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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test

Five studies, including a test with a read across substance, were used in a weight of evidence approach.

It is therefore not possible to chose a single key study for this endpoint.

The test substance was determined to be non-mutagenic in the bacterial reverse mutation assay in a weight of evidence approach.

In vitro micronucleus test in human lymphocytes

The test item is considered to be non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to the highest required concentration.

HPRT locus assay

Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-05 to 2017-06-29
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:
adopted 29 July 2016
Deviations:
yes
Remarks:
The test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.
Qualifier:
according to guideline
Guideline:
other: Regulation (EC) No 440/2008 of 30 May 2008: In vitro Mammalian Cell Micronucleus Test, No B.49; No L 193
Version / remarks:
Commission Regulation (EC) No 640/2012 of 06 July 2012
Deviations:
no
Principles of method if other than guideline:
The following alterations from the guidelines were performed:
A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Batch no.: 0013479406
Species / strain / cell type:
hepatocytes: huamn
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human lymphocytes
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Sex, age and number of blood donors: Experiment I: male donor (27 years old)
Experiment II: male donor (23 years old)
- Whole blood were used: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
With regard to the content (87.0 g/100 g) of the test item, 2299 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 14.9 to 2299 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
In the pre-test for toxicity, no precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using a Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity, no cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 2299 μg/mL were chosen as top treatment concentration for Experiment II.
Vehicle / solvent:
- Vehicle used: Water
- Justification for choice of vehicle:
The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
culture medium with 10.0% deionised water
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcin: without metabolic activation (continuous treatment)
Details on test system and experimental conditions:
METHOD OF APPLICATION: culture flasks

DURATION
- Exposure duration
Experiment I:
4 hours (with and without S9-mix)
Eperiment II
4 hours (with S9 mix)
20 hours (without S9 mix)

The cells were prepared 40 hours after start of treatment with the test item

- Fixation time (start of exposure up to fixation or harvest of cells): 40 hrs

Cytokinesis blocker: cytochalasin B

NUMBER OF REPLICATIONS: In each experimental group two parallel cultures were analysed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.
Fixative agent: ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively)

NUMBER OF CELLS EVALUATED:
At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
The frequency of micronucleated cells was reported as % micronucleated cells.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.


Rationale for test conditions:
The micronucleus assay will be considered acceptable if it meets the following criteria:
a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.
Evaluation criteria:
A test item can be classified as non-clastogenic and non-aneugenic if:
− the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.

A test item can be classified as clastogenic and aneugenic if:
− the number of micronucleated cells is not in the range of the historical laboratory control data and
− either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.
Statistics:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No

NUMBER OF CELLS WITH MICRONUCLEI: Please refer to the summary of results table

HISTORICAL CONTROL DATA
Percentage of micronucleated cells in human lymphocyte cultures (2015-2016)
Aqueous solvents: DMEM/Ham’s F12, Deionised water (10 % v/v) Organic solvents: DMSO (0.5 or 1.0 %), Acetone, Ethanol and THF (0.5 %)

Solvent Control without S9
Micronucleated cells in %

Pulse treatment (4/40)
No. of experiments: 78*
Mean: 0.60
95 % Ctrl limit: 0.08 – 1.12 0
1x SD: 0.26
Min – Max: 0.15 – 1.65

Continuous treatment (20/40)
No. of experiments: 79**
Mean: 0.57
95 % Ctrl limit: 0.12 – 1.03
1x SD: 0.23
Min – Max: 0.05 – 1.35

Solvent Control with S9
Micronucleated cells in %
Pulse treatment (4/40)
No. of experiments: 96*
Mean: 0.62
95 % Ctrl limit: 0.16 – 1.08
1x SD: 0.23
Min – Max: 0.15 – 1.30

Positive Control without S9
Micronucleated cells in %
Pulse treatment (4/40)
MMC
No. of experiments: 78
Mean: 12.48
95 % Ctrl limit: 1.44 – 23.52
1x SD: 5.52
Min – Max: 4.15 – 30.30

Continuous treatment (20/40)
Demecolcin
No. of experiments: 81
Mean: 3.72
95 % Ctrl limit: 1.43 – 6.01
1x SD: 1.15
Min – Max: 2.10 – 7.25

Positive Control with S9
Micronucleated cells in %
Pulse treatment (4/40)
CPA
No. of experiments: 165
Mean: 5.16
95 % Ctrl limit: 0.84 – 9.49
1x SD: 2.16
Min – Max: 2.10 – 11.90

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Table 1: Summary of results

Exp.

