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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Default
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 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
Pre-test: 10, 33, 67, 100, 333, 667, 1000, 3333, 6667, 10000 µg/plate, both with and without activation
Main test: 667, 1000, 3333, 6667 and 10000 µg/plate, both with and without activation
See also Table 1.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionized, distilled grade water.
- Justification for choice of solvent/vehicle: Ethyl lactate was serially diluted with deionized, distilled grade water to obtain the desired test concentrations.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2-aminoanthracene was used as positive control for all strains with activation
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: TA98 and TA1538, without activation
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: TA100 and TA1535, without activation
Positive control substance:
other: ICR-191
Remarks:
ICR-191 was used as positive control in TA1537 without activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation).


DURATION
- Preincubation period: no
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

NUMBER OF REPLICATIONS: Pre test: 1, main test: 3


NUMBER OF CELLS EVALUATED: Revertant colonies for a given tester strain and activation condition were counted either entirely by automated colony counter or entirely by hand.


DETERMINATION OF CYTOTOXICITY
- Method: other: The condition of the background bacterial lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.

Evaluation criteria:
For a test article to be considered positive, it must cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase in the number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article. In those cases where the observed dose-responsive increase in TA1537 or TA1538 revertants per plate is less than three-fold, the response must be reproducable.

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
Remarks:
see Table 2
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see Table 2
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The results of the dose range-finding study conducted in the presence and absence of rat liver microsomes indicate that no appreciable toxicity was observed up to 10000 µg per plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2:Results of Salmonella mutagenicity assay with ethyl lactate

Average Revertants Per Plate**± Standard Deviation

Liver Microsomes: None

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

TA1538

0*

 14 ± 5

  96 ± 20

 20 ± 3

    5 ± 1

     13 ± 3

667

 19 ± 2

 111 ± 6

  20 ± 6

    5 ± 1

     13 ± 3

1000

   9 ± 3

 104 ± 14

  22 ± 2

    7 ± 3

     14 ± 4

3333

 14 ± 2

 111 ± 12

  19 ± 1

    5 ± 1

     19 ± 2

6667

 17 ± 3

 107 ± 11

  17 ± 2

    8 ± 2

     12 ± 2

10000

 20 ± 7

    99 ± 6

  18 ± 5

    5 ± 2

     11 ± 2

Positive control

519 ± 53

  485 ± 12

281 ± 7

241 ± 23

   599 ± 37

Liver Microsomes: Rat Liver S-9

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

TA1538

0*

 23 ± 3

  105 ± 12

 12 ± 4

    9 ± 3

      22 ± 2

667

 34 ± 7

   99 ± 14

  15 ± 5

    6 ± 2

      20 ± 8

1000

 21 ± 1

  102 ± 9

  13 ± 6

    8 ± 3

      22 ± 7

3333

 27 ± 6

  102 ± 5

  13 ± 3

    7 ± 3

      21 ± 6

6667

 28 ± 5

  105 ± 13

  13 ± 3

  12 ± 5

      16 ± 5

10000

 28 ± 3

  102 ± 4

  12 ± 3

    7 ± 2

      21 ± 3

Positive control

950 ± 36

2358 ± 118

300 ± 4

 167 ± 7

  1387 ± 125

*Vehicle control with dionized, distilled grade water

** Mean of 3 plates

Applicant's summary and conclusion

Conclusions:
Ethyl lactate is not mutagenic
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA1538 of S. typhimurium were exposed to ethyl lactate at concentrations of 667, 1000, 3333, 6667 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (plate co-incubation).

Ethyl lactate was tested up to the limit concentration of 10000 µg/plate. There was no dose-related increase in the number of revertants in any of the test strains with and without activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.