 

 

 

34

 

 

 

 

333 µg

13

20

8

1.0

28

22

9

0.7

 

19

 

 

 

26

 

 

 

 

29

 

 

 

11

 

 

 

 

1000 µg

26

21

5

1.0

17

18

3

0.6

18

21

 

 

 

16

 

 

 

27

17

 

 

 

21

 

 

 

23

2500 µg

14

10

3

0.5

10

9

3

0.3

29

8

 

 

 

6

 

 

 

21

8

 

 

 

11

 

 

 

18

AAC 100 µg

366

384

22

18.3

 

 

 

 

 

378

 

 

 

 

 

 

 

 

409

 

 

 

 

 

 

 

 

2-AA 2.5 µg

 

 

 

 

778

683

87

23.6

 

 

 

 

 

607

 

 

 

 

 

 

 

 

665

 

 

 

 

(REV=revertants/plate, M=mean, SD=standard deviation, FAC=factor)

 

Table 10: E.coli WP2 uvrA

E.coli WP2 uvrA

Without S9 mix

With S9 mix

 

DOSE/PLATE

REV

M

SD

FAC

REV

M

SD

FAC

TITRE

DMSO

45

44

4

1.0

47

50

7

1.0

54

47

 

 

 

58

 

 

 

51

39

 

 

 

46

 

 

 

33

33 µg

33

39

7

0.9

48

44

9

0.9

 

47

 

 

 

34

 

 

 

 

36

 

 

 

51

 

 

 

 

100 µg

51

42

8

1.0

58

46

11

0.9

 

36

 

 

 

36

 

 

 

 

40

 

 

 

44

 

 

 

 

333 µg

48

44

7

1.0

44

44

6

0.9

 

36

 

 

 

39

 

 

 

 

47

 

 

 

50

 

 

 

 

1000 µg

33

27

6

0.6

51

36

15

0.7

 

21

 

 

 

36

 

 

 

 

28

 

 

 

21

 

 

 

 

2500 µg

34

25

8

0.6

27

26

7

0.5

24

19

 

 

 

19

 

 

 

26

23

 

 

 

32

 

 

 

29

5000 µg

29

28

4

0.6

24

23

4

0.5

32

24

 

 

 

19

 

 

 

47

32

 

 

 

26

 

 

 

21

4-NQO 5.0 µg

559

625

95

14.3

 

 

 

 

 

734

 

 

 

 

 

 

 

 

582

 

 

 

 

 

 

 

 

2-AA 60 µg

 

 

 

 

266

257

18

5.1

 

 

 

 

 

237

 

 

 

 

 

 

 

 

269

 

 

 

 

(REV=revertants/plate, M=mean, SD=standard deviation, FAC=factor)

 

Historical Negative Control Data

 

Table 11: TA 1535

Method

S9 mix

Negativ control

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

Water

02.03.-02.11.

397

10

458

18

5

SPT

-

DMSO

08.07.-03.11.

400

10

51

18

6

SPT

-

Acetone

01.96-01.11.

309

10

31

18

3

SPT

1:9

Water

11.02-02.11.

400

10

42

18

5

SPT

1:9

DMSO

08.07.-03.11.

400

10

58

18

6

SPT

1:9

Acetone

01.96.-01.11.

306

10

35

19

3

 

Method

S9 mix

Negativ control

Period

No. of plates

Min*

Max*

Mean*

SD

PIT

-

Water

08.01-03.11

400

9

36

18

4

PIT

-

DMSO

02.07-03.11

400

10

41

17

4

PIT

-

Acetone

01.96-02.11

261

10

32

8

3

PIT

1:9

Water

06.01-03.11

400

9

36

17

3

PIT

1:9

DMSO

01.07-02.11

400

10

45

17

5

PIT

1:9

Acetone

01.96-02.11

253

10

31

18

3

 

 

Table 12: TA 100

Method

S9 mix

Negativ control

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

Water

07.02-02.11

400

76

138

107

11

SPT

-

DMSO

08.07-02.11

400

80

160

107

14

SPT

-

Acetone

01.96-01.11

351

85

160

116

14

SPT

1:9

Water

05.02-02.11

400

81

155

113

14

SPT

1:9

DMSO

08.07-03.11

400

77

158

112

17

SPT

1:9

Acetone

01.96-01.11

351

81

172

119

17

 

