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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In a reverse gene mutation assay in bacteria (Ames Test), no mutagenicity was observed with or without metabolic activation. (Hoechst-Celanese, 1988)
Link to relevant study records
Reference
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:
1988-02-03 to 1988-02-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well-documented, scientifically acceptable study report
Qualifier:
no guideline available
Principles of method if other than guideline:
Test was performed according to the method published by Ames et al (Mutation Res., 31: 347-364, 1975)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254 induced rat liver)
Test concentrations with justification for top dose:
Pre-test: 0.018, 0.037, 0.073, 0.146, 0.293, 0.586, 1.17, 2.34, 4.69, 9.38, 18.8, 37.5, 75, 150 µL/plate
Main test: 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10 µL/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Substance was soluble in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix (TA 1535, TA 100)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9 mix (TA 1538, TA 98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Quinacrine mustard
Remarks:
without S9 mix (TA 1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: none

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Strains TA 1535, TA 1537, TA 1538
Data sets will be evaluated as positive if a dose response is observed over a minimum of three test concentrations and the increase in revertants is equal to or greater than three times the solvent control value at the peak of the dose response. The solvent control value should be within the normal range for evaluating the results.

Strain TA 98, TA 100
Data sets will be evaluated as positive if a dose response is observed over a minimum of three test concentrations and the increase in revertants achieves a doubling of the solvent control value at the peak of the dose response. The solvent control value should be within the normal range for evaluating the results.
Statistics:
Not applicable.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
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:
TEST-SPECIFIC CONFOUNDING FACTORS
No confounding factors were observed during the test.

RANGE-FINDING/SCREENING STUDIES: Doses for the mutagenesis assays ware selected from a preliminary study conducted on the test material at fourteen doses from 0.018 µL per plate to 150 µL per plate using the strain TA 100. In the preliminary study, the test material was toxic to the indicator strain at 9.38 µL per plate and above as evidenced by the reduced number of revertants, and/or the appearance of micro colonies on the minimal media plates, and/or the clearing of the background lawn.

MAIN TEST: Individual plate counts for the test material are given in the table under "Any other information on results incl. tables". Since the solvent control values were out of range with strain TA 98 in the non-activation and activation assays, the tests with this strain were repeated. The second trial was discarded as it was contaminated. The results with this strain from the third trial are given in the table under "Any other information on results incl. tables". The results of the assays conducted on the test material at dose level ranging from 0.050 µL per plate to 10.0 µL per plate in the absence and presence of metabolic activation did not exhibit increased numbers of his+ revertant colonies. The positive control treatments in both the non-activation and S9 activation assays induced large increases in the revertant numbers with all the indicator strains, which demonstrated the effectiveness of the S9 activation system and the ability of the test system to detect known mutagens.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Raw data

Dose (µL/plate)

Mean number of revertant colonies/ plate with different strains of Salmonella typhimurium

 

TA1535

TA1537

TA1538

TA98

TA100

 

 

 

Results with S9

Spontaneous Reversion

11

7

25

36

170

Positive control

248

492

1993

1808

2365

0.05

16

7

19

38

176

0.10

19

12

28

31

148

0.25

9

10

13

40

181

0.50

13

5

24

31

164

1.0

10

10

23

29

142

2.5

12

11

30

40

126

5.0

7

2

19

15

86

10.0

1

4

10

29

60

 

 

 

 

Results without S9

Spontaneous Reversion

26

14

17

37

169

Positive control

1409

1003

1665

1471

1207

0.05

26

11

12

30

190

0.10

20

9

12

27

149

0.25

17

5

13

32

161

0.50

23

10

15

32

183

1.0

25

11

16

30

151

2.5

34

9

19

34

144

5.0

21

4

29

24

105

10.0

21

1

8

9

36

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames Test

In a reverse gene mutation assay in bacteria, strains (TA 1535, TA 1537, TA 1538, TA 100, TA 98) of S. typhimurium were exposed to the test substance at concentrations of 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10 µL/plate in the presence and absence of mammalian metabolic activation. The mutation assay was conducted using one plate per dose level.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background. The test substance was not mutagenic in the S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 100 and TA98 under the experimental conditions with or without metabolic activation.

Florin et al. (1980) further supported the above results. 2-hydroxyacetophenone was tested in the spot test according to Ames et al. with and without metabolic activation at a concentration of 3 µmol/plate. The positive controls induced the appropriate responses in the corresponding strains.

The test substance was not mutagenic in the S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 100 and TA98 under the experimental conditions with or without metabolic activation.


Justification for selection of genetic toxicity endpoint
well-documented, scientifically acceptable study report

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled for mutagenicity under Regulation (EC) No 1272/2008, as amended for the seventh time in Regulation (EU) No 2015/1221.