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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Terephthalaldehyde
EC Number:
210-784-8
EC Name:
Terephthalaldehyde
Cas Number:
623-27-8
Molecular formula:
C8H6O2
IUPAC Name:
benzene-1,4-dicarbaldehyde
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch (Lot) Number: 180401
Expiry date: 01 April 2020 (retest date)
Physical Description: Light yellow powder
Purity/Composition: 99.6%
Storage Conditions: At room temperature

Method

Target gene:
histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichiacoli (E. coli) strain WP2uvrA
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa, gal, chl, bio, uvrB also plasmid pKM101 in TA98 and TA100
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254
Test concentrations with justification for top dose:
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of Terephthaldehyde used in the subsequent mutation assays was 5000 µg/plate or the level at which the test item inhibited bacterial growth.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191: TA1537 2.5µg/plate; All strains with MA, 2-aminoanthracene 1-15µg/pplate
Details on test system and experimental conditions:
Preparation of Test Item
No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. The test item formed a clear yellow solution in dimethyl sulfoxide.
The stock solution was treated with ultrasonic waves until the test item had completely dissolved.
Test item concentrations were used within 3 hours after preparation.
Any residual volumes were discarded.

Mutation Assay
At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.

The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture
(109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in dimethyl sulfoxide and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually (see deviation). Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without
S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
Statistics:
6. INTERPRETATION
No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table1          
Dose-Range Finding Test: Mutagenic Response of Terephthaldehyde in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay


Dose

(µg/plate)


Mean number of revertant colonies/3 replicate plates (
±S.D.) with one Salmonella typhimurium and one Escherichia coli strain.

 


TA100


WP2uvrA

 



 

 

Without S9-mix

 

Positive control

1002

±

120

 

1487

±

41

 

 

 

 

 

Solvent control

116

±

11

 

21

±

3

 

 

 

 

 

1.7

129

±

8

 

29

±

9

 

 

 

 

 

5.4

129

±

14

 

24

±

6

 

 

 

 

 

17

140

±

13

 

23

±

6

 

 

 

 

 

52

149

±

9

 

20

±

6

 

 

 

 

 

164

132

±

8

 

19

±

4

 

 

 

 

 

512

141

±

20

n

31

±

5

 

 

 

 

 

1600

120

±

11

s

23

±

7

n

 

 

 

 

5000

0

±

0

a NP

8

±

5

s NP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With S9-mix1

 

Positive control

1349

±

109

 

410

±

17

 

 

 

 

 

Solvent control

128

±

16

 

31

±

6

 

 

 

 

 

1.7

144

±

28

 

25

±

9

 

 

 

 

 

5.4

126

±

13

 

36

±

8

 

 

 

 

 

17

148

±

22

 

26

±

9

 

 

 

 

 

52

124

±

10

 

27

±

9

 

 

 

 

 

164

121

±

2

 

26

±

13

 

 

 

 

 

512

122

±

9

n

25

±

6

 

 

 

 

 

1600

114

±

11

s

22

±

1

 

 

 

 

 

5000

0

±

0

a NP

37

±

4

n NP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

Plate incorporation assay (5% S9)

NP

No precipitate

a

Bacterial background lawn absent

n

Normal bacterial background lawn

s

Bacterial background lawn slightly reduced

 


 

Table2          
Experiment 1: Mutagenic Response of Terephthaldehyde in the Salmonella typhimurium Reverse Mutation Assay


Dose

(µg/plate)


Mean number of revertant colonies/3 replicate plates (
±S.D.) with
different strains of Salmonella typhimurium.

 


TA1535


TA1537

 


TA98

 

 

Without S9-mix

 

Positive control

815

±

81

 

527

±

77

 

926

±

67

 

Solvent control

5

±

2

 

7

±

0

 

15

±

5

 

17

7

±

3

 

3

±

1

 

11

±

2

 

52

9

±

2

 

2

±

1

 

15

±

1

 

164

7

±

1

 

3

±

1

 

11

±

3

 

512

7

±

1

n

4

±

2

n

10

±

1

n

1600

6

±

3

s

2

±

1

s

9

±

3

s

5000

0

±

0

a NP

0

±

0

a NP

0

±

0

a NP

 

 

 

 

 

 

 

 

 

 

 

 

 

With S9-mix1

 

Positive control

265

±

21

 

262

±

19

 