Method

S9 mix

Negativ control

Period

No. of plates

Min*

Max*

Mean*

SD

PIT

-

Water

08.01-03.11

400

75

150

108

12

PIT

-

DMSO

02.07-03.11

397

79

153

105

12

PIT

-

Acetone

01.96-02.11

270

87

168

115

15

PIT

1:9

Water

08.01-03.11

400

80

151

113

13

PIT

1:9

DMSO

01.07-03.11

400

78

153

107

13

PIT

1:9

Acetone

01.96-02.11

246

90

181

119

17

 

 

Table 13: TA 1537

Method

S9 mix

Negativ control

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

Water

08.02-02.11

400

4

16

9

2

SPT

-

DMSO

07.07-03.11

400

5

16

9

2

SPT

-

Acetone

01.96-01.11

306

5

18

10

2

SPT

1:9

Water

08.02-02.11

400

5

17

10

2

SPT

1:9

DMSO

07.07-03.11

400

5

18

9

2

SPT

1:9

Acetone

01.96-01.11

306

5

20

11

2

 

Method

S9 mix

Negativ control

Period

No. of plates

Min*

Max*

Mean*

SD

PIT

-

Water

08.01-03.11

400

5

16

9

2

PIT

-

DMSO

02.07-03.11

700

5

16

9

2

PIT

-

Acetone

01.96-02.11

258

5

17

10

2

PIT

1:9

Water

06.01-03.11

400

5

16

10

2

PIT

1:9

DMSO

01.07-03.11

400

5

16

9

2

PIT

1:9

Acetone

01.96-02.11

249

5

20

10

2

 

 

Table 14: TA 98

Method

S9 mix

Negativ control

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

Water

08.02-02.11

400

17

53

29

5

SPT

-

DMSO

08.07-03.11

400

17

46

28

5

SPT

-

Acetone

01.96-01.11

312

19

52

29

5

SPT

1:9

Water

11.02-02.11

400

21

55

36

6

SPT

1:9

DMSO

08.07-03.11

400

18

53

34

6

SPT

1:9

Acetone

01.96-01.11

312

25

54

38

6

 

Method

S9 mix

Negativ control

Period

No. of plates

Min*

Max*

Mean*

SD

PIT

-

Water

06.01-03.11

400

18

48

28

5

PIT

-

DMSO

01.07-03.11

400

13

45

28

2

PIT

-

Acetone

01.96-02.11

261

18

45

28

5

PIT

1:9

Water

06.01-03.11

400

18

49

34

5

PIT

1:9

DMSO

12.06-03.11

400

14

55

33

6

PIT

1:9

Acetone

01.96-02.11

246

20

51

36

6

 

 

Table 15: WP2 uvrA

Method

S9 mix

Negativ control

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

Water

03.02-02.11

400

24

60

35

7

SPT

-

DMSO

05.07-03.11

400

24

61

38

7

SPT

-

Acetone

01.96-01.11

291

18

56

34

6

SPT

1:9

Water

03.02-02.11

397

24

59

40

7

SPT

1:9

DMSO

05.07-03.11

400

25

64

44

7

SPT

1:9

Acetone

01.96-01.11

291

15

61

38

7

 

Method

S9 mix

Negativ control

Period

No. of plates

Min*

Max*

Mean*

SD

PIT

-

Water

06.11-03.11

400

21

56

34

6

PIT

-

DMSO

12.06-03.11

400

24

57

36

7

PIT

-

Acetone

01.96-02.11

243

19

55

33

6

PIT

1:9

Water

01.01-03.11

400

25

70

39

7

PIT

1:9

DMSO

12.06-03.11

400

21

60

38

7

PIT

1:9

Acetone

01.96-02.11

243

20

58

37

7

 

* = revertants/plate                   PIT = preincubation test

SPT = standard plate test         SD = standard deviation

 

Historical Positive Control Data

 

Table 16: TA 1535

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

MMNG 5.0

05.08-03.11

400

505

1464

748

169

SPT

1:9

2-AA 2.5

05.08-03.11

400

77

955

164

96

 

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

PIT

-

MMNG 5.0

11.07-03.11

400

308

1680

689

162

PIT

1:9

2-AA 2.5

11.07-03.11

400

76

998

153

92

 

 

Table 17: TA 100

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

MMNG 5.0

05.08-03.11

400

318

1299

782

153

SPT

1:9

2-AA 2.5

05.08-03.11

400

509

2120

856

124

 

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

PIT

-

MMNG 5.0

12.07-03.11

400

505

1458

837

158

PIT

1:9

2-AA 2.5

11.07-03.11

400

501

1355

815

126

 

 

Table 18: TA 1537

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

AAC 100

04.08-03-11

400

214

981

393

71

SPT

1:9

2-AA 2.5

04.08-03.11

400

77

211

153

31

 

