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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
Study period:
1990-06-07 to 1991-05-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study according to OECD 471 (1983); Only four strains of Salmonella typhimurium were used.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only four strains of Salmonella typhimurium were used (TA 135, TA 1537, TA 100 and TA 98).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,7-methanooctahydro-1H-indene-diyldimethyl bis(2-carboxybenzoate)
EC Number:
407-410-2
EC Name:
4,7-methanooctahydro-1H-indene-diyldimethyl bis(2-carboxybenzoate)
Molecular formula:
C28H40O8
IUPAC Name:
2-[({5-[(2-carboxycyclohexanecarbonyloxy)methyl]tricyclo[5.2.1.0²,⁶]decan-8-yl}methoxy)carbonyl]cyclohexane-1-carboxylic acid; 2-[({8-[(2-carboxycyclohexanecarbonyloxy)methyl]tricyclo[5.2.1.0²,⁶]decan-4-yl}methoxy)carbonyl]cyclohexane-1-carboxylic acid
Constituent 2
Reference substance name:
4,7-methanooctahydro-1H-indene-diyldimethyl bis(2-carboxybenzoate)
IUPAC Name:
4,7-methanooctahydro-1H-indene-diyldimethyl bis(2-carboxybenzoate)
Test material form:
other: clear solid particles
Details on test material:
- Name of test material (as cited in study report): TCD Emulgator
- Physical state: solid
- Analytical purity: 95.7 %
- Purity test date: 1990-05-07
- Lot/batch No.:90003
- Stability under test conditions: A stability test in the solvent did not detect relevant changes in the percent of active ingredient
- Expiration date of the lot/batch: 1992-01-08
- Storage condition of test material: Refrigerator

Method

Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media:
The test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983).
0.1 mL compound, 0.1 mL bacteria, 0.5 mL S9 mix or buffer, 2.0 mL soft agar, 45 °C in water bath, max. 30 sec, transfer to Petri dish with solid agar.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
First test: 5000, 1000, 200, 40 and 8 µg/plate
Repeat test: 1200, 600, 300, 150, 75 and 37.5 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol for TCD Emulgator and DMSO or the positive controls
- Justification for choice of solvent/vehicle: according to information given by the internal sponsor
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: nitrofurantoin, 4-nitro-1,2- phenylene diamine, 2-aminoanthracene
Remarks:
without S9 mix: Sodium azide (only for TA 1535), nitrofurantoin (only for TA 100), 4-nitro-1,2-phenylene diamine (only for TA 1537 and TA98); with S9 mix: 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation)
(0.1 mL compound, 0.1 mL bacteria, 0.5 mL S9 mix or buffer, 2.0 mL soft agar. 45°C waterbath for max. 30 sec. and transfer to a petri dish with solid agar. Incubation at 37 °C for 48 hours and afterwards colony count using an automatic counter)
The bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37 °C and 90 rpm. These suspensions were used for the determination of mutant counts. No standardised procedure was employed to set the bacterial suspensions at a defined density of viable cells per milliliter, since the chosen method of incubation normally produces the desired density. The tests were performed both with and without S9 mix. The count was made after the plates had been incubated for 48 hours at 37 °C. If no immediate count was possible, plates were temporarily stored in a refrigerator.
First/pre-test:
The doses for the first trial were routinely determined on the basis of a standard protocol: 5000 μg per plate were used as the highest dose
Repeat test:
Based on the results from the pre-test doses ranging from 37.5 µg to 1200 µg/plate

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Four plates were used, both with and without S9 mix, for each strain and dose

NUMBER OF CELLS EVALUATED:
- The count was made after the plates had been incubated for 48 hours at 37 °C using an automatic counter. If no immediate count was possible, plates were temporarily stored in a refrigerator

DETERMINATION OF CYTOTOXICITY
- The toxicity of the substance was assessed in three ways. The first was a gross appraisal of background growth on the plates for mutant determination. Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9mix.

OTHER: Titer/viable cells
The numbers of viable cells were established when determining the titers. The dilution of bacterial suspensions used for the determination of titers was 1:1,000,000. Titers were determined under the same conditions as mutations, except that the histidine concentration in the soft agar was increased from 0.5 mM to 2.5 mM to permit the complete growth of bacteria.
Evaluation criteria:
The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratory's own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratory's experience.
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.
An assay not complying with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments . Even if the criteria for points (a), (b) and ( c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.

Assessment of results:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be at least twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.

Results and discussion

Test results
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:
Bacteriotoxic effects at 37.5 μg per plate and above.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Pre-test/repeat test:
5000, 1000, 200, 40 and 8 µg/plate were tested in the first test. Due to the substance's toxicity in the first test a second test under identical conditions as decribed were conducted with doses ranging from 37.5 μg to 1200 μg per plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No indication of a bacteriotoxic effect of TCD-emulsifier at 8 μg per plate. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of mean values without S9 mix
Table and group
µg/plate
Strain
TA1535 TA 100 TA 1537 TA 98
1-4        
0 21 75 5 26
8 26 68 7 32
40 26 79 7 32
200 24 85 9 28
1000 17 66 3 25
5000 14 -- - --
Na-azid 723      
NF   343    
4-NPDA     38 70
5-8        
0 8 79 10 22
37.5 9 85 8 22
75 9 87 7 19
150 11 86 5 23
300 10 93 8 18
600 12 84 9 18
1200 16 71 5 22
Na-azid 1056    
NF   422    
4-NPDA     36 60

Summary of mean values with S9 mix
Table and group
µg/plate
Strain
TA1535 TA 100 TA 1537 TA 98
1-4        
30% S9        
0 25 90 5 41
8 23 93 5 31
40 18 104 6 31
200 20 96 3 38
1000 19 81 3 4
5000 13 --- - --
2-AA 249 558 67 241
5-8        
30% S9      
0 15 113 9 48
37.5 16 134 10 55
75 16 115 7 49
150 20 124 6 49
300 20 116 12 44
600 19 122 7 34
1200 20 137 9 37
2-AA 125 698 74 366

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, no biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. TCD-emulsifier is considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 98 of S. typhimurium were exposed to TCD-emulsifier (95.7 %) in ethanol at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (first/pre-test) and at concentrations of 37.5, 75, 150, 300, 600, 1200 µg/plate (repeat test) in the presence and absence of mammalian metabolic activation. 

TCD-emulsifier was tested up to limit concentration (5000 µg/plate). Bacteriotoxic effects were reported from 37.5 µg per plate and above. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background at any concentration tested.

This study is classified as acceptable. The study satisfies the requirements of OECD test guideline 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.