 

Preparation

interval

 

Test item

concentration

in μg/mL

Proliferation

index

CBPI

Cytostasis

in %*

 

Micronucleated

cells

in %**

95% Ctrl limit

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

2.00

 

0.65

0.08 - 1.12

 

 

Positive control2

1.67

33.3

17.95S

 

 

 

751

2.00

0.2

0.6

 

 

 

1314

2.01

n.c

0.6

 

 

 

2299

2.02

n.c.

0.75

 

Exposure period 20 hrs without S9 mix

II

 

40 hrs

 

Solvent control1

2.02

 

0.55

0.12 - 1.03

 

 

Positive control3

1.59

42.3

4.95S

1.43 - 6.01

 

 

751

1.92

9.3

0.45

 

 

 

1314

1.95

6.9

0.45

 

 

 

2299

1.92

9.3

0.30

 

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1

2.07

 

0.90

0.16 – 1.08

 

 

Positive control4

1.91

15.0

4.00S

0.84 – 9.49

 

 

751#

2.08

n.c.

1.15

 

 

 

1314

2.07

n.c.

0.70

 

 

 

2299

2.06

0.8

0.45

 

II

40 hrs

Solvent control1

2.17

 

0.90

0.16 – 1.08

 

 

Positive control5

1.86

26.5

12.50S

0.84 – 9.49

 

 

751#

2.12

4.4

1.10

 

 

 

1314#

2.08

7.6

1.13

 

 

 

2299#

2.14

2.5

0.88

 


* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

SThe number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1Deion. water 10.0 % (v/v)

2MMC 0.8 μg/mL

3Demecolcin 100 ng/mL

4CPA 17.5 μg/mL

5CPA 15.0 μg/mL

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-06-01 to 2016-11-08
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:
28 Jul 2015
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian cell gene mutation test
Specific details on test material used for the study:
Batch no.: 0013479406
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Cell cycle length, doubling time or proliferation index: 12 - 16 hours
- Modal number of chromosomes: 20 chromosomes

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)

Culture medium
Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).
- Checked for Mycoplasma contamination: yes

Metabolic activation:
with and without
Metabolic activation system:
in induced with phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
The test substance was poorly soluble in culture medium at the end of 4 hours exposure period.
No cytotoxicity was observed in the pretest when tested up to the highest required concentration of 2350 μg/mL. Therefore, in the main experiments of this HPRT study concentrations at the border of solubility in culture medium were tested for gene mutations.

1st experiment: 4 hour exposure with and without S9-mix:
18.8, 37.5, 75.0, 150.0, 300.0, 600.0 μg/mL

2nd experiment: 4 hour exposure with and without S9-mix:
4.7, 9.4 ,18.8, 37.5, 75.0, 150.0 μg/mL



Vehicle / solvent:
- Vehicles used: Culture medium (Ham's F12)
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, culture medium (Ham's F12) was selected as vehicle.
Untreated negative controls:
yes
Remarks:
with and without S9 mix, were treated with culture medium without test substance in parallel to the other treatment groups
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: 20x10E6 cells in 40 mL)

DURATION
- Test substance incubation with test medium: approx. 20 – 24 hours after seeding
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): TG" medium: Ham's F12 medium containing stable glutamine and hypoxanthine) incubation for about 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): Day 7

SELECTION AGENT (mutation assays): Ham's F12 medium containing stable glutamine and hypoxanthine)

Fixation: Methanol

STAIN: Giesma

NUMBER OF REPLICATIONS: 2 (Cytotoxicity determination)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED: 200 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

- OTHER:
pH
The pH was measured at least for the top concentrations and for the negative controls with and without S9 mix.

Osmolality
Osmolality was measured in at least the top concentrations and the negative controls with and without S9 mix.

Solubility
Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.

Cell morphology
The test cultures of all test substance concentrations were examined microscopically for cell morphology and cellular attachment at the end of the exposure period, which is a further indication for cytotoxicity.

Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50 % (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (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 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 all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• 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 the following criteria are met:
• 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)
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report.
The dependent variable was the corrected mutant frequency 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 and the slope was greater than 0.
In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables.
However, both, biological and statistical significance are considered together.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity, in the 1st Experiment in the absence of S9 mix at the highest applied test substance concentration of 600 μg/mL, the survival (CE1) was clearly reduced to 17.1 % of control.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects
- Precipitation: In this study, in the absence and the presence of S9 mix, test substance precipitation in culture medium at the end of exposure period was observed at least at the highest applied test substance concentrations.