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

PIT

-

AAC 100

11.07-03.11

400

127

665

381

59

PIT

1:9

2-AA 2.5

11.07-03.11

400

71

196

130

21

 

 

Table 19: TA 98

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

NOPD 10

04.08-03.11

400

307

961

519

129

SPT

1:9

2-AA 2.5

05.08-03.11

400

463

1412

723

141

 

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

PIT

-

NOPD 10

11.06-03.11

400

314

1055

523

94

PIT

1:9

2-AA 2.5

10.07-03.11

400

218

1074

620

95

 

 

Table 20: WP2 uvrA

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

4-NQO 5.0

04.08-03.11

400

367

1613

839

198

SPT

1:9

2-AA 60.0

04.08-03.11

400

151

398

230

37

 

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

PIT

-

4-NQO 5.0

10.07-03.11

400

318

1466

658

134

PIT

1:9

2-AA 60.0

10.07-03.11

400

150

785

232

48

 

*         = revertants/plate

SPT    = standard plate test

PIT     = preincubation test     

SD       = standard deviation

2 -AA  = 2-aminoanthracene

MNNG= N-methyl-N'-nitro-N-nitrosoguanidine

AAC    = 9-aminoacridine

NOPD = 4-nitro-o-phenylendiamine

4-NQO= 4-Nitroquinoline-N-oxide

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 October 2011 to 10 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
adopted August 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Batch Number: 110007P040
Target gene:
hypoxanthine-guanine phosphoribosyl transferase
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Chinese hamster cell line V79 from Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line.

For cell lines:
- Absence of Mycoplasma contamination: screened
- Methods for maintenance in cell culture: Thawed stock cultures are propagated at 37 °C in 80 cm2 plastic flasks. About 5×105 cells
were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1 %). The cells were sub-cultured twice weekly.
- Cell cycle length, doubling time or proliferation index : 12 to 16 hours
- Modal number of chromosomes: 22
- Periodically checked for karyotype stabiliy: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
MEM containing Hank's salt supplemented with 10% FBS, 5µg/mL neomycin, 1% amphotericin B (Serum free medium was used during the 4h treatment), for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
The cell cultures were incubated at at 37 °C in a 1.5 % carbon dioxide atmosphere (98.5 % air).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: The S9 were prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU, weight approx. 220 – 320 g, Harlan Laboratories B.V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital and by peroral administrations of 80 mg/kg b.w. naphthoflavone each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules were kept at –20 °C for up to one week.
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. Cofactors were added to the S9 supernatant to reach following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-phosphate-buffer, pH 7.4.
- concentration of S9 mix and S9 in the final culture medium: Final protein concentration in the cultures: 0.75 mg/mL
- quality controls of S9: Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.
Test concentrations with justification for top dose:
4h without S9: 0, 0.3, 0.6, 1.3, 2.5 µg/mL (additional concentrations not chosen for analysis due to cytotoxicity: 3.8, 5.0 µg/mL)
4h with S9: 40, 80, 160, 240, 300 µg/mL
24h without S9: 0.32, 0.63, 1.3, 1.9, 2.5 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and relative non-toxicity to the cell cultures
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h and 24 h
- Harvest time after the end of treatment: 3 to 4 days

FOR GENE MUTATION:
- Expression time: 7 days
- Selection time: 8 days
- Selective agent: 6-thioguanine
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: Following the expression time cell culture flasks were seeded with about 3 - 5×105 cells each in medium
containing 6-TG. The cultures were incubated for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 10E+06 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

Evaluation of Results
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance was considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2.5 µg/mL (without S9), 300 µg/mL (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no effect
- Data on osmolality: no effect
- Precipitation/phase separation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation occurred at 312.5 µg/mL and above in the presence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
Two pre-tests were performed in order to determine the concentration range of the mutagenicity experiments. The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with respect to the current OECD Guideline 476. The first pre-test, performed in the concentration range from 39.1 to 5000 µg/mL, was not analysable in the absence of metabolic activation following 4 and 24 hours treatment due to exceedingly severe cytotoxicity down to the lowest concentration. Therefore, the pretest was repeated in a concentration range from 0.31 and 40.0 µg/mL without (4 and 24
hours treatment) metabolic activation. The general culture conditions and experimental conditions in this pre-tests were the same as described for the mutagenicity experiment below. In these pre-tests the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls.
Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

STUDY RESULTS
- Concurrent vehicle negative and positive control data : please refer to table no.1.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: please refer to table no.1.

