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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:
Study initiated on 23rd October 1990 and was completed (final report issued) on 15th April 1991. Experimental work started on 31st October 1990 and was completed on 24th January 1991.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in compliance with US FDA GLP Regulations (21CFR58), US EPA GLP (40CFR792 and 40CFR160) and OECD guidelines.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test item LB-7819-1 is Sodium Cocoyl Isethionate, 72.45%.

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
TA 98 (-S9) = 1, 3.3, 10, 33, 100 ug per plate
TA 98 (+S9) = 10, 33, 100, 333, 1000 ug per plate
TA 100 (-S9) = 1, 3.3, 10, 33, 100 ug per plate
TA 100(+S9) = 10, 33, 100, 333, 1000 ug per plate
TA 1535 (-S9) = 1, 3.3, 10, 33, 100 ug per plate
TA 1535 (+S9) = 10, 33, 100, 333, 1000 ug per plate
TA 1537 (-S9) = 1, 3.3, 10, 33, 100 ug per plate
TA 1537 (+S9) = 10, 33, 100, 333, 1000 ug per plate
TA 1538 (-S9) = 1, 3.3, 10, 33, 100 ug per plate
TA 1538 (+S9) = 10, 33, 100, 333, 1000 ug per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
Positive control used for strains TA98, TA100, TA1535, TA1537 & TA1538 all with S9 metabolic activation; all in concentration = 0.5ug/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Positive control used for TA98 & TA1538 (without S9 metabolic activation); both in concentrations = 1.0ug/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control used for TA100 & TA1535 (without S9 metabolic activation); both in concentrations = 1.0ug/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Positive control used for TA1537 (without S9 metabolic activation); in concentration = 0.25ug/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: 20 +/- 2 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable


SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable


NUMBER OF REPLICATIONS: 3 plates per dose (including vehicle & positive controls)


NUMBER OF CELLS EVALUATED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity was determined as >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. The reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. Conversely, cytotoxicity was determined by a reduction in the background lawn)


OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: none
Evaluation criteria:
For a test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article as specified below:
Strains TA1535, TA1537 and TA1538: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than three times the mean vehicle control value.
Strains TA98 and TA100: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than two times the mean vehicle control value.
Statistics:
For all replicate platings, the mean number of revertants per plate was calculated and the standard deviation around the mean was also calculated. The
results of these calculations are presented on the individual strain data forms.

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:
cytotoxicity
Remarks:
Cytotoxicity with S9 metabolic activation at >1000ug/plate; Cytotoxicity without S9 metabolic activation at >100ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity with S9 metabolic activation at >1000ug/plate; Cytotoxicity without S9 metabolic activation at >100ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: not applicable
- Water solubility: not applicable
- Precipitation: no effects
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: The Salmonella typhimurium Preincubation Reverse Mutation Assay with Confirmation on LB-7819-1 was performed in two phases. The first phase, the dose range-finding study, was used to establish the dose range over which the test article would be assayed. The second phase, the initial mutagenicity assay and the confirmatory assay, was used to evaluate the mutagenicity of the test article.

The maximum dose tested in the dose range-finding study was 10000 ug per plate. This dose was delivered to the test system as a suspension in water using a plating aliquot of 200 ul.
The results of the dose range-finding study of LB-7819-1 conducted in the presence and absence of microsomal enzymes indicate that because of toxicity to the test system, the appropriate maximum dose to be plated in the initial mutagenicity assay in the presence and absence of microsomal enzymes would be 1000 and 100 ug per plate, respectively.


COMPARISON WITH HISTORICAL CONTROL DATA: none


ADDITIONAL INFORMATION ON CYTOTOXICITY: The toxicity in the absence of microsomal enzymes was only marginal; therefore the maximum dose level in the absence of microsomal enzymes for the confirmatory assay was increased to 333 ug/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results of Salmonella Mutagenicity Assay in the Absence of Exogenous Metabolic Activation (Experiment B1)

Dose (ug)

Average Revertants Per Plate* +/- Standard Deviation

TA98

TA100

TA1535

TA1537

TA1538

0.0**

15 +/-0

92 +/- 4

9 +/- 2

5 +/- 3

9 +/- 2

1.0

20 +/- 5

100 +/- 6

8 +/- 1

5 +/- 2

6 +/- 2

3.3

19 +/- 5

86 +/- 9

10 +/- 4

3 +/- 1

7 +/- 1

10

17 +/- 6

118 +/- 14

10 +/- 5

6 +/- 1

9 +/- 1

33

11 +/- 3

77 +/- 8

6 +/- 5

4 +/- 1

4 +/- 1

100

14 +/- 3

73 +/- 6

6 +/- 1

2 +/- 1

3 +/- 1

Pos***

324 +/- 12

576 +/- 18

423 +/- 17

79 +/- 12

500 +/- 36

*Average and standard deviation from replicate platings

**0.0 = Vehicle plating aliquot of 200ul/plate

***Pos = Positive Control concentrations as specified in materials & methods (see above)

 

Summary of Results of Salmonella Mutagenicity Assay in the Presence of Exogenous Metabolic Activation (Experiment B1)

Dose (ug)

