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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1992-08-05, 1992-09-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Justification for Read Across is given in Section 13 of IUCLID

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
yes
Remarks:
2-aminoanthracene was used as only positive control with metabolic activation.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
other: UKEMS
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix and cofactors
Test concentrations with justification for top dose:
1.6, 8, 40, 200, and 1000 µg/plate (experiment 1) and 62.5, 125, 250, 500, and 1000 µg/plate (experiment 2)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: glutaraldehyde 25.0 μg/plate for TA102; 2-nitrofluorene 50.0 μg/plate for TA98
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene 5.0 µg/plate in DMSO, at least 1 strain
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours at 37 °C in nutrient broth (experiment 2 with metabolic activation).
- Exposure duration: 72 h.
- Expression time (cells in growth medium): 72 h.
- Selection time (if incubation with a selection agent): 72 h.
- Fixation time (start of exposure up to fixation or harvest of cells): 72 h.

SELECTION AGENT (mutation assays): growth in absence of histidine
Evaluation criteria:
The assay was considered valid if the following criteria were met:
i) the mean negative control counts fell within the normal range
ii) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
iii) no more than 5 % of the plates were lost through contamination or some other unforeseen event.
A test compound was considered to be mutagenic if:
i) the assay was valid
ii) Dunnett's test gave a significant response (p <0.01), and the data set showed a significant dose-correlation
iii) the positive responses described in (ii) were reproducible
Statistics:
For evaluation of test chemical and positive control data, the m-statistic was first calculated to check that the data were Poisson-distributed. Then, Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was then checked by linear regression analysis.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
presumably as the result of the increased exposure of the bacteria to the test agent due to the pre-incubation step.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: None
- Water solubility: low
- Precipitation: yes, at 1000 µg/plate (experiment 1) and at 500 and 1000 µg/plate (experiment 2)

RANGE-FINDING/SCREENING STUDIES: strain TA100 at 8, 40, 200, 1000, and 5000 µg/plate. No evidence of toxicity. Heavy precipitation of test agent was observed at concentrations of both 1000 and 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: comparable

ADDITIONAL INFORMATION ON CYTOTOXICITY: no evidence of toxicity was observed following these treatments, as would be indicated by a thinning of the background bacterial lawn or marked reductions in revertant numbers.
Remarks on result:
other: experiment 1

Any other information on results incl. tables

No substance treatment of any of the tester strains, either in the absence or presence of S-9 mix, resulted in a statistically significant (at the 1 % level using Dunnett's test) and dose-related increase in revertant numbers. The data obtained therefore gave no indication of an ability of the test agent to induce mutation.

Table: Revertants in S. typhimurium treated with the test substance, with and without metabolic activation (experiment 1):

Dose (µg/plate)

 TA98   

 TA100   

 TA1535   

 TA1537   

 TA 102   

 

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

 0

22.2±5.7

17.2±6.1

115.2±6.4

124±3.2

21.8±2.9

26.6±4.3

17.8±2.1

13±3.5

232.6±21.1

305.4±39.5

 1.6

27.7±5.1

24±7

105.7±6.7

119±29.5

26±1

29±1

15±2.6

10.3±3.1

298±50.7

362.3±7.8

 8

29.7±5.9

21.3±7.4

95.3±9.5

113.7±9.1

23.7±10.4

18.7±7.6

14.3±4.6

9±2.6

265.3±11.7

317.3±33.6

 40

25.3±6.7

21±6.1

113±2

124±23.8

17.7±2.5

23.7±1.2

16±5.3

11.7±3.5

267.7±39.7

333±28.6

 200

29.0±2.6

22.7±8.4

108.7±11.9

99.3±24

21.7±1.2

28±4.4

15±1

11.3±3.5

283.3±26.5

316.7±15.3

 1000

21.0±3.0

16.7±4.7

123.3±9.2

91±11

21.7±3.8

24±2.6

8.7±3.5

9.7±4

276±17.4

292±52.3

 2-NF

1645.3±111.9

 -

-

-

-

 -

 -

 -

 -

 ±

 NaN3

 -

 -

810.3 ±57.4

 -

463±57.7

 -

 -

 -

 -

 AAC

 -

 -

 -

 -

 -

 -

425.7±9.1

 -

 -

 -

 AAN

 -

1367.3±147

 -

1800.3±249.7 

 -

195.7±4

 -

 -

 -

 -

 GLU

 -

 -

 -

-

 -

 -

 -

 -

 477.7±45.8

 -

(Mean ± SD)

  

2-NF: 2-nitrofluorene;

NaN3: sodium azide

GLU: glutaraldehyde

AAC: 9-aminoacridine

AAN: aminoanthracene

Table: Revertants in S. typhimurium, treated with the test substance, with and without metabolic activation (experiment 2):

Dose (µg/plate)

 TA98   

 TA100   

 TA1535   

 TA1537   

 TA 102   

 

 

 

 

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

 -S9

 +S9

 

 

 

 0

29.4±5.0

28±4.5

111.6±12.7

103.6±10.8

22.2±2.8

21±1.6

15.4±3.8

13±2.2

319.2±48.9

357±19.4

 

 

 

 62.5

34.7±1.2

21±1.7

109±13.5

83.7±5.1

21.3±1.2

19.7±3.2

19.3±2.5

18.3±0.6

271±43.4

316±16.9

 

 

 

 125

36.7±7

28.7±6.1

113±10.5

87.3±10

16.3±3.5

11.3±4

16±0

13.7±2.1

222.3±11.9

311.3±13.4

 

 

 

 250

36.3±5.5

34.3±1.2

102.7±3.1

90±28.3

21.3±5.7

22.3±3.1

17±2

14.3±3.1

237.3±27.6

351.3±11.4

 

 

 

 500

19.3±9

24±4.6

90.3±1.5

70.7±2.1

15±5

21.3±3.8

15.3±2.1

16.3±4.5

292.3±29.8

162.7±11

 

 

 

 1000

25.3±11.0

18.7±2.5

101±13.9

75.3±3.8

16.3±2.5

21±2.4

14.3±6.1

10.3±1.2

232.7±17.7

190.7±11

 

 

 

 2-NF

1644±509.6

 -

-

-

-

 -

 -

 -

 -

 -

 

 

 

 NaN3

 -

 -

773.3 ±75.1

 -

479±49.4

 -

 -

 -

 -

 

 

 

 AAC

 -

 -

 -

 -

 -

 -

643.3±227.7

 -

 -

 -

 

 

 

 AAN

 -

2213.7±125.1

 -

1451.7±38.1 

 -

-

 -

 -

 -

 -

 

 

 

 GLU

 -

 -

 -

-

 -

 -

 -

 -

 715.3±89.4

 -

 

 

 

(Mean ± SD)

Applicant's summary and conclusion

Conclusions:
Negative with and without metabolic activation.
Executive summary:

The mutagenic potential of the substance was evaluated in the in-vitro gene mutation study in bacteria according to the OECD Guideline 471 and EU Method B.13/14. Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) strains were exposed to the substance in a range of 1.6 - 1000 µg/plate (experiment 1) and 62.5- 1000 µg/plate (experiment 2) both with and without metabolic activation. Positive control substances were tested in parallel.

Precipitation was noted at 1000 µg/plate (experiment 1) and at 500 and 1000 µg/plate (experiment 2). No evidence of cytotoxicity was observed.

No substance treatment of any of the tester strains, either in the absence or presence of S-9 mix, resulted in a statistically significant (at the 1 % level using Dunnett's test) and dose-related increase in revertant numbers

The substance was found negative with and without metabolic activation under the test conditions.