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Diss Factsheets

Administrative data

in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 27 January 2010 To 9 February 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
according to guideline
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): HMTBN 97%
- Physical state: clear yellowish liquid
- Analytical purity: 96.5 %
- Purity test date: 04 december 2009
- Lot/batch No.: 09 ADS 377
- Expiration date of the lot/batch: December 2011
- Stability under test conditions: no data
- Storage condition of test material: Refrigerated (ca. 4°C)


Target gene:
Histidine operon (S. typhimurium)
Tryptophan operon (E. coli)
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvrA (pKM101)
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and (,6-benzoflavone to stimulate mixed-function oxidases in the liver.
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500, 5000 µg (Plate incorporation assay)
50, 150, 500, 1500, 5000 µg (Pre-incubation assay)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Ths test substance is known to be soluble in water.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: Without S9 mix: Sodium azide: 2 µg/plate for strains TA100 and TA1535; 9-Aminoacridine : 50 µg/plate for strain TA1537; 2-Nitrofluorene : 2 µg/plate for strain TA98; 4-Nitroquinoline-1-oxide : 2 µg/plate for strain WP2 uvrA (pKM101)
Details on test system and experimental conditions:
- in agar (plate incorporation) for the first test,
- preincubationfor the second test.

- Preincubation period: 30 minutes
- Exposure duration: ca 72 hours

- Method: relative total growth

Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenicactivity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.
No statistical analysis has been performed.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvrA (pKM101)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
in strains TA 98, TA1535 and TA1537 at 5000 µg/plate
Vehicle controls validity:
Untreated negative controls validity:
not examined
Positive controls validity:
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

Interpretation of results (migrated information):
negative with metabolic activation
ambiguous without metabolic activation

Under the conditions of this test, HMTBN 97% showed no evidence of mutagenic activity.
Executive summary:

In this in vitro assessment of the mutagenic potential of HMTBN 97%, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to HMTBN 97% diluted in water. Water was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a plate incorporation assay, modified to include a delayed-plating procedure, since it was expected that there would be degradation of the test substance under the conditions of test; the second included a pre-incubation stage. Concentrations of HMTBN 97% up to 5000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. Toxicity (observed as a reduction in revertant colony numbers of at least 50% relative to the

vehicle controls) was obtained in strains TA98, TA1535 and TA1537 following exposure to HMTBN 97% at 5000 μg/plate in one or both tests.

No evidence of mutagenic activity was seen at any concentration of HMTBN 97% in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical

control range of the laboratory.

It is concluded that HMTBN 97% showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.