In detail, in both experiments in the absence of S9 mix test substance precipitation was observed macroscopically in culture medium at the end of treatment at 150.0 μg/mL and above.
In the 1st Experiment in the presence of S9 mix precipitation in culture medium at the end of treatment was observed macroscopically from the lowest applied concentration of 18.8 μg/mL onward. These raw data recordings were in disagreement with the data of the pretest, and it was assumed that these data did not reflect the real conditions. However, assessment of precipitation with the unaided eye in cell culture flasks in the presence of S9 mix is somehow difficult. In the 2nd Experiment in the presence of S9 mix precipitation in culture medium at the end of treatment was observed macroscopically at 150 μg/mL as expected. To clarify the solubility issue an additional solubility testing was performed. At this trial, test substance precipitates in culture medium 4 hours after start of treatment were observed macroscopically from 75 μg/mL onward either in the absence or presence of S9 mix.
Finally, based on the observations in the pretest, the 2nd Experiment and an additional solubility testing the border of solubility has been expected in the range of 75.0 to 293.8 μg/mL.
Thus, it has to be considered that the lowest applied concentrations in the 1st Experiment in the presence of S9 mix did not show precipitation, and, thus, the requirements of the current OECD Guideline 476 were fulfilled.


RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the purity of the test substance 2350.0 μg/mL was used as top concentration both with and without S9 mix at 4-hour exposure time.
The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for at least some selected doses.
In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.
In addition, precipitation of the test substance was not observed macroscopically in the stock solution (Test group: 2350 μg/mL). By the end of treatment test substance precipitation occurred in culture medium at concentrations of 293.8 μg/mL and above in the absence and presence of S9 mix.
After 4 hours treatment in the absence and presence of S9 mix cytotoxicity was not observed as indicated by a reduced relative cloning efficiency of about or below 20 %.

HISTORICAL CONTROL DATA
- Positive historical control data:

Summary (all experimental conditions)
Period: January 2014 - December 2015

Without S9 mix: 300 - 400 μg/mL ethyl methanesulfonate (EMS)

Corrected Mutant Frequency*
Exposure period: 4 hrs
Mean: 138.82
Minimum: 42.47
Maximum: 360.93
Standard Deviation: 86.31
95% Lower Control Limit: 0.00
95% Upper Control Limit: 325.03
No. of Experiments: 19

With S9 mix: 1.25 μg/mL 7.12-Dimethylbenz[a]anthracene (DMBA)
Corrected Mutant Frequency*
Exposure period: 4 hrs
Mean: 98.14
Minimum: 21.52
Maximum: 189.14
Standard Deviation: 48.01
95% Lower Control Limit: 0.00
95% Upper Control Limit: 202.20
No. of Experiments: 18

* = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)


- Negative (solvent/vehicle) historical control data:
Summary (all vehicles. all experimental conditions)
Period: March 2016 – August 2016

Without and with S9 mix - all vehicles*
Corrected Mutant Frequency**
Exposure period: 4 hrs
Mean: 2.39
Minimum: 0.00
Maximum: 9.93
Standard Deviation: 2.33
95% Lower Control Limit: 0.00
95% Upper Control Limit: 7.19
No. of Experiments: 37

* = culture medium. water 10 % (v/v). DMSO 1 % (v/v). acetone 1 % (v/v)
** = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Summary (all vehicles)
Period: March 2016 – August 2016

Without S9 mix - all vehicles*
Corrected Mutant Frequency**
Exposure period: 4 hrs
Mean: 2.53
Minimum: 0.00
Maximum: 6.48
Standard Deviation: 1.78
95% Lower Control Limit: 0.00
95% Upper Control Limit: 6.36
No. of Experiments: 19

With S9 mix - all vehicles*
Corrected Mutant Frequency**
Exposure period: 4 hrs
Mean: 2.25
Minimum: 0.00
Maximum: 9.93
Standard Deviation: 2.85
95% Lower Control Limit: 0.00
95% Upper Control Limit: 8.43
No. of Experiments: 18

* = culture medium. water 10 % (v/v). DMSO 1 % (v/v). acetone 1 % (v/v)
** = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Table 1: Summary of results – experimental parts without S9 mix

 

Exp.

Exposure period [h]

Test groups [µg/mL]

S9 mix

Prec.*

Genotoxicity**

MFcorr.

[per 106cells]

Cytotoxicity***

CE1[%]

CE2[%]

1

4

Negative control

-

n.d.

0.66

100.0

100.0

18.8

-

-

3.79

104.6

95.1

37.5

-

-

2.86

102.7

103.3

75.0

-

-

1.66

83.3

98.7

150.0

-

+

1.52

78.7

86.6

300.0

-

+

2.45

41.4

80.3

600.0

-

+

2.13

17.1

92.5

Positive control1

-

n.d.

205.86S

67.7

72.8

2

4

Negative control

-

n.d.

1.36

100.0

100.0

4.7

-

-

n.c.

94.3

n.c.

