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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline, and in compliance with GLP. Read-across to the registered substance is considered scientifically justified.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
: not tested in the absence of metabolic activation
Principles of method if other than guideline:
Method: Clive et al
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
0-1.2 µl Dequest 2000/ml (calculated by previous reviewer as 0-0.78 µl active acid/ml). These concentrations were positive in the previous experiment.
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no information
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with activation
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2 days


SELECTION AGENT (mutation assays): 5ug/ml trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 6x10E06 (1st experiment); 9x10E06 (2nd experiment)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)
Evaluation criteria:
Positive result: Dose-related increase in number of mutant colonies and mutant frequency at one or more concentrations of greater than or equal to twice control levels with an RTG of greater than 10%.
Statistics:
No statistical analysis of results was carried out.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
not determined
Remarks:
Concentrations based on data from previous experiment
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: test solution was neutralised to eliminate pH effects. Slight drop in pH seen at highest concentration but approximate fall in 0.08pH units only
- Precipitation: white precipitate reported at concentrations greater than 0.61µl/ml

RANGE-FINDING/SCREENING STUDIES: not conducted as this is a follow-up experiment

COMPARISON WITH HISTORICAL CONTROL DATA: no data

Any other information on results incl. tables

In the first experiment all treated cultures had higher RTGs than the control culture and a dose-related increase in mutant frequency was seen (see Table 1). The experiment was discounted because of the low control growth.

Table 1: Results of Mammalian Mutagenicity assay 1 with tester strain L5178Y in presence of metabolic activation

Concentration

µl/ml

Mutant frequency

x10E-06

RTG

%

0*

23

100

0.49

22

195

0.51

33

134

0.77

48

149

0.96

59

133

1.2

58

121

In the second experiment no increases in mutant frequency and no cytotoxicity were seen. It had been proposed that a selective advantage of mutants in the presence of test substance was the reason for the increase in revertants observed in the previous experiment. No effects on levels of spontaneous mutants were seen when cells were plated immediately after treatment. This finding does not support the proposed reason for the previously observed positive result.


Table 2: Results of Mammalian Mutagenicity assay 2 with tester strain L5178Y in presence of metabolic activation

Concentration

µl/ml

Mutant frequency

x10E-06

RTG

%

0*

101

100

0.61

91

100

0.77

112

74

0.96

98

92

1.2

100

88

1.5

112

103

Positive control

659

46



Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation

A neutralized solution of ATMP acid was tested for mutagenicity to mammalian cells under GLP. No increase in the number of revertants was observed in the presence of metabolic activation when tested up to the solubility limit. The test results indicate that the positive findings in the previous experiment with un-neutralised ATMP acid were an artefact of pH. It is concluded that ATMP acid is not mutagenic to mammalian cells under the conditions of this test.