Average Revertants Per Plate* +/- Standard Deviation

TA98

TA100

TA1535

TA1537

TA1538

0.0**

21 +/- 3

118 +/- 24

8 +/- 2

6 +/- 1

14 +/- 3

10

25 +/- 4

109 +/- 21

9 +/- 2

6 +/- 6

13 +/- 4

33

20 +/- 3

127 +/- 5

9 +/- 3

8 +/- 1

10 +/- 3

100

22 +/- 6

109 +/- 11

10 +/- 4

8 +/- 1

10 +/- 5

333

18 +/- 4

95 +/- 10

8 +/- 5

7 +/- 1

1 +/- 0

1000

11 +/- 2

60 +/- 13

4 +/- 2

1 +/- 1

0 +/- 1

Pos***

953 +/- 18

1071 +/- 24

88 +/- 10

126 +/- 8

1223 +/- 14

*Average and standard deviation from replicate platings

**0.0 = Vehicle plating aliquot of 200ul/plate

***Pos = Positive Control concentrations as specified in materials & methods (see above)

 

Summary of Results of Salmonella Mutagenicity Assay in the Absence of Exogenous Metabolic Activation (Experiment B2)

Dose (ug)

Average Revertants Per Plate* +/- Standard Deviation

TA98

TA1535

TA1538

0.0**

17 +/- 2

7 +/- 3

7 +/- 1

3.3

14 +/- 2

6 +/- 2

6 +/- 4

10

14 +/- 2

7 +/- 1

6 +/- 2

33

17 +/- 4

8 +/- 1

3 +/- 1

100

16 +/- 3

9 +/- 2

2 +/- 1

333

12 +/- 4

6 +/- 1

0 +/- 0

Pos***

296 +/- 27

144 +/- 27

417 +/- 32

*Average and standard deviation from replicate platings

**0.0 = Vehicle plating aliquot of 200ul/plate

***Pos = Positive Control concentrations as specified in materials & methods (see above)

 

Summary of Results of Salmonella Mutagenicity Assay in the Presence of Exogenous Metabolic Activation (Experiment B2)

Dose (ug)

Average Revertants Per Plate* +/- Standard Deviation

TA98

TA1535

TA1538

0.0**

23 +/- 3

8 +/- 4

9 +/- 3

10

18 +/- 3

9 +/- 2

7 +/- 2

33

21 +/- 4

6 +/- 3

9 +/- 3

100

20 +/- 9

13 +/- 4

8 +/- 2

333

16 +/- 4

10 +/- 4

3 +/- 3

1000

11 +/- 4

7 +/- 2

0 +/- 1

Pos***

1058 +/- 144

47 +/- 8

745 +/- 140

*Average and standard deviation from replicate platings

**0.0 = Vehicle plating aliquot of 200ul/plate

***Pos = Positive Control concentrations as specified in materials & methods (see above)

 

 

 

Summary of Results of Salmonella Mutagenicity Assay in the Absence of Exogenous Metabolic Activation (Experiment B3)

Dose (ug)

Average Revertants per Plate* +/- Standard Deviation

TA100

TA1537

0.0**

132 +/- 15

3 +/- 2

3.3

116 +/- 16

3 +/- 2

10

128 +/- 19

4 +/- 3

33

111 +/- 17

6 +/- 3

100

89 +/- 11

3 +/- 2

333

85 +/- 9

2 +/- 2

Pos***

658 +/- 19

1846 +/- 16

*Average and standard deviation from replicate platings

**0.0 = Vehicle plating aliquot of 200ul/plate

***Pos = Positive Control concentrations as specified in materials & methods (see above)

 

Summary of Results of Salmonella Mutagenicity Assay in the Presence of Exogenous Metabolic Activation (Experiment B3)

Dose (ug)

Average Revertants per Plate* +/- Standard Deviation

TA100

TA1537

0.0**

142 +/- 5

8 +/- 3

10

133 +/- 13

5 +/- 3

33

147 +/- 16

7 +/- 1

100

146 +/- 10

5 +/- 2

333

128 +/- 23

4 +/- 2

1000

102 +/- 1

2 +/- 2

Pos***

715 +/- 22

68 +/- 8

*Average and standard deviation from replicate platings

**0.0 = Vehicle plating aliquot of 200ul/plate

***Pos = Positive Control concentrations as specified in materials & methods (see above)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with or without metablolic activation

The results of the Salmonella typhimurium Preincubation Reverse Mutation Assay with Confirmation on LB-7819-1 indicate that under the conditions of this study, the test article LB-7819-1 (Sodium Cocoyl Isethionate) did not cause a positive response with any of the tester strains in the presence and absence of microsomal enzymes prepared from Aroclor-induced rat liver.
MA
Executive summary:

In Experiment T9509-Bl, no positive responses were observed with any of the tester strains in the presence and absence of microsomal enzymes. Since the toxicity in the absence of microsomal enzymes was only marginal, the maximum dose level in the absence of microsomal enzymes for the confirmatory assay was increased to 333 ug/plate.

In Experiment T9509-B2, no positive responses were observed with tester strains TA98, TA1535 and TA1538 in the presence and absence of microsomal enzymes. Tester strains TA100 and TA1537 were not evaluated or reported due to unacceptable titer values.

These two strains were retested in Experiment T9509-B3 each in the presence and absence of microsomal enzymes.

In Experiment T9509-B3, no positive responses were observed with tester strains TAIOO and TA1537 in the presence and absence

of microsomal enzymes. A 2.0-fold, non-dose responsive increase was observed with tester strain TA1537 in the absence of microsomal enzymes. However, dose responsive increases of this magnitude with this particular strain (less than 3.0-fold) and non-dose responsive increases are not evaluated as positive.

All criteria for a valid study were met as described in the protocol.