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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

GLP compliant OECD 471 study: negative with and without metabolic activation

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jul 30 - Aug 27, 2020
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:
other: Japanese Guidelines for this test system
Version / remarks:
1993
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)
TRP operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor (pKM101)
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa + R-factor (pKM101)
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2
Details on mammalian cell type (if applicable):
trp-, uvrA
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from beta-Naphthoflavone/Phenobarbital-pretreated rats with standard co-factors
Test concentrations with justification for top dose:
THF was selected as solvent for the current experiment and 5000 µg/plate was chosen as the appropriate maximum test material concentration.
Vehicle / solvent:
Name: Tetrahydrofuran (THF)
CAS: 109-99-9
Batch: K50942531904
Supplier: Merck KGaA, Darmstadt, Germany
Final concentration: 25 µL/plate
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: Daunomycin
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
Bacterial strains were tested in accordance with the plate incorporation method. 3 parallel plates were used for each concentration step of the test material. The incubation of plates was performed at 36-38°C for 2 days. Liver S9 mix from rats pre-treated with beta-naphthoflavone/phenobarbital was used as the metabolic activation system. Two experimental series were performed, containing 10% S9 ind the 1st and 20% S9 in the 2nd series.
Rationale for test conditions:
according to Guideline
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 (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) 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 second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met

Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed.

Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was considered, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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:
no cytotoxicity, but tested up to precipitating concentrations
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:
no cytotoxicity, but tested up to precipitating concentrations
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:
no cytotoxicity, but tested up to precipitating concentrations
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

First Series

 

Metabolic Activation

Test
Material

Concentr. [µg/plate]

 

Revertants per plate (Mean ± SD)

 

 

 

 

 

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

 

Without activation

THF

 

37 ± 6

119 ± 21

27 ± 3

14 ± 2

37 ± 8

 

 

Art. 139838

5.00

38 ± 3

110 ± 11

31 ± 7

12 ± 4

37 ± 4

 

 

 

15.8

47 ± 2

129 ± 31

24 ± 4

12 ± 3

45 ± 7

 

 

 

50.0

46 ± 9

122 ± 10

27 ± 6

16 ± 2

40 ± 9

 

 

 

158

36 ± 3

112 ± 14

28 ± 6

17 ± 2

39 ± 7

 

 

 

500

41 ± 1 M E

122 ± 8 M E

23 ± 1 M E

13 ± 3 M E

36 ± 7 M E

 

 

 

1580

36 ± 5 M E

103 ± 6 M E

19 ± 6 M E

13 ± 5 M E

35 ± 9 M E

 

 

 

5000

31 ± 10 M E

106 ± 3 M E

19 ± 3 M E

11 ± 1 M E

33 ± 3 M E

 

 

4-NOPD

20.0

379 ± 11

 

 

 

 

 

 

4-NOPD

60.0

 

 

 

77 ± 3

 

 

 

NaN3

2.00

 

1407 ± 25

774 ± 41

 

 

 

 

NQO

2.00

 

 

 

 

2068 ± 398

With

activation

THF

 

44 ± 3

147 ± 18

20 ± 3

12 ± 4

40 ± 5

 

 

Art. 139838

5.00

46 ± 4

132 ± 12

16 ± 2

11 ± 2

39 ± 6

 

 

 

15.8

44 ± 13

136 ± 17

22 ± 5

13 ± 4

44 ± 5

 

 

 

50.0

49 ± 3

147 ± 14

23 ± 2

15 ± 3

44 ± 5

 

 

 

158

55 ± 2

153 ± 10

23 ± 3

10 ± 2

44 ± 4

 

 

 

500

35 ± 6 M E

142 ± 6 M E

21 ± 3 M E

10 ± 3 M E

38 ± 16 M E

 

 

 

1580

38 ± 3 M E

131 ± 13 M E

19 ± 4 M E

8 ± 2 M E

41 ± 3 M E

 

 

 

5000

38 ± 2 M E

126 ± 8 M E

17 ± 5 M E

10 ± 2 M E

40 ± 1 M E

 

 

2-AA

2.00

461 ± 130

642 ± 88

 

 

 

 

 

2-AA

5.00

 

 

213 ± 29

712 ± 121

 

 

 

2-AA

10.0

 

 

 

 

251 ± 23

 

 


 

Second Series

Metabolic Activation

Test
Material

Concentr. [µg/plate]

 

Revertants per plate (Mean ± SD)

 

 

 

 

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without activation

THF

 

33 ± 4

122 ± 8

24 ± 1

12 ± 3

34 ± 9

 

Art. 139838

15.8

28 ± 9

130 ± 7

26 ± 10

13 ± 1

47 ± 10

 

 

50.0

32 ± 4

124 ± 9

23 ± 2

8 ± 1

35 ± 7

 

 

158

39 ± 6

113 ± 21

23 ± 3

8 ± 2

36 ± 7

 

 

500

30 ± 6 M E

108 ± 11 M E

25 ± 3 M E

10 ± 3 M E

39 ± 4 M E

 

 

1580

27 ± 8 M E

115 ± 13 M E

37 ± 4 M E

9 ± 2 M E

33 ± 4 M E

 

4-NOPD

20.0

436 ± 27

 

 

 

 

 

4-NOPD

60.0

 

 

 

103 ± 18

 

 

NaN3

2.00

 

1372 ± 23

774 ± 29

 

 

 

NQO

2.00

 

 

 

 

1494 ± 108

With

 activation

THF

 

37 ± 4

155 ± 17

15 ± 3

11 ± 3

45 ± 6

 

Art. 139838

15.8

35 ± 4

146 ± 17

16 ± 3

9 ± 2

50 ± 7

 

 

50.0

47 ± 12

144 ± 5

15 ± 3

12 ± 2

46 ± 8

 

 

158

43 ± 7

150 ± 16

13 ± 4

11 ± 2

40 ± 8

 

 

500

36 ± 10 M E

137 ± 8 M E

15 ± 5 M E

8 ± 1 M E

49 ± 9 M E

 

 

1580

35 ± 7 M E

139 ± 5 M E

12 ± 3 M E

8 ± 2 M E

45 ± 5 M E

 

2-AA

2.00

172 ± 24

446 ± 48

 

 

 

 

2-AA

5.00

 

 

117 ± 13

275 ± 38

 

 

2-AA

10.0

 

 

 

 

202 ± 31

 

 

 

Key to Positive controls

Key to Plate Postfix Codes

2-AA

2-Aminoanthracene

4-NOPD

4-Nitro-o-phenylendiamin

NaN3

Sodium azide

NQO

4-Nitroquinoline-N-oxide

E

Precipitation until end of experiment

M

manual count

 

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.
Executive summary:

 


Objective


The present study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix).


 


Study Design


The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with β-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively.


 


Results


Solvent and positive control treatments were included for all strains. The mean numbers of re-vertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.


Following treatment of all bacteria tester strains with the test material in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed.


 


Conclusion


With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the provided information there is no need to classified according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.

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