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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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was designed to comply with the OECD Guideline No. 471 (Bacterial Reverse Mutation test, 1997), the OECD Principles of Good Laboratory Practice (ENV/MC/CHEM(98)17, revised in 1997) and with the Chemicals Act of the Federal Republic of Germany (“Chemikaliengesetz-ChemG”, Gesetz zum Schutz vor gefährlichen Stoffen).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2010

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

Constituent 1
Chemical structure
Reference substance name:
Chloroacetic anhydride
EC Number:
208-794-2
EC Name:
Chloroacetic anhydride
Cas Number:
541-88-8
Molecular formula:
C4H4Cl2O3
IUPAC Name:
2-chloroacetyl 2-chloroacetate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Chloroacetic anhydride
- Physical state: white to light brown powder
- Analytical purity: 98,99 %
- Impurities (identity and concentrations):
chloroacetic acid: 0,81%
chloroacetyl chloride: 0,14%
each unspecified: 0,04% and 0,02%
- Purity test date: 12 June 2009
- Lot/batch No.: CHP-20-10196-111
- Expiration date of the lot/batch: 16 June 2011
- Storage condition of test material: At room temperature in the dark (ambient humidity)
- Stability under test conditions: Chloroacetic anhydride was dissolved in dimethylsulfoxide (DMSO). The solution was prepared freshly,
i.e. on the same day of plating. The stability of the test substance in the vehicle can be assumed.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
All strains, Plate Test
100 µg/plate
250 µg/plate
500 µg/plate
1000 µg/plate
2000 µg/plate

TA 98 and TA 100, Preincubation
50 µg/plate
100 µg/plate
250 µg/plate
500 µg/plate
1000 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO was used as solvent vehicle and negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Confirmation of Tester Strain Genotypes:
Tester strain cultures were checked for their typical genetic markers when frozen permanents were prepared or when the number of spontaneous
revertants fell outside the normal range.
The R-factor strains (TA 98, TA 100 and TA 102) were routinely tested for the ampicillin resistance factor because the plasmid is somewhat unstable and can be lost during subculturing. The optical density (OD) at 600 nm of each bacterial culture was determined.

JUSTIFICATION OF TEST CONCENTRATIONS
Chloroacetic anhydride was bacteriotoxic in the screening Ames test starting at 500 μg/plate using the preincubation method. Therefore,
2000 μg/plate and 1000 μg/plate was investigated as the highest concentration using the plate test and preincubation conditions,
respectively.

METABOLIC ACTIVATION:
S9
Batch (Protein): 2334 (36.7 mg/mL); 2494 (35.5 mg/mL)
Species/Tissue: Rat/Liver, Aroclor 1254 induced
Manufacturer: MolTox, Boone, NC (USA)
Storage: ≤ -70 °C
Immediately before use the thawed S9 fraction was mixed with the following solutions to achieve the S9 mixture according to Ames et al.
Na-phosphate buffer (pH 7.4) 100 mmol/L
Glucose-6-phosphate 5 mmol/L
NADP 4 mmol/L
MgCl2 8 mmol/L
KCl 33 mmol/L
S9 fraction 10 % (v/v)

