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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471: negative

OECD 476: positive (-S9), negative (+S9)

OECD 473: positive (-S9)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Aug - 18 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1993
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon, trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 induced by ß-Naphthoflavone/phenobarbital
Test concentrations with justification for top dose:
1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate (with and without S9 mix)
2nd series: 50, 158, 500, 1580, 5000 µg/plate (with and without S9 mix)
Vehicle / solvent:
- Solvent used: DMSO, final concentration 10 µL per plate
- Justification for choice of solvent: solubitlity properties of the test item, non toxicity to bacteria
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: 2-aminoanthracene 2-10 µg/plate, daunomycin 1 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2-3 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
-S9 concentration: 1st series 10%, 2nd series 20%

Evaluation criteria:
A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid AND
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA98, TA100, WP2 uvrA) or 3-fold (TA1535, TA1537) as compared to the concurrent negative controls is observed.
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a 2nd independent experiment.
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is considered negative or non-mutagenic in this assay if
- the assay is considered valid AND
- non of the above criteria are met
Statistics:
Not performed as not mandatory for this test system.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Following treatment with the test item, no precipitation on the agar plates occurred. No toxicity to the bacteria was observed.
Under the experimental conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item un the absence and presence of S9-mix.
According to the criteria for negative and positive results as predetermined in the study plan, the test item was not mutagenic under the described experimental conditions.

Table 1: Summary of Experiment 1

Metabolic Activation

Test material

Concentr. (µg/plate)

Revertant Colony Counts (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

DMSO

 

40 +/- 7

112 +/- 15

29 +/- 6

7 +/- 3

27 +/- 7

Test item

5

34 +/- 6

115 +/- 17

27 +/- 4

4 +/- 1

29 +/- 4

15.8

34 +/- 4

127 +/- 17

25 +/- 4

6 +/- 2

27 +/- 3

50

32 +/- 8

111 +/- 13

22 +/- 4

8 +/- 2

29 +/- 2

158

33 +/- 2

115 +/- 17

34 +/- 5

8 +/- 1

28 +/- 7

500

35 +/- 3

135 +/- 3

22 +/- 1

7 +/- 4

33 +/- 6

1580

33 +/- 2

113 +/- 19

23 +/- 2

6 +/- 4

28 +/- 4

5000

34 +/- 10

122 +/- 8

23 +/- 5

7 +/- 2

33 +/- 5

DAUN

1

257 +/- 37

 

 

 

 

NaN3

2

 

1724 +/- 63

911 +/- 4

 

 

9-AA

50

 

 

 

1282 +/- 397

 

NQO

2

 

 

 

 

1618 +/- 171

With Activation

DMSO

 

40 +/- 9

126 +/- 11

21 +/- 4

11 +/- 2

37 +/- 5

Test item

5

45 +/- 4

121 +/- 3

25 +/- 11

11 +/- 2

29 +/- 6

15.8

43 +/- 11

110 +/- 10

15 +/- 6

6 +/- 5

37 +/- 4

50

36 +/- 2

99 +/- 26

16 +/- 6

7 +/- 1

39 +/- 13

158

32 +/- 7

102 +/- 15

19 +/- 4

10 +/- 3

38 +/- 11

500

40 +/- 3

115 +/- 4

23 +/- 10

9 +/- 6

38 +/- 9

1580

37 +/- 6

107 +/- 9

22 +/- 8

9 +/- 1

27 +/- 3

5000

43 +/- 3

114 +/- 5

25 +/- 5

9 +/- 2

33 +/- 4

2-AA

2

817 +/- 123

1486 +/- 107

 

 

2-AA

5

 

 

187 +/- 12

466 +/- 14

 

2-AA

10

 

 

 

 

301 +/- 5

Table 1: Summary of Experiment 2

Metabolic Activation

Test material

Concentr. (µg/plate)

Revertant Colony Counts (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

DMSO

 

37 +/- 7

109 +/- 18

19 +/- 4

8 +/- 4

27 +/- 6

Test item

5

--

--

--

--

--

15.8

--

--

--

--

--

50

35 +/- 5

118 +/- 29

21 +/- 6

8 +/- 1

30 +/- 5

158

35 +/- 2

113 +/- 10

16 +/- 5

7 +/- 4

33 +/- 3

500

40 +/- 8

118 +/- 3

25 +/- 7

8 +/- 4

26 +/- 12

1580

39 +/- 6

115 +/- 15

19 +/- 3

7 +/- 2

34 +/- 3

5000

40 +/- 4

129 +/- 21

19 +/- 6

6 +/- 4

30 +/- 7

DAUN

1

205 +/- 22

 

