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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethylhydroxylamine
- Analytical purity: 86.733% in water
- Lot/batch No.: 2905012

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (from Sprague-Dawley rats, treated by  Aroclor 1254)
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
distilled water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9-mix . sodium azide (TA1535 and TA100, 1µg/plate), 2-nitrofluorene (TA98, 0.5 µg/plate), mitomycin C (TA102, 0.5 µg/plate), 9-aminoacridine (TA 1537, 50µg/plate). With S9-mix: 2-anthramine (all strains, 2 µg/plate except 10 µg/plate for TA102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) excepted for the 2nd assay with S9 (preincubation)

DURATION
-  Pre-incubation time: 60 minutes
- Pre-incubation temperature: 37°C
- Incubation time: 48 to 72 hours
- Incubation temperature: 37°C

NUMBER OF REPLICATIONS:  3/dose

EXAMINATION:
- Bacterial toxicity (performed on TA98, TA100 and TA102 at 0, 10, 100,  500, 1000, 2500 and 5000 µg/plate)
- Number of revertants / plate. 
Two identical assays were performed.
Evaluation criteria:
- Positivity criteria for genotoxicity:
. number of revertants in the vehicle control is consistent with  laboratory historical data,
. number of revertants in the positive control is higher than that of  vehicle control and is consistent with laboratory historical data,
. number of revertants at least twice that of negative control revertants,
. reproducibility of the positive response.
Statistics:
Not appropriate

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA1537, TA 98, TA 100, TA 102
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 examined
Positive controls validity:
valid
Additional information on results:
All the dose-levels are expressed as active substance, taking into  account the purity of 86.733%.

BACTERIAL TOXICITY
No toxicity was noted towards the three strains used, with and without  S9-mix.

GENOTOXIC EFFECTS
The number of revertants for the vehicle and positive controls was as  specified in the acceptance criteria. The study was therefore considered  valid.  Since the test substance was freely soluble and non-toxic, the highest  dose-level was 5000 µg/plate, according to the criteria specified in the  international guidelines.  The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000  µg/plate, for both mutagenicity experiments with and without S9 mix.  In the TA 1535 strain in the first experiment without S9 mix, a 2-fold  increase in the number of revertants was noted at 1250 µg/plate. This  increase was not reproducible (not observed in the second experiment  performed under the same experimental conditions). In addition the  revertant values obtained remained clearly within the vehicle historical  range (3-23). Therefore, this slight increase in revertants was not  considered as relevant. 

PRECIPITATION
No precipitate was observed in the Petri plates

TEST-SPECIFIC CONFOUNDING FACTORS
none

Applicant's summary and conclusion

Conclusions:
DIETHYLHYDROXYLAMINE does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The potential of diethylhydroxylamine to induce reverse mutation in Salmonella typhimurium was evaluated in an OECD TG #471 study.Diethylhydroxylamine was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to 312.5, 625, 1250, 2500 and 5000 µg/plate of diethylhydroxylamine in distilled water (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. No toxicity was noted towards all the strains used, both with and without S9 mix. Diethylhydroxylamine did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five tester strains. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. In conclusion, diethylhydroxylamine did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.