- Genotoxicity results: please refer to table no. 1.

HISTORICAL CONTROL DATA
Please refer to table no. 2

Table 1 Summary of genotoxicity and cytotoxicity results

 

 

 

 

 

S9

mix

 

relative

cloning

efficiency I

%

relative

cell

density

%

relative

cloning

efficiency II

%

mutant

colonies/

106cells

 

 

induction

factor

 

relative

cloning

efficiency I

%

relative

cell

density

%

relative

cloning

efficiency II

%

mutant

colonies/

106cells

 

 

induction

factor

 

 

conc.

PS

 

µg/mL

 

 

 

 

Column

1

2

3

4

5

6

7

8

9

10

11

12

13

Experiment 1 / 4 h treatment

 

 

 

culture I

culture II

Solvent control with DMSO

 

 

-

100.0

100.0

100.0

18.9

1.0

100.0

100.0

100.0

5.8

1.0

Positive control (EMS)

150.0

 

-

92.6

140.2

98.2

75.7

4.0

70.7

93.5

91.3

165.1

28.3

Test item

0.3

 

-

97.8

1182

97.0

9.0

0.5

94.2

136.9

102.6

12.6

2.2

Test item

0.6

 

-

98.0

1222

87.8

16.7

0.9

88.9

160.7

104.8

15.1

2.6

Test item

1.3

 

-

75.5

116.4

84.0

24.2

1.3

75.6

119.4

108.3

30.3

5.2

Test item

2.5

 

-

12.7

1012

84.4

6.5

0.3

33.1

97.1

93.9

17.3

3.0

Test item

3.8

 

-

0.0

culture was not continued*

24.5

3.5

culture was not continued*

Test item

5.0

 

-

0.0

culture was not continued*

214

culture was not continued*

Solvent control with DMSO

 

 

+

100.0

100.0

100.0

7.5

1.0

100.0

100.0

100.0

9.2

1.0

Positive control (DMBA)

1.1

 

+

75.4

122.7

89.3

485.8

64.8

74.9

102.6

70.6

630.6

68.5

Test item

20.0

 

+

97.0

culture was not continued**

96.2

culture was not continued**

Test item

40.0

 

+

95.7

100.5

93.9

15.1

2.0

97.5

104.6

82.3

5.0

0.5

Test item

80.0

 

+

94.7

80.3

98.4

7.9

1.1

95.6

116.6

78.3

21.5

2.3

Test item

160.0

 

+

92.6

86.8

105.1

8.1

1.1

94.6

91.4

87.4

13.3

1.4

Test item

240.0

PS

+

24.0

125.0

93.7

20.8

2.8

92.3

101.1

78.3

10.0

1.1

Test item

300.0

PS

+

0.0

30.3

97.1

7.9

1.1

84.3

90.4

76.1

4.0

0.4

Experiment II / 24 h treatment

 

 

 

culture I

culture II

Solvent control with DMSO

 

 

-

100.0

100.0

100.0

21.3

1.0

100.0

100.0

100.0

3.6

1.0

Positive control (EMS)

150.000

 

-

119.6

80.3

84.1

347.6

16.4

108.6

102.5

82.3

308.6

85.1

Test item

0.039

 

-

108.6

culture was not continued**

94.5

culture was not continued**

Test item

0.079

 

-

84.6

culture was not continued**

87.2

culture was not continued**

Test item

0.160

 

-

82.9

culture was not continued**

76.0

culture was not continued**

Test item

0.32

 

-

88.8

84.7

99.6

11.0

0.5

79.5

77.8

96.3

16.4

4.5

Test item

0.63

 

-

83.0

93.5

104.4

15.9

0.7

73.4

89.4

111.7

10.5

2.9

Test item

1.3

 

-

73.3

77.8

109.3

11.3

0.5

76.2

90.7

99.1

7.4

2.1

Test item

1.9

 

-

57.9

69.7

112.8

12.8

0.6

46.3

95.1

137.5

4.9

1.3

Test item

2.5

 

-

20.0

432

112.8

27.1

1.3

18.3

73.5

155.1

8.2

2.3

Experiment II / 4 h treatment

 

 

 

 

 

Solvent control with DMSO

 

 

+

100.0

100.0

100.0

15.9

1.0

100.0

100.0

100.0

3.9

1.0

Positive control (DMBA)

1.1

 

+

61.7

70.9

66.5

485.0

30.6

73.8

69.5

85.0

479.5

122.2

Test item

20.0

 

+

95.2

culture was not continued**

102.7

culture was not continued**

Test item

40.0

 

