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Administrative data

in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-20 to 2018-02-09
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st July 1997
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:
Test material form:


Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- Source of cells: MOLTOX, INC., NC 28607, USA (TA98, 1535 and 102), Xenometrix AG, Switzerland (TA100 and 1537)

- Type and identity of media: Nutrient medium: 8 g Nutrient Broth and 5 g NaCl per litre, plus 125 µL ampicillin (10 mg/mL) for TA98, TA100, TA102); Agar Plates: Vogel-Bonner Medium E agar plates contain per litre 15 g Agar Agar, 20 mL Vogel-Bonner salts and 50 mL glucose solution (40%); Overlay Agar: The overlay agar contains per litre: 7.0 g Agar Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H20 and 12.2 mg biotin
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (see box "Any other information on materials and methods incl. tables"; Results: see box "Any other information on results incl tables", Table 2). Two independent main experiments were performed with the following concentrations:
31.6, 100, 316, 1000, 2500 and 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, AppliChem Lot No. 0001027819
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
Distilled water
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
sodium azide
TA100 and TA1535 (10 µg/plate), without S9
Untreated negative controls:
Distilled water
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
TA98 (10 µg/plate) and TA1537 (40 µg/plate), without S9
Untreated negative controls:
Distilled water
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
TA102 (1 µL/plate), without S9
Untreated negative controls:
Distilled water
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
other: 2-aminoanthracene
All strains (2.5 µg/plate), except TA102 (10 µg/plate), with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, Experiment I), pre-incubation (Experiment II)
- Cell density at seeding (if applicable): approx. 10^9 cells/mL, 100 µL/plate

- Experiment I :
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate: 100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 μL Bacteria suspension, 2000 μL Overlay agar.
- Experiment II:
For the pre-incubation method 100 µL of the test solution was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.

- Preincubation period (Experiment II): 60 min at 37 °C
- Exposure duration: 48 h in the dark at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/concentration level including the controls

Cytotoxicity is considered either as a clearing or diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
Evaluation criteria:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, TA102)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2014 -2016) (see box “Any other information on material and methods incl. tables”, Table 1)
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA102
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. Precipitation of the test item was observed in all tester strains used in experiment I and II (with and without metabolic activation). In experiment I precipitation of the test item was found at a concentration of 5000 µg/plate (with and without metabolic activation) and at concentrations of 2500 µg/plate and higher (with and without metabolic activation) in experiment II. No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 4,4’-Dimethoxytrityl chloride at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
Remarks on result:
other: Experiment I, precipitation was observed in all tester strains at 5000 µg/plate

Any other information on results incl. tables

Results of the pre-experiment:

Table 2: Results of the pre-experiment

Substance Dose (µg/plate) TA98
Mutation Factor*
 Mutation Factor*
without S9 with S9 without S9 with S9

Solvent Control (DMSO)

1.0 1.0 1.0 1.0
4-NOPD 10.0 28.6 - - -
NaN3 10.0 - - 6.8 -
2-AA 2.50 - 55.8 - 20.7
Test Item 3.16 0.9 1.3 0.9 1.0
10.0 1.0 1.0 1.1 0.9
31.6 0.9 1.2 0.9 0.9
100 0.6 1.0 0.8 0.8
316 0.8 0.9 0.9 0.7
1000 0.8 1.0 0.7 0.7
2500 0.6 1.2 0.6 0.6
5000 0.8 1.0 0.8 0.8

Applicant's summary and conclusion

Under the experimental conditions reported, 4,4'-Dimethoxytrityl chloride did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 4,4'-Dimethoxytrityl chloride is considered to be non-mutagenic according to CLP criteria in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria conducted according to OECD 471, strains TA98, TA100, TA102, TA1535 and TA1537 of S. typhimurium were exposed to 4,4'-Dimethoxytrityl chloride (99.73% purity) dissolved in DMSO at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains and both experiments (plate incorporation and pre-incubation). Based on the results, 4,4'-Dimethoxytrityl chloride is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.