9.4

-

-

2.17

92.3

93.6

18.8

-

-

0.67

116.7

101.7

37.5

-

-

0.76

97.0

88.8

75.0

-

-

4.26

74.0

87.5

150.0

-

+

2.97

35.7

80.0

Positive control1

-

n.d.

111.88S

67.0

102.7

 

*       Macroscopically visible precipitation in culture medium at the end of exposure period

**      Mutant frequency MFcorr.: mutant colonies per 106cells corrected with the CE2value

***    Cloning efficiency related to the respective vehicle control

S       Mutant frequency statistically significant higher than corresponding control values

n.c.   Culture was not continued since a minimum of only four analysable concentrations is required

n.d.   Not determined

1       EMS 400 μg/mL

2       DMBA 1.25 μg/mL

 

Table 1 continued: Summary of results – experimental parts with S9 mix

 

Exp.

Exposure period [h]

Test groups [µg/mL]

S9 mix

Prec.*

Genotoxicity**

MFcorr.

[per 106cells]

Cytotoxicity***

CE1[%]

CE2[%]

1

4

Negative control

+

n.d.

1.07

100.0

100.0

18.8

+

+

1.43

113.2

99.3

37.5

+

+

1.45

102.7

98.2

75.0

+

+

1.04

92.6

102.8

150.0

+

+

1.75

101.9

102.5

300.0

+

+

5.28S

74.8

94.3

600.0

+

+

0.40

81.4

89.3

Positive control2

+

n.d.

221.50S

82.9

76.2

2

4

Negative control

+

n.d.

3.51

100.0

100.0

4.7

+

-

n.c.

112.2

n.c.

9.4

+

-

2.39

106.3

110.1

18.8

+

-

1.20

91.0

110.1

37.5

+

-

2.18

106.9

100.4

75.0

+

-

4.78

70.5

91.7

150.0

+

+

1.46

78.1

90.4

Positive control2

+

n.d.

95.81S

71.5

94.3

 

*       Macroscopically visible precipitation in culture medium at the end of exposure period

**      Mutant frequency MFcorr.: mutant colonies per 106cells corrected with the CE2value

***    Cloning efficiency related to the respective vehicle control

S       Mutant frequency statistically significant higher than corresponding control values

n.c.   Culture was not continued since a minimum of only four analysable concentrations is required

n.d.   Not determined

1       EMS 400 μg/mL

2       DMBA 1.25 μg/mL

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1980-11-05 to 1980-11-07
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
five strains tested (not including E. coli), only mixture tested with purity below 20 %
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Lot/batch No.: 9084282
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA1537, TA1538, TA 98, TA100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix, Aroclor 1254 induced
Test concentrations with justification for top dose:
the substance was tested at 1, 10, 100, 500 and 1000 µL/plate.
Vehicle / solvent:
- Vehicle/solvent used: water
Untreated negative controls:
yes
Remarks:
S-9 fraction without test substance, all strains tested
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
other: with metabolic activation: 2-Aminoanthracene (all strains); without metabolic activation: N-methyl-N-nitro-N-nitrosoguanidine (TA 1535, TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C
- Expression time (cells in growth medium): 48 hours at 37 °C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS:3

NUMBER OF CELLS EVALUATED: all revertant cells were counted

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The following criteria were used in the evaluation and reporting of the mutagenic potential of the test material:
a. the spontaneous revertant levels for each strain when used in either the direct plate assay or the activated plate assay must be within the acceptable limits as defined by the historical data,
b. all sterility controls must be negative,
c. all positive controls must demonstrate that the indicator strains are functional with known mutagens as evidenced by an increase of at least three times the number of revertant colonies per plate as the spontaneous revertant controls,
d. to be considered positive for mutagenic activity, the test material should exhibit a dose reponse effect (increasing numbers of revertant colonies with increased amounts of the test sample),
e. for strains TA 1535, TA 1537, TA 1538 and TA 98, the test sample should produce a positive dose response over the concentrations with the lowest increase in revertants/plate greater than or equal to 3x the solvent control value or the S-9 fraction control value, as applicable, to be considered mutagenic, and
f. to be considered mutagenic for strain TA 100, the test sample should produce a positive dose response over three concentrations with at least one dose producing an increase in revertants/plate greater than or equal to 3-5x the solvent control value or the S-9 fraction control value, as applicable.
Statistics:
Least squares linear regression analysis was used to compute the "best fit" regression line of dose reponse on dose level for the test sample. These regression lines represent the strength of dose response obtained against each bacterial indicator strain. For each response line
y = mx + b,
the sample regression coefficient, m, was tested for a statistically significant deviation from the value zero (0) which would indicate mutagenic, activity (3). The null hypothesis and the alternative hypothesis are stated below:
H0:m = 0, the data do not suggest mutagenic activity
HA: m>0, the data suggest increasing mutagenic activity dose
Significance level = 0.05
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
The sample was mutagenic at concentrations of 100, 500, and 1000 µL. A dose-response relationship was exhibited.
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
The sample was mutagenic at concentrations of 100, 500, and 1000 µL. A dose-response relationship was exhibited.
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
The sample was mutagenic at concentrations of 100, 500, and 1000 µL. A dose-response relationship was exhibited.
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
The sample was mutagenic at concentrations of 100, 500, and 1000 µL. A dose-response relationship was exhibited.
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
The sample was mutagenic at concentrations of 500 and 1000 µL. A dose-response relationship was exhibited.
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Number of revertants per plate (mean of three plates),