+

87.8

81.3

120.0

9.3

0.6

107.1

85.4

99.5

7.3

1.9

Test item

80.0

 

+

89.4

95.5

93.7

19.5

1.2

90.1

86.3

94.3

11.0

2.8

Test item

160.0

 

+

82.8

86.8

98.7

10.4

0.7

95.0

80.8

101.2

20.1

5.1

Test item

240.0

 

+

59.6

76.5

107.9

8.2

0.5

100.1

84.4

96.0

21.7

5.5

Test item

300.0

PS

+

0.0

12.7

81.1

30.5

1.9

99.9

80.0

90.4

13.7

3.5

 

* culture was not continued due to exceedingly severe cytotoxic effects

** culture was not continued as only four concentrations are required by the guideline

PS phase separation visible at the end of treatment

EMS: ethylmethane sulfonate

DMBA: 7,12-dimethylbenz(a)anthracene

 

Table 2 Historical control data

 

Number of mutant colonies per 106cells

without metabolic activation (4 hours treatment time)

 

Positive control EMS 150 µg/mL

Solvent control (medium, water, acetone DMSO, ethanol, THF)

Range:

65.0 - 1386.4

4.4 - 42.9

Mean value:

181.8

16.7

Standard deviation:

170.5

8.2

Number of studies:

59

60

with metabolic activation {4 hours treatment time)

 

Positive control DMBA 1.1 -2.0 µg/mL

Solvent control (medium, water, acetone DMSO, ethanol, THF)

Range:

64.3 - 2634.0

3.3 - 45.3

Mean value:

939.4

17.1

Standard deviation:

406.1

7.6

Number of studies:

59

59

without metabolic activation (24 hours treatment time)

 

Positive control EMS 75 - 300 µg/mL

Solvent control (medium, water, acetone DMSO, ethanol, THF)

Range:

51.6 - 1925.5

2.6 - 40.3

Mean value:

358.2

18.0

Standard deviation:

246.6

8.2

Number of studies:

48

48

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

MNT in vivo: negativ (RA, OECD 474, GLP, BASF 2002)
MNT in vivo: negativ (RA, OECD 474, GLP, CrayValley 2001)
MNT in vivo: negativ (RA, OECD 474, GLP, Cytec 2006)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 July 2002 to 08 August 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
19 May 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Lot/batch No.: B 02011
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: about 29 g
- Assigned to test groups randomly: yes, under following basis: randomized plan prepared with an appropriate computer program
- Housing: Makrolon cages, type MI, housed individually from start of the treatment until the end of the test
- Diet: ad libitum
- Water: ad libitum
- 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/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the hydrolytical sensitivity of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 12.5 g/100 ml, 25.0 g/100 mL and 50.0 g/100 mL.
- Amount of vehicle: 4 mL/kg bw.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment.
Duration of treatment / exposure:
single dose
Frequency of treatment:
once, single dose
Post exposure period:
24 or 48 hours
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals per dose for the 24 hour sacrifices and 5 animals per dose for the 48 hour sacrifices
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPP) and vincristine sulphate (VCR)
- Justification for choice of positive controls: The stability of CPP and VCR is well-defined under the selected conditions, since both positive control articles are well-defined clastogens and aneugens respectively.
- Route of administration: The positive controls, both, dissolved in purified water were administered to male animals once orally or intraperitoneally each in a volume of 10 ml/kg body weight.
- Doses / concentrations: 20 mg CPP//kg body weight for clastogenic effects and 0.15 mg VCR/kg body weight for aneugenic effects.
Tissues and cell types examined:
In general, 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored .
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline were survived by all animals (male and female) without any clinical signs. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the híghest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control groups,the 24-hour sacrifice interval was investigated only.

DETAILS OF SLIDE PREPARATION:
The two femora were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained. The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- Number of small micronuclei (dD/4) (d = diameter of micronucleus, D= cell diameter)

The size of micronuclei may indicate the possible mode of action of the test substance, i .e . a clastogenic or a spindle poison effect. Slides were coded before microscopic analysis. Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative values can occur.
Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i .e. >=2000 polychromatic erythrocytes.
- The proportion of cells with micronuclei in negative control animals was within the normal range of the historical control data.
- The two positive control chemicals induced a significant increase in the number of cells containing small and large micronuclei within the range of the historical control data or above.

