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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:
from november 16th, 1988 to February 7th, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to the OECD guideline n°471.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
A c. 700 g sample of the test substance, a white powder designated
AF—366, was received from Fuji Photo Film B.V., Tilburg, The Netherlands
on September 19, 1988. The test substance was stored at ambient
temperature and in the dark, until use.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
The S—9 batch of October 11, 1988 contained 0.94 µmol cytochrome P—450 per gram protein.
Test concentrations with justification for top dose:
0 µg per plate,
61.7 µg per plate,
185.2 µg per plate,
555.6 µg per plate,
1666.7 µg per plate,
5000.0 µg per plate.
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: 1.0 µg per plate with the strains TA 1535 and TA 100, in the absence of S-9 mix.
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: 2.0 µg per plate with the stains TA 1538 and TA 98 in the absence of S-9 mix.
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: 80.0 µg per plate with the stain TA 1537 in the absence of S-9 mix.
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2.0 µg per plate with the stains TA 1535, TA 1538, TA98 and TA 100, 5.0 µg per plate with strain 1537 in the presence of S-9 mix.
Details on test system and experimental conditions:
Toxicity test
A preliminary test with 6 concentrations of the test substance and the
vehicle (DM50) as negative control was carried out to assess the toxicity
of the test substance for S. typhimurium TA 1535, TA 1537, TA 1538, TA 98
and TA 100, both in the absence and in the presence of the S—9 mix. The
toxicity test was carried out between November 16 and December 13, 1988.
Just prior to use, a stock solution containing 100.0 mg of the test
substance per ml was prepared in DMSO under gentie heating in a waterbath
at 50°C. For the other dose levels a series of stepwise dilutions — by a
factor 10 — in DMSO were prepared from the stock solution.
Briefly, the toxicity test was carried out as follows. To 2 ml molten top
agar (containing 0.6% Difco agar, 0.5% NaCl and 0.05 mM L—histidine.HCl/
0.05 mM biotin), maintained at 46°C, were added in this sequence: 0.1 ml
of a fully grown culture of the appropriate tester strain, 0.1 ml of the
appropriate solution of the test substance and 0.5 ml S—9 mix, if any. The
ingredients were thoroughly mixed and the mix was immediately poured onto
minimal glucose agar plates (1.5% Bacto—Difco agar in Vogel and Bonner
medium E with 2% glucose). After incubation for three days at 37°C, the
background lawn of bacterial growth was examined microscopically and the
his+ revertants were counted by hand.
The resuits show that the test
substance was not toxic for any of the Salmonella typhimurium strains,
either in the absence or in the presence of the S—9 mix. At the highest
concentration used (10.0 mg per plate) the test substance precipitated in
the plates. A slight precipitate was seen at a concentration of 1.0
mg/plate.
In view of these observations, 5.0 mg/plate was chosen as the highest
concentration for the mutagenicity assay, both in the absence and in the
presence of the S—9 mix.

Mutagenicity assay
The mutagenicity assays were carried out according to a protocol entitled
“Protocol for the examination of the mutagenic activity of AF—366 in the
Ames test”, which was approved by the sponsor on October 6, 1988. In both
experiments the plate—incorporation method with the histidine requiring
S. typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100 as
indicator strains was applied. This assay has been described in detail by
Ames et al. (1975) and by Maron and Ames (1983). The mutagenicity assays
were carried out between January 31 and February 7, 1989.
Appropriate solutions of the test substance, containing 0, 0.6, 1.9, 5.6,
16.7 and 50.0 mg/ml, were prepared in DM50 just prior to use. The repeat
experiment was carried out with the same concentrations.
The reference mutagens used as positive controls were:
— sodium azide: 1.0 µg per plate with the strains TA 1535 and TA 100, in
the absence of the S—9 mix
— 2—nitrofluorene; 2.0 µg per plate with the strains TA 1538 and TA 98, in
the absence of the S—9 mix
— 9—aminoacridine: 80.0 µg per plate with strain TA 1537, in the absence
of the S—9 mix
— 2—aminoanthracene: 2.0 µg per plate with the strains TA 1535, TA 1538,
TA 98 and TA 100, 5.0 pg per plate with strain TA 1537, in the presence
of the S—9 mix.
Briefly, the mutagenicity test was carried out as follows. To 2 ml molten
top agar, maintained at 46°C, were added
in this sequence: 0.1 ml of the appropriate tester strain, 0.1 ml of the
appropriate solution of the test substance or the controls and 0.5 ml of
the S—9 mix, if any. The ingredients were thoroughly mixed and the mix was
immediately poured onto minimal glucose agar plates.
All determinations were made in triplicate. The plates were
incubated at 37°C for three days. Then the his+ revertants were counted by
hand. The background lawn of bacterial growth was exainined
microscopically.
S—9 and S—9 mix were checked for sterility.
The viable count of each culture was made by plating appropriate dilutions
of the cultures on nutrient broth agar plates. Each culture was exainined
for the nuxnber of spontaneous revertants, histidine requirement and sen—
sitivity to ampicillin, crystal violet and UV radiation.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Additional information on results:
Evaluation of test results
A positive response in the assay system is taken to be a two—fold or
greater increase in the mean nuinber of revertant colonies appearing in the
test plates over and above the background spontaneous reversion rate
observed with the vehicle, together with evidence of a dose—response.

Results
From the resuits obtained in both experiments it appeared that incubation
of the test substance with the bacteria did not increase the number of
his+ revertants with S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 or
TA 100, either in the absence or in the presence of the S—9 mix.
At the two higher concentrations used, the test substance precipitated in
the plates.
The positive controls used in the present assays gave the expected strong
increase in the number of his+ revertants, both in the absence and in the
presence of the S—9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

From the above findings it is concluded that AF—366 did not show mutagenic
activity in Salmonella typhimurium TA 1535, Tl 1537, TA 1538, T 98 or
TA 100 either in the absence or in the presence of the S—9 mix, under the
conditions employed in this evaluation.