strain

TA 1535

strain

TA 1537

Strain

TA 1538

strain

TA 98

strain

TA 100

conc. [µL]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

water

13.7

13.7

4.3

4.3

15.7

15.7

32

32

133.7

133.7

1

14.7

14.3

6.3

12.3

17.7

30

23

48

141

122.3

10

20.3

13.3

13.3

8.3

27.3

31

37.3

45.7

137.7

137.3

100

85.3

104

68.7

71

87

118

117.3

138.7

217.7

236.7

500

388

389.7

319

343

453.7

453.7

403.7

482

506.3

556

1000

638

600

562.3

594.7

749.7

738.7

683.7

804

765

771

N-methyl-N-nitro-N-nitrosoguanidine 5 µg

281.7

 

 

 

 

 

 

 

784

 

2-Nitrofluorene

0.5 µg

 

 

 

 

1104.3

 

920

 

 

 

9-Aminoacridine

100 µg

 

 

49.7

 

 

 

 

 

 

 

S-9 fraction

(negative control) 50 µL

 

 

 

 

 

 

 

 

 

 

2-Aminoanthracene

2.5 µg

 

362

 

234.3

 

1746.7

 

1922.3

 

1771.3
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1981-04-20 to 1981-05-01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
five strains tested (not including E. coli)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Lot/batch No.: 10494871
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA1537, TA1538, TA 98, TA100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix, Aroclor 1254 induced
Test concentrations with justification for top dose:
the substance was tested at 1, 10, 100, 500 and 1000 µg/plate.
Vehicle / solvent:
- Vehicle/solvent used: water
Untreated negative controls:
yes
Remarks:
S-9 fraction without test substance, all strains tested
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
other: with metabolic activation: 2-Aminoanthracene (all strains); without metabolic activation: N-methyl-N-nitro-N-nitrosoguanidine (TA 1535, TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C
- Expression time (cells in growth medium): 48 hours at 37 °C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS:3

NUMBER OF CELLS EVALUATED: all revertant cells were counted

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Statistics:
Least Squares linear regression analysis was used to compute the "best fit" regression line of dose reponse on dose level for the test sample. These regression lines represent the strength of dose response obtained against each bacterial indicator strain . For each response line y= mx+b, the sample regression coefficient, m, was tested for a statistically significant deviation from the value zero (0) which would indicate mutagenic activity (3). The null hypothesis and the alternative hypothesis are:
H0: m=0, the data do not suggest mutagenic activity.
Ha: m>0, the data suggest increasing mutagneic activity
Significance level= 0.05
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Number of revertants per plate (mean of three plates),

strain

TA 1535

strain

TA 1537

Strain

TA 1538

strain

TA 98

strain

TA 100

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

water

19.3

19.3

5.0

5

18.7

18.7

38

38

136

141.3

1000

16.3

21.3

5

10.7

12.7

41

39.7

69

132.3

158.3

500

24

22.7

3.7

9.7

17.7

43

36.7

65

159.7

159

100

23.3

20

7.3

9.3

14.3

39.7

43.3

64.3

136.3

152

10

16.7

19

5.7

6.7

15.7

26.3

47.3

54

157.7

145.7

1

20.7

24

3

6

16.3

27.7

57.7

59.3

171.7

136

N-methyl-N-nitro-N-nitrosoguanidine 5 µg

1012.3

 

 

 

 

 

 

 

1740

 

2-Nitrofluorene

0.5 µg

 

 

 

 

1741.7

 

1545

 

 

 

9-Aminoacridine

100 µg

 

 

497.7

 

 

 

 

 

 

 

S-9 fraction

(negative control)

 

17

 

8

 

22.3

 

53.7

 

152.3

2-Aminoanthracene

2.5 µg

 

355.7

 

261.7

 

1636.7

 

2116

 