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals.
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time.
- The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN.
The number of micronuclei in polychromatic erythrocytes was analyzed.
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians . Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p <=0.05, ** for p<=0.01) were printed with the group means in the tables. This test was performed one-sided.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control and within the range of historical control data.
With 19.7%° the positive control substance cyclophosphamide for clastogenicity, led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei.
With 61 .0%° the positive control vincristine for spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i.e . 14 .2%°.
- Ratio of PCE/NCE: No inhibition of erythropoesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Summary tables

Table 1:Polychromatic and normochromatic erythrocytes

 

Interval: 24 hours

Total No. of MN (‰) in

Interval: 48 hours

Total No. of MN (‰) in

PCE’s

NCE’s

PCE’s

NCE’s

PCE’s

NCE’s

PCE’s

NCE’s

Vehicle

DMSO

10000

2955

0.9

1.0

10000

2801

0.8

0.0

 

500 mg/kg

10000

2492

1.9*

1.2

 

 

 

 

 

1000 mg/kg

10000

2875

1.0

2.1

 

 

 

 

 

2000 mg/kg

10000

3540

1.3

1.1

10000

2812

1.7*

0.4

CCP

20 mg/kg

10000

3201

19.7**

0.9

 

 

 

 

VCR

0.15 mg/kg

10000

3749

61.0**

0.5

 

 

 

 

Wilcoxon Test (one-sided): * : p <= 0.05, ** : p <= 0.01

A pairwise comparison of each dose group with the vehicle control group.

 

Table 2: Polychromatic erythrocytes: differentiation between small and large micronuclei

 

Interval: 24 hours Total No. of Cells (‰) with

Interval: 48 hours Total No. of MN (‰) with

Of PCE’s

MN. D<D/4

MN.d=>D/4

Of PCE’s

MN. D<D/4

MN.d=>D/4

Vehicle

DMSO

10000

0.9

0.0

10000

0.8

0.0

 

500 mg/kg

10000

1.9*

0.0

 

 

 

 

1000 mg/kg

10000

1.0

0.0

 

 

 

 

2000 mg/kg

10000

1.3

0.0

10000

1.7*

0.0

CPP

20 mg/kg

10000

19.7**

0.0

 

 

 

VCR

0.15 mg/kg

10000

46.8**

14.2**

 

 

 

Wilcoxon Test (one-sided): * : p <= 0.05, ** : p <= 0.01

A pairwise comparison of each dose group with the vehicle control group.

 

 

Historical Negative Control Data / Mice (NMRI)

 

Table 3: Route Oral

2000 evaluated Polychromatic Erythrocytes per Animal

Means of 5 Animals per Group

Vehicle Control Water

micronuclei in polychromatic erythrocytes ‰

small

large

total

mean

1.4

0.0

1.4

min

0.6

0.0

0.6

max

2.4

0.2

2.6

SD

0.5

0.1

0.6

n

32

 

Vehicle control Olive oil

micronuclei in polychromatic erythrocytes ‰

small

large

total

mean

1.2

0.0

1.2

min

0.8

0.0

0.8

max

1.8

0.1

1.8

SD

0.4

0.0

0.4

n

17

 

Table 4: Route Oral

1000 evaluated Polychromatic Erythrocytes per Animal

Means of 5 Animals per Group

Vehicle Control DMSO

micronuclei in polychromatic erythrocytes ‰

small

large

total

mean

1.5

0.0

1.5

min

1.0

0.0

1.0

max

2.0

0.1

2.1

SD

0.3

0.0

0.4

n

12

 

Historical Positive Control Data / Mice (NMRI)

 

Table 5: Cyclophosphamide 20 mg/kg bw

2000 evaluated Polychromatic Erythrocytes per Animal

Means of 5 Animals per Group

Route oral

micronuclei in polychromatic erythrocytes‰

small

large

total

mean

12.8

0.1

12.9

min

8.7

0.0

8.9

max

17.9

0.9

17.9

SD

2.4

0.2

2.4

n

6

 

Table 6: Vincristine 0.15 mg/kg bw

1000 / 2000 evaluated Polychromatic Erythrocytes per Animal

Means of 5 Animals per Group

2000 PCEs, Route intreperitoneal

micronuclei in polychromatic erythrocytes ‰

small

large

total

mean

60.5

11.9

72.2

min

21.9

3.8

26

max

101.9

22.3

113.7

SD

17

4.8

19.8

n

51.0

 

