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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
Study period:
May-November 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 471
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
none
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: Dowanol-DPnB (n-butoxypropoxypropanol
or dipropylene glycol normal-butyl
ether). CAS # 29911-28-2
Appearance: Clear liquid.
Batch No.: XZ 95411.00
Purity: More than 95%
Specific Gravity: 0.91 kg/liter
Solubility in water: 5% in water; soluble in DMSO
(determined by NOTOX)
Stability: Not specified.
Storage: Ambient temperature in the dark.
Administered as: Dilution in culture medium. DMSO was
used to help dissolve DPnB in the
culture medium for the samples tested
with S-9.

Method

Target gene:
histidine mutation
Species / strain
Species / strain / cell type:
other: Salmonella/microsome test. Strains TA98, TA100, TA1535, TA1537, TA1538.
Details on mammalian cell type (if applicable):
n/a
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 
Test concentrations with justification for top dose:
100, 333, 1000, 3330, 5000 µg/plate
Vehicle / solvent:
Dimethylsulphoxide (DMSO)
Controls
Negative solvent / vehicle controls:
yes
Remarks:
0.1 ml DMSO
Positive controls:
yes
Remarks:
see table 1 below for details
Details on test system and experimental conditions:
Frozen stock cultures of Salmonella typhimurium (from Bruce Ames, U California, Berkeley) were transferred to nutrient rich broth (Oxoid No. 3) and incubated at 37°C until reaching an optical density of 0.4 at 700 nm (or approximately 109 cells/ml).  This was done for each of the five tester strains (TA 98, 100, 1535, 1537, & 1538).  To 3 ml of liquefied top agar (45°C) was added: 1) 0.1 ml of fresh bacterial culture and 2) either 0.1 ml of a dilution of the test material in DMSO or 0.5 ml of a dilution of the test material in S-9 supernatant. The vortexed liquefied agar containing the test material was then poured into selective agar plates.  The plates were incubated in the dark at 37°C during which time histidine independent revertant colonies developed.  Colonies were counted with an Arteck Model 880 colony counter (or manually).  Results were considered positive if the number of colonies exceeded twice background for any of the strains at any dose and if a dose-response relationship was observed in any strain, with or without S-9 activation.  In addition the positive response had to be reproducible in a second experiment.  Results were considered negative if the revertant counts did not exceed background for any tester strain and the negative response is reproducible in a second experiment.
Evaluation criteria:
The validity of the assay was assessed by determining that 1) negative and positive control revertant counts fell within historical control counts and 2) toxicity did not interfere with interpretation of results.
Statistics:
Mean and standard deviation of the revertants were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: Strains TA98, TA100, TA1535, TA1537, TA1538.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DPnB was not toxic to the test organisms at concentrations up to and including 5000 µg/plate.  DPnB did not cause mutations in the Ames plate assay with or without S-9 metabolic activation.  TA98 did show revertant counts that just met or barely exceeded twice background at two non-consecutive dose levels.  Because there was no dose-response and because this result was not repeated in a second assay, the results with TA98 were considered negative.  No other strain, with or without S-9 activation showed an increase in revertant counts.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

DPnB did not cause mutations in the Ames plate assay with or without S-9 metabolic activation.
Executive summary:

Frozen stock cultures of Salmonella typhimurium (from Bruce Ames, U California, Berkeley) were transferred to nutrient
rich broth (Oxoid No. 3) and incubated at 37°C until reaching an optical density of 0.4 at 700 nm (or
approximately 109 cells/ml).  This was done for each of the five tester strains (TA 98, 100, 1535, 1537, & 1538).  To 3
ml of liquefied top agar (45°C) was added: 1) 0.1 ml of fresh bacterial culture and 2) either 0.1 ml of a dilution
of the test material in DMSO or 0.5 ml of a dilution of the test material in S-9 supernatant. The vortexed liquefied
agar containing the test material was then poured into selective agar plates.  The plates were incubated in the
dark at 37°C during which time histidine independent revertant colonies developed.  Colonies were counted with an
Arteck Model 880 colony counter (or manually).  Results were considered positive if the number of colonies exceeded twice
background for any of the strains at any dose and if a dose-response relationship was observed in any strain, with
or without S-9 activation.  In addition the positive response had to be reproducible in a second experiment. 
Results were considered negative if the revertant counts did not exceed background for any tester strain and the negative
response is reproducible in a second experiment.

The validity of the assay was assessed by determining that 1) negative and positive control revertant counts fell within historical control counts and 2) toxicity did not interfere with interpretation of results.

100, 333, 1000, 3330, 5000 µg/plate of DPnB was tested. DPnB was not toxic to the test organisms at concentrations up to and including 5000 µg/plate.  DPnB did not cause mutations in the Ames plate assay with or without S-9 metabolic activation.  TA98 did show revertant counts that just met or barely exceeded twice background at two non-consecutive dose levels.  Because there was no dose-response and because this result was not repeated in a second assay, the results with TA98 were considered negative.  No other strain, with or without S-9 activation showed an increase in revertant counts.

The report did not specify which positive control agents were tested with each tester strain.  0.1 ml DMSO was used
as a vehicle to solubilize DPnB for the non-activation portion of the study.

In conclusion, DPnB did not cause mutations in the Ames plate assay with or without S-9 metabolic activation.

This study was identified as key for this toxicity endpoint because of the methods followed (which were documented in
the report).  The report included GLP and Quality Assurance statements, signed by the Study Director and Head of the QA
Unit, respectively.  The cell lines used, test substance concentrations and dose spacing (several dose levels
including negative control and a high dose of 5,000 µg/plate), time exposed to the test and control agents,
metabolic activation system, number of replicates, the number of plates scored, and scoring criteria all followed
or exceeded guidance as specified in OECD Guideline 471 "Bacterial Reverse Mutation Test".  The positive control
agents gave the expected results showing that the cell line was responsive to reverse mutation.