Diethylbenzene

Genetic toxicity: in vitro

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:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted similar to OECD TG-471 and in accordance to GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Remarks:

Not specified in report.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diethylbenzene

EC Number:

246-874-9

EC Name:

Diethylbenzene

Cas Number:

25340-17-4

Molecular formula:

C10H14

IUPAC Name:

diethylbenzene

Constituent 2

Reference substance name:

o-diethylbenzene

EC Number:

205-170-1

EC Name:

o-diethylbenzene

Cas Number:

135-01-3

Molecular formula:

C10H14

IUPAC Name:

1.2-diethylbenzene

Details on test material:

IUCLID4 Test substance: as prescribed by 1.1 - 1.4

DEB (m,p,o isomers)
Purity 98%
Compound name: Dowtherm*J
Lot# MM871021
Appearance: Clear liquid

Method

Target gene:

TA98- hisD3052, TA100-hisG46, TA1535- hisG46, and TA1537-hisC3076

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

The tester strains were originally obtained from Dr. Bruce N. Ames, University of California, Berkeley, CA,
and are currently maintained in our laboratory as frozen permanents at -100°C or below.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Exogenous metabolic activation was provided by male  Sprague-Dawley rat liver S-9 preparations from Aroclor 1254.

Test concentrations with justification for top dose:

0 (DMSO), 5.0, 15.8, 50.0, 158.0, 500.0 1580.0 and 5000.0 µg/plate.

Vehicle / solvent:

DEB was dissolved in DMSO and incubated with the tester strains in  suspension culture for 30 min prior to the addition of soft agar and  plating-out.    

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA100 & TA1535 (non-activated)- sodium azide, TA98 (non-activated)- 2-nitrofluorene, TA1537 (non-activated)-ICR-191 and TA98, 100, 1535 and 1537 (activated)- 2-anthramine

Details on test system and experimental conditions:

IUCLID4 Type: Ames test
All dose  levels along with the positive and negative controls were assayed in  triplicate.

Mutagenicity Assay
The pre-incubation modification of the standard Ames plate incorporation assay (Yahagi et al., 1975) was employed to optimize the contact between the bacteria and test chemical. Pre-incubation of the bacteria and the metabolic activation system is required for chemicals such as dimethylnitrosamine to elicit a positive response in the Ames test (Bartsch et al . , 1976). The bacteria, test chemical, and, if necessary, an activation mixture were incubated in sterile 12 x 75 mm tightly capped culture tubes in a gyratory incubator (300 RPM) at 30C for 30 minutes. After the 30 minute incubation, 2 ml of supplemented top agar were added, the overlay poured onto plates, and the plates were placed in a 37°C incubator for approximately 2 days. All negative and positive controls were also pre-incubated. The total volume of incubation mixture was 0.7 ml and included:
(a) 0.5 ml of S-9 activation mixture or 0.5 m l of 0.2 M sodium phosphate buffer, pH 7.4,
(b). 0.1 ml of an overnight bacterial culture, and
(c) 0.1 m l of test chemical solution or solvent.

Test Concentrations
The test material was dissolved in dimethyl sulfoxide (DMSO). The test material was shown to be stable in DMSO for at least 4 h (Campbell, 1988).
The negative control (solvent) plates consisted only of bacterial culture, 0.5 m l of 0.2 M sodium phosphate buffer or 0.5 ml S-9 activation mixture,
and 0.1 ml of solvent. All positive controls were prepared in DMSO except , for sodium azide, which was prepared in distilled water. All dose levels
along with ,the positive and negative controls were assayed in triplicate. The test agent was initially assayed in TA100 at various dose levels. The
concentrations tested in the other three strains were based upon the toxicity observed in TA100.

Colony Counting
The revertant colonies were counted either manually or using an Artek automatic colony counter, Model 880. The counter is calibrated periodically by using computer-generated " artificial plates" consisting of a specified number of randomly placed dots within a circle the size of a petri dish.
Transparencies of the "artificial plates" are counted in the same manner as a routine bacterial plate. A correction factor was determined to compensate for the area not scanned by the counter (i.e., dish edge) and overlapping colonies. This correction factor was used to automatically adjust
the observed number of colonies on each plate to more accurately reflect the actual number of colonies present.

Evaluation criteria:

Overtly toxic concentrations of the test chemical are easily visualized as showing no-bacterial growth on the plate (i.e ., absence of background
lawn). Lower levels of toxicity may be seen as a thin or sparse bacterial lawn, a reduction in the number of revertants, or the appearance of microcolonies (overgrown background lawn). Positive and negative controls are run concurrently with the test chemical , and appropri ate responses for
these controls are prerequisites for evaluating the response o f the bacteria to the test chemical.

A test chemical is considered a bacterial mutagen if both the mean number of revertant colonies observed is at least three times higher than the mean of the negative (solvent) control and it produces a dose response in relation to concentrations tested. If a chemical produces reproducible revertant rates in excess of 3X over background, but no definitive dose response relationship, it is considered to be a presumptive bacterial mutagen. Increased revertant rates at a single dose near the highest toxic concentration will be examined critically for biological significance. If a
chemical produces reproducible revertant rates greater than 2X but less than 3X over the negative controls at several concentrations, the results
are considered to be equivocal or inconclusive. Test substances failing to meet the above criteria are considered non-mutagenic in this system.

Statistics:

None

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

=/>50ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

=/>50ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

=/>50ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

=/>50ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Initially, DEB was assayed in TA100 at dose levels of 5.0, 15.8, 50 158.0,  500.0, 1580.0, and 5000.0 µg/plate.  In the absence of S-9, excessive  bacterial toxicity was observed at concentrations of 50 µg/plate and  above.  Hence, the highest concentration tested in all subsequent assays  was limited to 15.8 µg/plate.

There was no evidence of mutagenic activity for the test chemical in  TA98, TA1000, and TA1537.  In TA1535, the mutation frequency in the  absence of S-9 at some test chemical concentrations were found to be  doubled.  However, such an effect was non-reproducible in a repeat assay.   Hence, the increases seen in the initial assay were interpreted to be  unrelated to treatment.

The responsiveness of the tester strains to mutagens is clearly  demonstrated by the marked increases in revertant frequency in positive  control treatments,

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce a mutagenic response in any of the tester strains as judged by the frequency of histidine-dependent (his+) revertants. Hence, the test material was classified as negative in the AMES test under the experimental conditions used.

Executive summary:

Dowtherm*J heat transfer fluid (diethyl benzene) was evaluated in the Salmonel1a/mammalian-microsome bacterial mutagenicity assay (Ames test ) using the preincubation modification of the standard assay. The test material was assayed in the presence and absence of an externally supplied metabolic activation system

(S-9) using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535, and TA1537. The test material did not induce a mutagenic response in any of the tester

strains as judged by the frequency of histidine-independent (hist) revertants. Hence, the test material was classified as negative in the Ames test under the

experimental conditions used.