1931.3
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1981-04-20 to 1981-05-01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Fve strains tested (not including E. coli), only tested in mixture
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Lot/batch No.: 259
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA1537, TA1538, TA 98, TA100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix, Aroclor 1254 induced
Test concentrations with justification for top dose:
the substance was tested at 10, 100, 1000, 5000 and 10000 µg/plate.
Vehicle / solvent:
- Vehicle/solvent used: water
Untreated negative controls:
yes
Remarks:
S-9 fraction without test substance, all strains tested
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
other: with metabolic activation: 2-Aminoanthracene (all strains); without metabolic activation: N-methyl-N-nitro-N-nitrosoguanidine (TA 1535, TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C
- Expression time (cells in growth medium): 48 hours at 37 °C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS:3

NUMBER OF CELLS EVALUATED: all revertant cells were counted

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Statistics:
Least Squares linear regression analysis was used to compute the "best fit" regression line of dose reponse on dose level for the test sample. These regression lines represent the strength of dose response obtained against each bacterial indicator strain . For each response line y= mx+b, the sample regression coefficient, m, was tested for a statistically significant deviation from the value zero (0) which would indicate mutagenic activity (3). The null hypothesis and the alternative hypothesis are:
H0: m=0, the data do not suggest mutagenic activity.
Ha: m>0, the data suggest increasing mutagneic activity
Significance level= 0.05
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
10000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
10000 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Number of revertants per plate (mean of three plates),

strain

TA 1535

strain

TA 1537

Strain

TA 1538

strain

TA 98

strain

TA 100

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

water

14

14

3.7

3.7

12.3

12.3

38

38

141.7

141.7

10000

t

t

t

t

t

t

29.7

33

88.7

90

5000

9.3

6

3

3

t

10.3

35

43.3

99.3

104.3

1000

10

9.3

5

4

13.3

12

30

39.3

91.7

123.3

100

11

13

3.3

8

15.3

12.3

40

54.3

117.3

159.7

10

9.7

18.3

3

5.3

12

13

42.7

43.3

129.7

159.3

N-methyl-N-nitro-N-nitrosoguanidine 5 µg

1229

 

 

 

 

 

 

 

1114.3

 

2-Nitrofluorene

0.5 µg

 

 

 

 

1387.7

 

1526

 

 

 

9-Aminoacridine

100 µg

 

 

705.3

 

 

 

 

 

 

 

S-9 fraction

(negative control) 50 µL

 

19.7

 

6.3

 

15.3

 

44.3

 

154.7

2-Aminoanthracene

2.5 µg

 

117.7

 

211.7

 

1408.3

 

956.7

 

1256.3

t: toxicity

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1981-04-20 to 1981-05-01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Five strains tested (not including E. coli), only tested in mixture
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Lot/batch No.: 1084823
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA1537, TA1538, TA 98, TA100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix, Aroclor 1254 induced
Test concentrations with justification for top dose:
the substance was tested at 0.001, 0.01, 0.5, 1, 5 and 10 µL/plate
Vehicle / solvent:
- Vehicle/solvent used: water
Untreated negative controls:
yes
Remarks:
S-9 fraction without test substance, all strains tested
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
other: with metabolic activation: 2-Aminoanthracene (all strains); without metabolic activation: N-methyl-N-nitro-N-nitrosoguanidine (TA 1535, TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C
- Expression time (cells in growth medium): 48 hours at 37 °C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS:3

NUMBER OF CELLS EVALUATED: all revertant cells were counted

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The following criteria were used in the evaluation and reporting of the mutagenic potential of the test material:
a. the spontaneous revertant levels for each strain when used in either the direct plate assay or the activated plate assay must be within the acceptable limits as defined by the historical data,
b. all sterility controls must be negative,
c. all positive controls must demonstrate that the indicator strains are functional with known mutagens as evidenced by an increase of at least three times the number of revertant colonies per plate as the spontaneous revertant controls,
d. to be considered positive for mutagenic activity, the test material should exhibit a dose reponse effect (increasing numbers of revertant colonies with increased amounts of the test sample),
e. for strains TA 1535, TA 1537, TA 1538 and TA 98, the test sample should produce a positive dose response over the concentrations with the lowest increase in revertants/plate greater than or equal to 3x the solvent control value or the S-9 fraction control value, as applicable, to be considered mutagenic, and
f. to be considered mutagenic for strain TA 100, the test sample should produce a positive dose response over three concentrations with at least one dose producing an increase in revertants/plate greater than or equal to 3»5x the solvent control value or the S-9 fraction control value, as applicable.
Statistics:
Least squares linear regression analysis was used to compute the "best fit" regression line of dose reponse on dose level for the test sample. These regression lines represent the strength of dose response obtained against each bacterial indicator strain. For each response line y = mx + b, the sample regression coefficient, m, was tested for a statistically significant deviation from the value zero which would indicate mutagenic activity. The null hypothesis and the alternative
hypothesis are stated below:
H0 m = 0, the data do not suggest mutagenic activity
HA: m > 0, the data suggest increasing mutagenic activity

