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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report date:
1982

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) adipate
EC Number:
203-090-1
EC Name:
Bis(2-ethylhexyl) adipate
Cas Number:
103-23-1
Molecular formula:
C22H42O4
IUPAC Name:
bis(2-ethylhexyl) adipate
Details on test material:
- Name of test material (as cited in study report): DEHA
- Physical state: clear colourless liquid
- Lot/batch No.: 6109

Method

Target gene:
Histidine operon: his C, D and G.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: lipopolysaccharide coat deficient. For TA98 and TA100, the strain contains a resistent transfer factor plasmid pKM101.
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: lipopolysaccharide coat deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 mix activation system
Test concentrations with justification for top dose:
7 doses of 0.15 µl to 150 µl per plate. The assays were conducted using three plates per dose level.
Vehicle / solvent:
Solvent used: N,N Dimethyl Formamide.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: As positive control for nonactivation for strains TA-1535 and TA-100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: As positive control for nonactivation for strains TA-1538 and TA-98.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: As positive control for nonactivation for strain TA-1537.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine as positive control for activation of all strains
Details on test system and experimental conditions:
Media:
The bacterial strains were cultured in Oxoid Media #2 (nutrient Broth). The selective medium was Vogel Bonner Medium E with 2% glucosel. The overlay agar consisted of 0.6% purified agar with 0.5 mM histidine, 0.05 mM biotin and 0.1 M NaCl.

Determination of cytotoxicity:
To a sterile test tube containing 2.0 ml of overlay agar (placed in a 43ºC-45ºC water bath) the following was added:
* 0.1 ml to 0.2 ml of a solution of the test material to give the appropriate dose.
* 0.2 ml of 10-6 dilution of overnight culture.
* 0.5 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured on to nutrient agar plates. After the overlay agar was set, the plates were incubated at 37ºC for approximately 24 hours. The number of colonies growing an the plates were counted and recorded.

Mutagenicity testing:

Nonactivation assay:
To a sterile 13 x 100 mm test tube placed in a 43ºC water bath the following was added in order:
(a) 2.00 ml of 0.6% agar containing 0.05 mM histidine and 0.05 mM biotin.
(b) 0.05 ml of a solution of the test chemical to give the appropriate dose.
(c) 0.1 ml - 0.2 lnl of indicator organism(s).
(d) 0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates. After the top agar has set, the plates were incubated at 37ºC for approximately 2 days. The number of his+ revertant colonies growing on the plates was counted and recorded.

Activation assay:
The activation assay was run concurrently with the nonactivation assay. The only difference was the addition of 0.5 ml of S9 mix to the tubes in place of 0.5 ml of phosphate buffer which is added in nonactivation assays. All other details are similar to the procedure for nonactivation assays.
Evaluation criteria:
Evaluation criteria for Ames Assay:
Because the procedures used to evaluate the mutagenicity of the test material were semiquantitative, the criteria used to determine positive effects were inherently subjective and were based primarily on a historical data base. Most data sets were evaluated using the following criteria:
(1) Strains TA-1535, TA-1537 and TA-1538
If the solvent control value was within the normal range, a test material producing a positive response equal to three times the solvent control value was considered mutagenic.
Strains TA-98 and TA-100
Il the solvent control value was within the normal range, a test material producing a positive response equal to twice the solvent control value for TA-98 and TA-100 was considered mutagenic. The following normal range of revertants for solvent controls were generally considered acceptable:
TA-1535: 8-30
TA-1537: 4-30
TA-1538: 10-35
TA-98: 20-75
TA-100: 80-250

Statistics:
Statistical methods were not currently used, and evaluation was based on the criteria included in the protocol.

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
Remarks:
No significant toxicity was observed at doses ranging from 0.02 µl to 150 µl per plate using strain TA-100.
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No significant toxicity was observed at doses ranging from 0.02 µl to 150 µl per plate using strain TA-100.
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material Di-2-ethyl hexyl adipate (DEHA) did not exhibit genetic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions according to the evaluation criteria.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

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