 

 

 

NaN3

2

 

1586 +/- 50

877 +/- 9

 

 

9-AA

50

 

 

 

1176 +/- 244

 

NQO

2

 

 

 

 

1530 +/- 159

With Activation

DMSO

 

50 +/- 10

106 +/- 7

20 +/- 3

11 +/- 3

36 +/- 5

Test item

5

--

--

--

--

--

15.8

--

--

--

--

--

50

42 +/- 1

113 +/- 10

19 +/- 6

12 +/- 6

37 +/- 8

158

45 +/- 4

112 +/- 14

13 +/- 1

14 +/- 2

36 +/- 2

500

45 +/- 7

108 +/- 16

16 +/- 4

9 +/- 5

37 +/- 11

1580

39 +/- 8

109 +/- 12

20 +/- 1

13 +/- 2

30 +/- 2

5000

47 +/- 17

105 +/- 6

16 +/- 4

7 +/- 4

33 +/- 5

2-AA

2

275 +/- 23

439 +/- 11

 

 

2-AA

5

 

 

132 +/- 25

89 +/- 8

 

2-AA

10

 

 

 

 

205 +/- 23

Key to Positive Controls

NaN3                Sodium azide

2-AA                2-Aminoanthracene

9-AA                9-Aminoacridine

DAUN              Daunomycin

NQO                4-Nitroquinoline-N-oxide

Conclusions:
The test item is considered not mutagenic in bacteria according to OECD Guideline 471.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Apr - 15 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other:
Details on mammalian cell type (if applicable):
human lymphocyte cultures prepared from the pooled blood of three Female
donors
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation
was obtained from Molecular Toxicology Incorporated, USA where it was prepared
from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as
lyophilized S-9 mix (MutazymeTM), stored frozen at <-20°C, and thawed and
reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each
batch was checked by the manufacturer for sterility, protein content, ability to convert
ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome
P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Test concentrations with justification for top dose:
0, 40, 70, 100 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic events if:
1. A statistically significant increase in the proportion of cells with structural
aberrations (excluding gaps) at one or more concentrations was observed (p≤0.05)
2. The incidence of cells with structural aberrations (excluding gaps) at such a
concentration that exceeded the normal range in both replicate cultures
3. A concentration-related increase in the proportion of cells with structural
aberrations (excluding gaps) was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were
met.
The test article was considered negative in this assay if none of the above criteria
were met.
Results which only partially satisfied the above criteria were to be dealt with on a
case-by-case basis. Evidence of a concentration-related effect was considered useful
but not essential in the evaluation of a positive result (Scott et al., 1990). Biological
relevance was taken into account, for example consistency of response within and
between concentrations, or effects occurring only at high or very toxic concentrations,
and the types and distribution of aberrations.

Scott D, Dean B J, Danford N D and Kirkland D J (1990). Metaphase chromosome
aberration assays in vitro. Basic Mutagenicity Tests; UKEMS recommended
procedures. Kirkland D J (Ed), pp 62-86
Statistics:
Standard statistical methods have been applied for data processing.
Key result
Species / strain:
mammalian cell line, other: human lymphocytes
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
Species / strain:
mammalian cell line, other: human lymphocytes
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
Conclusions:
The test material induced structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested for 20+0 hours in the absence of a rat liver metabolic activation system (S-9) under the experimental conditions described.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Apr - 31 Oct, 2018
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 using the Hprt and xprt genes)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
tk +/-
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation
was obtained from Molecular Toxicology Incorporated, USA where it was prepared
from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as
lyophilized S-9 mix (MutazymeTM), stored frozen at <-20°C, and thawed and
reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each
batch was checked by the manufacturer for sterility, protein content, ability to convert
ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome
P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Test concentrations with justification for top dose:
1st series -S9: 0, 80, 90, 100, 110, 120, 150 µg/mL
1st series +S9: 0, 100, 110, 120, 130, 150, 180 µg/mL

2nd series -S9: 0, 80, 100, 120, 150, 180, 210, 240, 270, 300, 350 µg/mL
2nd series +S9: 0, 80, 100, 120, 150, 180, 240, 270, 300, 350, 400 µg/mL