Significance level = 0.05
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Number of revertants per plate (mean of three plates),

strain

TA 1535

strain

TA 1537

Strain

TA 1538

strain

TA 98

strain

TA 100

conc. [µL]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

water

19.3

19.3

5

5

18.7

18.7

38

38

141.3

141.3

1

25

19

6.3

5.7

9.7

42

45.7

64.3

144

141

0.5

24

19

6.3

8.7

16

38.3

46

57

149.7

145.7

0.1

21

17.3

9

3.3

16.3

30

50.7

55

156.3

144.7

0.01

22

16

11

9.3

16

21.3

42

46.3

140

155.3

0.001

26.3

21

4.7

9

18.7

24

40.3

55.3

141.7

132.3

N-methyl-N-nitro-N-nitrosoguanidine 5 µg

1012.3

 

 

 

 

 

 

 

1740

 

2-Nitrofluorene

0.5 µg

 

 

 

 

1741.7

 

 

1545

 

 

 

 

9-Aminoacridine

100 µg

 

 

497.7

 

 

 

 

 

 

 

S-9 fraction

(negative control) 50 µL

 

17

 

8

 

22.3

 

 

53.7

 

 

152.3

2-Aminoanthracene

2.5 µg

 

355.7

 

261.7

 

1636.7

 

 

2116

 

 

1931.3

 

 

Table 2: Concentrations 1, 5 and 10 µL/plate, strains TA 1538 and TA 98Number of revertants per plate (mean of three plates)

Strain

TA 1538

strain

TA 98

conc. [µL]

-MA

+MA

-MA

+MA

water

12.3

12.3

38

38

10

13

15.7

45.3

50.3

5

15.3

16

39

55

1

15.7

16.7

49

59

N-methyl-N-nitro-N-nitrosoguanidine 5 µg

 

 

 

 

2-Nitrofluorene

0.5 µg

1387.7

 

 

1526

 

 

S-9 fraction

(negative control) 50 µL

 

15.3

 

 

44.3

 

2-Aminoanthracene

2.5 µg

 

1408.3

 

 

1427.7

 

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990-02-14 to 1990-03-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Five strains tested but not including E. coli
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Lot/batch No.: Op. 604/89
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat livers (Aroclor 1254 induced)
Test concentrations with justification for top dose:
10.0; 100.0; 333.3; 1000; and 5000 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: deionized water
- Justification for choice of solvent/vehicle: good solubility of the test substance in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine (TA 1537, TA 1538, TA 98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 hours at 37 °C
- Expression time (cells in growth medium): 72 hours at 37 °C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertant cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No appropriate statistical method is available.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1: Number of revertants per plate (mean of three plates), experiment 1

strain

TA 1535

strain

TA 1537

Strain

TA 1538

strain

TA 98

strain

TA 100

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Negative control

6

7

5

5

19

18

18

23

73*

82

Solvent control

7

8

4

5

16

19

17

22

79

75

10

8

7

5

5

14

18

19

26

77

68

100

5

5

4

6

15

17

19

26

71

74

333.3

5

8

5

3

18

20

19

21

72

57

1000

6

6

4

6

16

18

17

27

69

53

5000

5

7

2

5

17

20

19

26

73

70

Positive control sodium azide (10 µg)

738

 

146

 

1475

 

760

 

656

 

Positive control 2-Aminoanthracene (10 µg)

 

200

 

37

 

78

 

1440

 

1236

*Unexpected low revertant rate

 

 

Table 2: Number of revertants per plate (mean of three plates), experiment 2

strain

TA 1535

strain

TA 1537

Strain

TA 1538

strain

TA 98

strain

TA 100

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Negative control

21

16

6

8

15

17

15

30

75

99

Solvent control

21

17

6

9

21

23

15

27

78

88

10

29

21

7

6

22

16

18

27

71

79

100

19

22

7

9

21

30

14

24

66

84

333.3

16

21

4

10

21

30

13

31

73

88

1000

18

20

5

10

22

32

15

32

67

94

5000

17

16

6

8

24

22

16

43

71

80

Positive control sodium azide (10 µg)

1335

 

217

 

1345

 

1522

 

1291

 

Positive control 2-Aminoanthracene (10 µg)

 

65

 

107

 

547

 

1667

 

573

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

Five Ames test reports are used for a weight of evidence approach to determine the genetic toxicity of the test substance.