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
Batch: CLX 598/1
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 5-7 weeks old
- Weight at study initiation: 27.2-34.8
- Housing: housed in labelled polycarbonate cages containing purified sawdust
- Diet: standard pelleted laboratory animal diet; ad libitum
- Water: tap-water; ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: corn oil
- Concentration of test material in vehicle: 250 mg/10mL; 125 mg/10mL and 62.5 mg/10mL
Details on exposure:
The mice received a single intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of the test item. The route of administration was chosen to maximize the chance of the test article reaching the target tissue. The dosing volume was 10 mL/kg body weight.
Duration of treatment / exposure:
single dose
Frequency of treatment:
once, single dose
Post exposure period:
24h, 48h treatment groups
48h positiv conrol
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5; two groups per dosage
Control animals:
yes, concurrent vehicle
Positive control(s):
- positiv control: cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes.
Details of tissue and slide preparation:
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.
Evaluation criteria:
The test substance is considered positive in the micronucleus test if:
It Induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.
Statistics:
Wilcoxon Rank Sum Test; two-sided test at P < 0.05
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
lethargic and rough coat; no abnormalities at 62.5 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow.
- Ratio of PCE/NCE: The animals of the 250 mg/kg b.w. group showed slight decreases in the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time compared to the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time of the animals treated with the lowest dose group of 62.5 mg/kg body weight. This phenomenon, reflects a toxic effect of the test substance on the erythropoiesis, implying that it reached the bone marrow cells of the mouse.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Lot/batch No.: 5010121200
Species:
mouse
Strain:
other: Crl:CD-l(ICR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: 5-8 wks
- Weight at study initiation: 23-30 g
- Housing: Groups of up to seven in solid-floor polypropylene cages with wood-flake bedding
- Diet: Certified Rat and Mouse Diet Code 5LF2, BCM, IPS Ltd., London, UK, ad libitum
- Water: Ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 30-70 %
- Air changes: 15 per h
- Photoperiod: 12 h dark / 12 h light
Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: Arachis oil
- Lot/batch no.: SN365
Duration of treatment / exposure:
Single treatment
Frequency of treatment:
Single treatment
Post exposure period:
24 h (for 100, 50 and 25 mg/kg bw dose groups); 48 h (for 100 mg/kg bw dose group)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
7
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
Tissue: Femoral bone marrow
Cell types: Erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the results of a range-finding toxicity test, the maximum tolerated dose of the test material, 100 mg/kg bw, with the clinical signs of hunched posture and ptosis was selected for use in the main test, with 50 and 25 mg/kg as the lower dose levels.

TREATMENT AND SAMPLING TIMES: 24 or 48 h following dosing

DETAILS OF SLIDE PREPARATION: Both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCEblue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
Evaluation criteria:
A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 h kill times when compared to their corresponding control group. A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
The data was analysed following a √(x +1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No significant effects except hunched posture and ptosis
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Maximum tolerated dose: 100 mg/kg bw i.p. No toxicity was observed by oral route, therefore, systemic absorption could not be confirmed using this dose route.
- Clinical signs of toxicity in test animals: In animals dosed with the test material via the intraperitoneal route premature deaths (animals killed in extremis) occurred at and above 200 mg/kg bw, and clinical signs were observed at and above 100 mg/kg bw as follows: Hunched posture, ptosis, lethargy, ataxia, increased salivation, piloerection, decreased respiratory rate, laboured respiration, hypothermia, splayed gait, prostration and elevated tail.
- Other: The test material showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in test groups when compared to the concurrent vehicle control groups. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.
- Ratio of PCE/NCE: There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48 h test material groups when compared to their concurrent vehicle control groups. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred. There were no statistically significant increases in the frequency of micronucleated PCEs in any of the dose groups when compared to their concurrent vehicle control groups.
- Other: Mortality data and clinical observations: There were no premature deaths seen in any of the dose groups. Following clinical signs were observed in animals dosed with the test material at 100 mg/kg bw in both the 24 and 48 h groups: Hunched posture and ptosis.

Table 1: Micronucleus Test - Summary of Group Mean Data

Treatment Group Number of PCE with Micronuclei per 2000 PCE PCE/NCE Ratio
Group Mean SD Group Mean SD
Vehicle Control (Arachis oil)
10 mL/kg bw
48 h Sampling Time
1.1 1.2 0.83 0.38
Vehicle Control (Arachis oil)
10 mL/kg bw
24 h Sampling Time
1.1 1.2 0.76 0.23
Positive Control
50 mg/kg bw
24 h Sampling Time
86.4*** 27.8 1.35 0.27
Test substance
100 mg/kg bw
48 h Sampling Time
1.3 0.8 0.9 0.33
Test substance
100 mg/kg bw
24 h Sampling Time
1.4 1.9 0.78 0.34
Test substance
50 mg/kg bw
24 h Sampling Time
0.1 0.4 0.89 0.26
Test substance
25 mg/kg bw
24 h Sampling Time
1.9 1.6 0.71 0.51

where,

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standard deviation

*** = P < 0.001

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

Additional information

Genetic toxicity in vitro

 