The maximum concentrations selected for testing in Mutation Experiment 1 were
based on the toxicity information obtained in the Range-Finder Experiment. In the
Range-Finder, excessive toxicity (<10% RS) was observed in the absence of S-9 and
an appropriate limit concentration (10-20% RS) was achieved in the presence of S-9.
Concentrations chosen for testing in Experiment 1 were selected to allow for any
marginal shifts in toxicity but no toxicity at all was observed at any concentration
tested for either treatment condition. No significant increases in MF were observed in
Mutation Experiment 1. There was a significant linear trend in the presence of S-9
(p<0.05) but in the absence of any significant increases in MF this was considered not
biologically relevant. As an appropriate limit concentration was not achieved, the
experiment could not be considered sufficiently robust and an additional experiment
was conducted (Mutation Experiment 2).
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
see below
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the
negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend
analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper
limit of the last 20 studies in the historical negative control database (mean MF +/-
2 standard deviations.
The test article was considered positive in this assay if all of the above criteria are
met.
The test article was considered negative in this assay if none of the above criteria are
met.
Results that only partially satisfied the assessment criteria described above were
considered on a case-by-case basis. Positive responses seen only at high levels of
cytotoxicity required careful interpretation when assessing their biological relevance.
Extreme caution was exercised with positive results obtained at levels of RS lower
than 10%.
Statistics:
Standard statistical methods have been applied for data processing.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
Species / strain:
mouse lymphoma L5178Y cells
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

Range-Finder Experiment - 3 Hour Treatment in the Absence and Presence of S-9

Concentration
/ [µg/mL]
- S9
+ S9
%RS
%RS
0
100
100
62.5
43
59
125
3
18
250
0
0
500
0
0
1000
0
0
2000
0
0


Mutation Experiment 1 Summary of Data - 3 Hour treatment in the Absence and Presence of S-9



Concentration / [µg/mL] %RS MF (day 7) Concentration / [µg/mL] %RS MF (day 7)
0
100
5.96
0
100
3.56
80
140
4.19 NS 100
96
3.84 NS
90
120
5.88 NS 110
95
5.26 NS
100
134
3.95 NS 120
96
3.34 NS
110
140
4.30 NS 130
101
5.13 NS
120
125
5.57 NS 150
102
6.34 NS
150
125
5.89 NS
180
93
4.46 NS
NQO 0.15
84
25.22
B[a]P 2
39
45.72
NQO 0.2
77
30.54
B[a]P 3
33
29.07



Mutation Experiment 2 Summary of Data - 3 Hour treatment in the Absence and Presence of S-9



Concentration / [µg/mL] %RS MF (day 7) Concentration / [µg/mL] %RS MF (day 7)
0
100
2.05
0
100
3.47
80
88
2.88 NS 80
66
6.17 NS
100
92
4.09 NS 100
89
3.29 NS
120
71
4.47 NS 120
83
2.62 NS
150
75
6.61 *
150
63
1.85 NS
180
64
2.65 NS 180
54
3.86 NS
210
46
2.62 NS 210
49
5.69 NS
240
42
3.94 NS 240
43
5.70 NS
270
27
5.90 *
270
31
4.70 NS
300
19
9.20 *#
300
26
2.73 NS
350
14
5.54 *
350
11
7.32 #
NQO 0.15
19
31.77
B[a]P 2 39
14.64
NQO 0.2
23
28.15
B[a]P 3 25
19.97


%RS Percent relative survival adjusted by post treatment cell counts
NS Not significant
*, **, *** Test for linear trend: χ2 (one-sided), significant at 5%, 1% and 0.1% level respectively
* Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level
# MF value exceeds the upper limit of the last 20 studies in the historical negative control database
(mean MF±2 standard deviations)

Conclusions:
It was concluded that the test material did induce mutation at the hprt locus in mouse lymphoma L5178Y cells when tested for 3 hours in the absence of a rat liver metabolic activation system (S-9), under the experimental conditions described. In the same test system, the test material did not induce biologically relevant increases in mutation when tested up to the limit of toxicity for 3 hours in the presence of S-9.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Remarks:
In vivo mammalian somatic cell study: gene mutation and chromosome aberrations
Data waiving:
exposure considerations
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776.