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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 27 - May 21, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): HR 99/9040020/1
- Physical state: clear viscous liquid
- Analytical purity: 98.5%

Method

Target gene:
Different loci of the bacterial histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
supernatatnt of rat liver homogenates (S9) from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
0, 50, 150, 500, 1500, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: commonly used solvent
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide; 2-nitrofluorene; 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: not applicable
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 to 72 hours


SELECTION AGENT (mutation assays): histidine



NUMBER OF REPLICATIONS: 3

NUMBER OF EXPERIMENTS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
According to OECD TG 471
Statistics:
X²-test

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
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:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment AM026991: Induction of revertants by HR 99/9040020/1 (2-Ethylhexyl 4-methoxycinnamate);

without metabolic activation

 

Substance

Concentration

(µg/plate)

Number of revertants/plate

(mean of 3 replicates±standard deviation)

TA98

TA100

TA102

TA1535

TA1537

Control

0

34±3

145±8

359±22

16±3

9±4

Solvent control

0

25±4

137±13

337±4

24±3

12±4

 

HR 99/9040020/1

50

29±4

127±7

303±28

22±6

8±3

HR 99/9040020/1

150

28±6

128±12

301±17

18±3

12±5

HR 99/9040020/1

500

29±3

130±15

312±24

21±3

11±3

HR 99/9040020/1

1500 - P

29±5

141±1

315±23

19±5

12±4

HR 99/9040020/1

5000 - P

30±5

129±12

306±12

19±4

9±3

 

Sodium azide

0.5

 

336±15

 

 

 

Sodium azide

0.7

 

 

 

453±41

 

2-nitrofluorene

2.5

331±12

 

 

 

 

9-aminoacridine

50

 

 

 

 

273±64

Mitomycin C

0.15

 

 

1061±38

 

 

 

Experiment AM026991: Induction of revertants by HR 99/9040020/1 (2-Ethylhexyl 4-methoxycinnamate);

with metabolic activation

 

Substance

Concentration

(µg/plate)

Number of revertants/plate

(mean of 3 replicates±standard deviation)

TA98

TA100

TA102

TA1535

TA1537

Control

0

30±2

119±8

339±2

24±3

13±5

Solvent control

0

33±3

105±10

315±44

20±5

10±3

 

HR 99/9040020/1

50

37±7

124±4

344±7

26±7

9±4

HR 99/9040020/1

150

47±6

132±10

352±10

25±5

10±1

HR 99/9040020/1

500

31±10

104±2

366±22

17±3

12±5

HR 99/9040020/1

1500 - P

41±1

146±22

348±11

19±4

12±3

HR 99/9040020/1

5000 - P

33±5

124±10

322±17

16±8

15±4

 

2-amino-anthracene

0.7

777±27

763±26

 

 

 

2-amino-anthracene

2.0

 

 

1192±96

631±52

414±34

 

 

 

 

Experiment AM026992: Induction of revertants by HR 99/9040020/1 (2-Ethylhexyl 4-methoxycinnamate);

without metabolic activation

 

 

Substance

Concentration

(µg/plate)

Number of revertants/plate

(mean of 3 replicates±standard deviation)

TA98

TA100

TA102

TA1535

TA1537

Control

0

29±7

114±9

240±9

23±2

5±2

Solvent control

0

23±6

115±13

251±15

24±7

9±4

 

HR 99/9040020/1

50

23±4

118±8

241±19

25±3

10±6

HR 99/9040020/1

150

21±5

115±5

233±12

24±5

9±3

HR 99/9040020/1

500

21±5

103±8

238±5

24±3

10±3

HR 99/9040020/1

1500 - P

28±4

114±9

233±18

27±3

11±6

HR 99/9040020/1

5000 - P

22±1

105±9

234±9

28±4

10±2

 

Sodium azide

0.5

 

468±37

 

 

 

Sodium azide

0.7

 

 

 

542±44

 

2-nitrofluorene

2.5

495±47

 

 

 

 

9-aminoacridine

50

 

 

 

 

238±32

Mitomycin C

0.15

 

 

671±18

 

 

 

 

Experiment AM026992: Induction of revertants by HR 99/9040020/1 (2-Ethylhexyl 4-methoxycinnamate);

with metabolic activation

 

Substance

Concentration

(µg/plate)

Number of revertants/plate

(mean of 3 replicates±standard deviation)

TA98

TA100

TA102

TA1535

TA1537

Control

0

44±9

96±6

273±16

19±4

14±4

Solvent control

0

35±4

86±4

276±14

15±3

13±2

 

HR 99/9040020/1

50

46±7

98±6

288±50

14±6

16±5

HR 99/9040020/1

150

40±7

94±10

279±36

13±3

22±3

HR 99/9040020/1

500

35±5

101±7

292±7

15±3

16±4

HR 99/9040020/1

1500 - P

46±7

100±10

320±10

20±4

13±4

HR 99/9040020/1

5000 - P

35±9

103±12

273±9

16±6

13±4

 

2-amino-anthracene

0.5

928±40

637±19

 

 

 

2-amino-anthracene

0.7

 

 

1301±85

635±34

222±11

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 of S. typhimurium  were exposed to 2-Ethylhexyl 4-methoxycinnamate (HR 99/9040020/) dissolved in DMSO, at concentrations of 0, 50, 150, 500, 1500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation, i.e. supernatant of rat liver homogenates (S9) from Aroclor 1254-induced male Sprague-Dawley rats.   2-Ethylhexyl 4-methoxycinnamate was tested up to insoluble concentration. Precipitation occurred at 1500 and 5000 µg/plate without signs of bacteriotoxicity at any tested dose level. HR 99/9040020/1 did not increase the number of revertants in any tester strain in a dose-related manner, with or without metabolic activation.The negative controls performed as expected.The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background with the test substance.   This study is classified as acceptable because it was conducted equivalent to the OECD Test Guideline No. 471 and under GLP conditions, and because the documentation is sufficient for assessment. This study therefore satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation)  

Conclusion:

2-Ethylhexyl 4-methoxycinnamate was negative in a valid bacterial cell mutation assay.