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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from 1998-08-10 to 1998-08-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according 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
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tri (hexyl, octyl, decyl) citrate
- Substance type: pure active substance
- Physical state: liquid
- Storage condition of test material: room temperature in a closed container

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (80 mg/kg bw) and ß-Naphtoflavone (100 mg/kg bw) induced S9 liver microsomal fraction of male Wistar rats
Test concentrations with justification for top dose:
33.3 / 100.0 / 333.3 / 1000.0 / 2500.0 / 5000.0 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 10 µg/plate for S. typhimurium strains TA 1535, TA 100; 4-nitro-o-phenylene-diamine, 10 µg/plate for S. typhimurium strain TA 98 and 40 µg/plate for S. typhimurium strain TA 1537; methyl methane sulfonate, 1 µL/plate for E. coli strain WP2 u
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2.5 µg/plate for all strains
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation in two independent experiments


DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours at least


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: detected by a reduction in the number of revertants, a clearing of diminution of tht background lawn or by the degree of survival of treated cultures


OTHER EXAMINATIONS:
- Data recording: The colonies were counted using an Artek-counter Modell 880 ( Artek Systems Corpoporation, BIOSYS GmbH, Karben, Germany) at maximum sensitivity. If precipitation of test item precluded automatic counting the revertant colonies were counted by hand.


OTHER: The properties of the S. typhimurium and E. coli strains with regard to membrane permeability, ampicillin resistance and normal spontaneous mutation rates were checked regularly to Ames et al.
Evaluation criteria:
A test was considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the control plates without S9 mix were within the range of spontaneous reversion frequencies
- corresponding background growth on both negative control and test plates was observed
- the positive controls showed a distinct enhancement over the control plates

A test item was considered as mutagenic if a dose-related increase in the number of revertants occured and/or a reproducible biologically relevant positive response for at least one the test points occured in at least one strain with or without metabolic activation.
A biologically relevant increase was described as:
- if in strain TA 100 the number of reversions was at least twice as high
- if in strains TA 1535, TA 1537, TA 98 and WP2 uvr A was at least three times higher
than the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants was regarded as an indication of possible existing mutagenic potential of the test item regardless of whether the highest dose induced the above described enhancement factors or not.
Statistics:
no statistical evaluation performed

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
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: no data
- Precipitation: nothing mentioned
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: Yes, performed using the same experimental conditions as described for the plate incorporation test to detemine the toxicity of the test item. 8 concentrations were tested for toxicity and induction of mutations with 3 plated each. No toxicity or genotoxicity was observed.


COMPARISON WITH HISTORICAL CONTROL DATA: nothing mentioned


ADDITIONAL INFORMATION TO CYTOTOXICITY: Normal background growth was observed up to the highest concentration of the test item with and without metabolic activation in all stains used.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table #1:Number of revertants per plate (mean of 3 plates) of the plate incorporation test

 

[Strain TA 1535]

[Strain TA 1537]

[Strain TA 98]

Conc.
[µg/plate]

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

0*

 12±2

 9±2

 -

 11±1

 7±3

 -

 40±7

 59±21

 -

33.3

 10±2

 8±3

 no

 8±3

 9±2

 no

 59±3

 64±8

 no

100.0

 11±1

 10±2

 no

 10±4

 6±2

 no

 57±12

 65±10

 no

333.3

 10±3

 9±3

 no

 15±8

 9±2

 no

 47±2

 67±6

 no

1000.0

 11±5

 11±1

 no

 11±4

 7±2

 no

 38±10

 67±14

 no

2500.0

 11±6

 8±4

 no

 8±2

 4±1

 no

 62±7

 67±4

 no

5000.0

 9±1

 7±1

 no

 9±4

 6±2

 no

 42±5

 69±9

 no

Positive control

1258±101

212±16

-

224±16

225±18

-

1090±34

247±151

-

*solvent control with DMSO

Table #2:Number of revertants per plate (mean of 3 plates) of the plate incorporation test

 

[Strain TA 100]

[Strain WP2 uvrA]

Conc.
[µg/plate]

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

0*

 125±17

 118±13

 -

 33±7

 60±9

 -

 

 

 

33.3

 119±10

 123±12

 no

 42±19

 49±2

 no

 

 

 

100.0

 123±9

 124±8

 no

 37±12

 59±3

 no

 

 

 

333.3

 130±4

 125±18

 no

 38±8

 52±6

 no

 

 

 

1000.0

 135±9

 138±12

 no

 42±7

 58±11

 no

 

 

 

2500.0

 134±10

 128±3

 no

 39±6

 66±10

 no

 

 

 

5000.0

 146±12

 122±10

 no

 36±3

 54±7

 no

 

 

 

Positive control

1321±8

2315±155

-

807±47

769±21

-

*solvent control with DMSO

Table #3:Number of revertants per plate (mean of 3 plates) of the preincubation test

 

[Strain TA 1535]

[Strain TA 1537]

[Strain TA 98]

Conc.
[µg/plate]

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

0*

 16±4

 13±2

 -

 11±2

 10±2

 -

 37±3

 50±5

 -

33.3

 15±5

 11±4

 no

 13±2

 11±2

 no

 51±7

 51±2

 no

100.0

 11±4

 12±3

 no

 14±5

 10±1

 no

 49±2

 64±6

 no

333.3

 17±1

 14±2

 no

 13±3

 12±4

 no

 50±4

 60±13

 no

1000.0

 15±1

 12±2

 no

 17±4

 10±3

 no

 39±6

 52±5

 no

2500.0

 17±9

 11±4

 no

 8±3

 8±1

 no

 33±6

 50±12

 no

5000.0

 13±8

 14±5

 no

 14±3

 10±4

 no

 44±10

 60±14

 no

Positive control

1044±14

143±5

-

298±11

135±17

-

1163±58

1128±82

-

*solvent control with DMSO

Table #4:Number of revertants per plate (mean of 3 plates) of the preincubation test

 

[Strain TA 100]

[Strain WP2uvrA]

Conc.
[µg/plate]

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

¿ MA

+ MA

Cytotoxic
(yes/no)

0*

 101±17

 113±12

 -

 27±4

 50±8

 -

 

 

 

33.3

 90±10

 110±8

 no

 31±8

 33±7

 no

 

 

 

100.0

 84±6

 108±18

 no

 31±1

 33±5

 no

 

 

 

333.3

 99±23

 94±4

 no

 36±5

 37±4

 no

 

 

 

1000.0

 99±7

 104±10

 no

 27±5

 36±2

 no

 

 

 

2500.0

 115±8

 115±6

 no

 32±5

 39±2

 no

 

 

 

5000.0

 144±30

 98±13

 no

 30±9

 41±15

 no

 

 

 

Positive control

1335±103

1198±118

-

210±28

223±2

-

*solvent control with DMSO

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The reported data of this mutagenicity test showed that the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Tri (hexyl, octyl, decyl) citrate was considered to be non-mutagenic in this bacterial reverse mutation assay.