In the first study, the substance was tested as a concentrate at doses of 1, 10, 100, 500 and 1000 µg/plate. Five Salmonella typhimurium strains were tested (TA 1535, TA 1537, TA 1538, TA 98, TA 100). The test was conducted with and without metabolic activation (S-9 mix, Aroclor 1254 induced). As positive controls, the following substances were used: with metabolic activation: 2-Aminoanthracene (all strains); without metabolic activation: N-methyl-N-nitro-N-nitrosoguanidine (TA 1535, TA 100), 9-Aminoacridine (TA 1537), 2-Nitrofluorene (TA 1538, TA 98). As a result, the number of revertant colonies (mean of three replicates per concentration) was not greater than in the solvent controls. No cytotoxicity was observed in the test concentrations. As a conclusion, the tested substance was not mutagenic in this assay.

In the second study, a preparation of the test substance (below 20 % purity) was tested at concentrations of 10, 100, 1000, 5000 and 10000 µg/plate. The test followed the same procedure. Genetic toxicity was not observed either, but cytotoxic effects were found in strains TA 1535 and TA 1538 at concentrations of 10000 µg/plate and in strain TA 1538 at a concentration of 5000 µg/plate.

Another bacterial reverse mutation assay was conducted using the five Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. Bacteria were exposed to a test substance preparation (less than 20% of test substance) in concentrations of 1, 10, 100, 500, 1000 µL/plate without and with metabolic activation (S9 mix from rat livers, Aroclor 1254 induced). As a result, the sample was mutagenic to strains TA 1535, TA 1537, TA 1538, and TA 98 at concentrations of 100, 500 and 1000 µL, and to strain TA 100 at concentrations of 500 and 1000 µL. A dose-response relationship was exhibited by each strain.

The same substance preparation was tested at concentrations of 0.001, 0.01, 0.1, 0.5 and 1 µL/plate, using the Salmonella strains TA1535, TA 1537, TA 1538, TA 98, and TA 100 both without and with the addition of the metablic activation system (S-9). Due to a possible dose-related response, the test material was further tested at 1, 5, and 10 µL/plate using Salmonella strains TA 1538 and TA 98. Under the conditions of this investigation, the substance was found to be non-mutagenic both without and with the addition of the metabolic activation system

In a further bacterial reverse mutation assay a read-across substance (Similar Substance 01) was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The read across substance was tested at the following concentrations: 10; 100; 333.3; 1000; and 5000 µg/plate. No toxic effects, normally evidenced by a reduction in the number of revertants, occurred in the test groups with and without metabolic activation in all strains used. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. A slight increase in the number of revertant colonies was observed in strain TA 98 at 5000 µg/plate in the presence of metabolic activation in experiment II. This effect is considered not to be relevant as it was not reproduced in the independent experiment. Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration- dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies. In conclusion the read across substance did not induce point mutations or frameshifts in the genome of the strains used.

Conclusion

In conclusion, only in one study a positive result was shown. The other three studies with the test substance or the read across substance did not reveal a positive result, the test material was thus concluded to be non-mutagenic in the bacterial reverse mutation assay.

In vitro micronucleus test in human lymphocytes

The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments according to OECD 487 and GLP.

In each experimental group two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.

In this study in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied and evaluated concentration. In Experiment I and II in the absence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment I in the presence of S9 mix, however, one single increase in the number of micronucleated cells (1.15 %) above the historical control data (0.16 –1.08 %) was observed after treatment with 751 μg/mL. In Experiment II in the presence of S9 mix, increases in the number of micronucleated cells above the historical control data (0.16 –1.08 %) were observed after treatment with 751 and 1314 μg/mL (1.10 and 1.13 %). Since all values were not statistically significant and no dose-dependency was observed, the findings were considered as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, the test item is considered to be non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to the highest required concentration.

In vitro gene mutation test in CHO cells (HPRT Locus Assay)

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according to OECD 476 and GLP.

Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested:

1st Experiment

without S9 mix

0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 μg/mL

with S9 mix

0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 μg/mL

2nd Experiment

without S9 mix

0; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 μg/mL

with S9 mix

0; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 μg/mL

 

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

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.

Dose selection for genotoxicity testing was based on the solubility properties of the test substance in culture medium. The highest tested concentrations in both main experiments showed clear test substance precipitates in culture medium macroscopically at the end of exposure period.

In this study, in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations, except in the 1st Experiment in the absence of S9 mix.

Based on the results of the 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.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The test substance and read-across substance was determined to be non-mutagenic in the Ames test. In the in vitro micronucleus test the substance was determined to be non-clastogenic and non-aneugenic. In the HPRT locus assay the substance is not mutagenic in CHO cells. As a result the substance is not considered to be classified and labelled for genotoxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.