976

±

166

 

Solvent control

7

±

5

 

4

±

2

 

16

±

5

 

17

8

±

2

 

4

±

2

 

10

±

3

 

52

8

±

4

 

1

±

0

 

12

±

3

 

164

5

±

1

 

4

±

2

 

13

±

3

 

512

10

±

2

n

3

±

0

n

11

±

4

n

1600

8

±

2

s

2

±

1

s

10

±

2

s

5000

0

±

0

a NP

0

±

0

a NP

0

±

0

a NP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

Plate incorporation assay (5% S9)

NP

No precipitate

a

Bacterial background lawn absent

n

Normal bacterial background lawn

s

Bacterial background lawn slightly reduced

 


 

Table3          
Experiment 2: Mutagenic Response of Terephthaldehyde in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

 


Dose

(µg/plate)


Mean number of revertant colonies/3 replicate plates (
±S.D.) with
different strains of Salmonella typhimurium and one Escherichia coli strain.

 


TA1535


TA1537

 


TA98


TA100


WP2uvrA

 

Without S9-mix

 

Positive control

745

±

14

 

809

±

36

 

1036

±

55

 

1064

±

63

 

1519

±

42

 

Solvent control

9

±

4

 

4

±

2

 

14

±

2

 

166

±

10

 

21

±

1

 

25

8

±

4

 

2

±

2

 

9

±

5

 

150

±

2

 

 

-

 

 

50

11

±

2

 

2

±

1

 

9

±

5

 

151

±

14

 

 

-

 

 

100

13

±

8

 

5

±

5

 

8

±

3

 

147

±

6

 

24

±

10

 

250

10

±

4

 

4

±

1

 

13

±

6

 

160

±

8

 

23

±

7

 

500

8

±

4

n

3

±

1

n

4

±

4

n

142

±

16

n

21

±

6

 

2500

0

±

0

a NP

0

±

0

a NP

0

±

0

a NP

0

±

0

a NP

30

±

9

n

5000

 

-

 

 

 

-

 

 

 

-

 

 

 

-

 

 

24

±

3

s NP

 

 

With S9-mix1

 

Positive control

232

±

24

 

418

±

23

 

503

±

85

 

1254

±

208

 

312

±

15

 

Solvent control

12

±

3

 

4

±

3

 

17

±

2

 

125

±

4

 

33

±

2

 

25

14

±

2

 

5

±

3

 

15

±

1

 

118

±

36

 

 

-

 

 

50

6

±

1

 

4

±

1

 

20

±

5

 

135

±

11

 

 

-

 

 

100

10

±

2

 

8

±

2

 

15

±

1

 

132

±

4

 

34

±

8

 

250

12

±

5

 

3

±

2

 

17

±

9

 

126

±

24

 

30

±

4

 

500

11

±

4

n

4

±

1

n

19

±

7

n

114

±

16

n

30

±

1

 

2500

 

 

e NP MC

 

 

e NP MC

 

 

e NP MC

 

 

 

e NP MC

24

±

11

 

5000

 

-

 

 

 

-

 

 

 

-

 

 

 

-

 

 

53

±

5

n NP

 

 

1

Plate incorporation assay (10% S9)

MC

Microcolonies

NP

No precipitate

a

Bacterial background lawn absent

e

Bacterial background lawn extremely reduced

n

Normal bacterial background lawn

s

Bacterial background lawn slightly reduced

-

Not tested

 

 

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this study it is concluded that Terephthaldehyde is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The objective of this study was to determine the potential of Terephthaldehyde and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichiacoli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 180401 of Terephthaldehyde was a light yellow powder. The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the tester strains TA100 and WP2uvrA. Terephthaldehyde did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in tester strain TA100 at dose levels of 1600 and 5000 μg/platein the absence and presence of S9-mix and in tester strain WP2uvrA at the dose level of 5000 μg/plate in the absence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 17 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Terephthaldehyde did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants an/or a reduction of the bacterial background lawn, was observed in all three tester strains at the dose levels of 1600 and 5000 µg/plate.

In a follow-up experiment of the assay with additional parameters,the test item was tested at a concentration range of 25 to 2500 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98 and TA100 and at 100 to 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix in tester strain WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix,except in tester strainWP2uvrA in the presence of S9-mix. 

Terephthaldehyde did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that Terephthaldehyde is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.