Gene mutation in bacteria

The test substance was not mutagenic in a standard plate test and in a pre-incubation Ames test with and without metabolic activation (BASF, 2012). In the standard plate test, concentration up to 5000 μg/plate were tested in Salmonella typhimurium strains TA1535, TA 1537, TA 98 and TA 100 and E.coli WP2 uvrA. For the pre-incubation test, E.coli were also exposed to up to 5000 µg/plate, while up to 2500 µg/plate were used with TA tester strains. S9 fraction was prepared from the liver of male Sprague-Dawley rats, treated with a single dose of phenobarbital and β-naphthoflavone. Cytotoxicity was observed at and above 2500 µg/plate in the standard plate test and at and above 333 µg/plate in the pre-incubation assay, depending on test strain and test conditions.

 

Gene mutation in mammalian cells

The test substance was not mutagenic in a HPRT assay using chinese hamster V79 cells (BASF, 2012). Duplicate cell cultures were exposed to the test substance for either 4 hours (with and without S9) or 24 hours (without S9). The dose range was limited by the cytotoxic properties of the test substance to 0.3 - 2.5 µg/plate (without S9) and 40 - 300 µg/plate (with S9). After an expression phase of 7 days and a selection period of 8 days, mutant colonies were counted. S9 fraction was prepared from the liver of male Sprague-Dawley rats, treated with a single dose of phenobarbital and β-naphthoflavone.

 

Cytogenicity/ micronucleus assay in mammalian cells

The test substance induced a dose-dependent and statistically significant increase in the number of cells containing micronuclei in the absence of S9 mix and is therefore considered to have a chromosome-damaging (clastogenic) effect or to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence of metabolic activation (BASF, 2019).

Two independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats (exogenous metabolic activation).

In two independent experiments the cells were exposed for 4 hours in the absence and presence of S9 to the following concentration ranges: first experiment: 5.6 - 342.9 µg/mL (without S9), 32.7 - 2000 µg/mL (with S9), and second experiment 0.5 - 30.0 µg/mL (without S9). A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within the historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC) and Cyclophosphamide (CPA), led to the expected increase in the number of cells containing micronuclei. Cytotoxicity indicated by clearly reduced proliferation index (CBPI) was observed at least at the highest applied test substance concentration in all experimental parts of this study.

 

Genetic toxicity in vivo

 

Cytogenicity/ micronucleus assays

No in vivo cytogenetic studies exist for the test item, but three studies according to OECD guidelines and GLP are available for the structural analogue Propylidynetrimethanol, ethoxylated, esters with acrylic acid (CAS no. 28961-43-5).

 

This substance did not induce chromosomal damage (clastogenicíty) or spindle poison effects (aneugenic activity) in the micronucleus test method according to OECD guideline 474 and GLP (BASF, 2002), when administered once orally in DMSO to NMRI mice at dose levels of 500 mg/kg, 1000 mg/kg and 2000 m g/kg body weight. The administration of the test substance was tolerated by all animals without any clinical signs. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2,000 mg/kg body weight and in the vehicle controls. The animals exposed to 1,000 mg/kg or 500 mg/kg body weight and those of the positive control group were all sacrificed after 24 hours. 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded. All dose groups showed rates of micronuclei that were close to the range of the concurrent negative control and within the range of the historical control data. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected. Both of the positive control chemicals, i .e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

 

This negative result was confirmed in two further MNT studies in vivo according to OECD 474 and GLP (CrayValley 2001, Cytec 2006), in which the substance was administered intraperitoneally in doses up to 250 mg/kg bw to NMRI or Crl:CD-l(ICR)BR mice, respectively. The first study showed a slight decrease in the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time in mice treated with 250 mg/kg bw. In the second study clinical signs (hunched posture and ptosis) were seen in mice treated with 100 mg/kg bw., the highest dose used in that study. Especially the observation from the first study imply systemic uptake of the test substance and that it reaches the bone marrow cells of mice.

 

In conclusion, the test substance is not considered to induce gene mutation as determined from gene mutation assays in bacteria and mammalian cells. Although a chromosome damaging potential in the absence of metabolic activation was observed in an in vitro micronucleus assay, no evidence of a mutagenic effect was seen for structural analogues when tested in whole animal clastogenicity/aneuploidy studies. It is therefore concluded that the test substance is not mutagenic.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on gene mutation, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.