Plate test
0.1 mL of the vehicle, test substance or positive control solution, 0.1 mL of a bacterial shaking culture (6 h, exponential phase) and 0.5 mL phosphate buffer or S9 mix were added to 2 mL soft agar. After vortexing, these mixtures were overlaid immediately onto minimal medium plates in triplicate
(n = 6 for the negative control).
Preincubation
0.1 mL of the vehicle, test substance or positive control solution, 0.5 mL phosphate buffer or S9 mix and 0.1 mL of a bacterial culture were
preincubated and shaken for 20 min at 37°C.
Then 2 mL soft agar were added to the mixture and after vortexing overlaid onto minimal medium plates in triplicate(n=6 for negative control).
The tests were performed in the presence and absence of rat liver microsomal enzymes (S9 mix).
Evaluation criteria:
Revertant his+ colonies were counted using an ARTEK Counter 880 (non-computerized) after incubation at 37°C for 2 days (TA 102: 3 days) and the values were listed manually in a word document. For all replicate platings, the mean number of revertants per test concentration was
calculated. The condition of the background bacterial lawn (residual growth on minimum histidine) was evaluated macroscopically for evidence of
bacteriotoxicity induced by the test substance. If extreme thinning or complete lack of the microcolony lawn compared to the negative, vehicle
control plates was observed, no revertants were counted. Evidence of test substance precipitates in the agar overlay was recorded.
Evaluation Criteria:
A reproducible, concentration-dependent increase in the number of revertants of at least one tester strain over the vehicle control value and/or
outside the historical control range is indicative of genotoxic activity.
Assay acceptance criteria:
The assay was considered valid since the following criteria were met:
All tester strains exhibit a characteristic number of spontaneous revertants per plate. The addition of the metabolic activation system did not alter
significantly the number of spontaneous revertants per plate and therefore the numbers were combined and given as ranges (see below). These
ranges were taken from about 210 experiments (not filed in the raw data) conducted in our laboratory.
TA 1535: 5 - 22
TA 1537: 2 - 29
TA 98: 12 - 68
TA 100: 54 - 197
TA 102: 252 - 531
In addition, the reference mutagens induced a distinct increase in the number of revertants, reflecting also the activity of the metabolizing system.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Plate Test
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 500 μg/plate without S9, >= 1000 μg/plate with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Plate Test
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 1000 μg/plate with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Preincubation
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no bacteriotoxicity up to 1000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
SOLUBILITY AND CYTOTOXICITY:
Chloroacetic anhydride did not precipitate up to 2000 μg/plate but it was bacteriotoxic primarily at 500 μg/plate in absence and at 1000 μg/plate in presence of S9 mix in the plate test. There was no bacteriotoxicity up to 1000 μg/plate in the preincubation test.
MUTAGENICITY
The OD600 of the individual overnight bacterial cultures varied between 2.0 and 3.1 (raw data). These values correspond to a bacterial titer of ca. 100 millions/0.1 mL. Chloroacetic anhydride did not increase the number of revertant colonies in different tester strains of S. typhimurium in presence and absence of a metabolic activation system compared to the negative control when tested up to the bacteriotoxic concentrations. Furthermore,
chloroacetic anhydride did not increase the number of revertant colonies in TA 98 and TA 100 in the preincubation test. It is possible that an impurity
in the batch (purity 97.4%, darkbrown, solidified melt) used for the screening Ames study was responsible for the slight increase in TA 98 (1000 μg/plate with S9 mix) and in TA 100 (500 μg/plate without S9 mix) in that study using the preincubation method. The validity of this study is given since
the vehicle control plates showed spontaneous revertants in different tester strains of S. typhimurium at frequencies similar to those described in the literature and within the historical control range experienced in our laboratory. All of the positive control mutagens NaN3, 9-AA, 2-NF, MMC and
2-AA showed the expected strain specific responses with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenic activity of Chloroacetic anhydride in S. typhimurium Plate Test (Summary)

Without metabolic activation

μg/plate

Mean Revertants/Plate

S. typhimurium

TA 1535

TA 1537

TA 98

TA 100

TA 102

Negative Control

DMSO

5

3

22

69

397

Chloroacetic anhydride

 

 

 

 

 

100

7

5

27

80

401

250

9

3

18

71

402

500

6

1 T

15 T

58 T

338

1000T

1

1

0

34

299

2000T

0

0

0

0

95

Positive Controls

 

 

 

 

 

NaN3      5

1198

-

-

1295

-

9-AA      50

-

196

-

-

-

2-NF      10

-

-

1495

-

-

MMC     0.5

-

-

-

-

1305

With metabolic activation

μg/plate

Mean Revertants/Plate

S. typhimurium

TA 1535

TA 1537

TA 98

TA 100

TA 102

Negative Control

DMSO

7

4

26

82

446

Chloroacetic anhydride

 

 

 

 

 

100

8

5

23

88

494

250

8

4

29

87

603

500

7

4

23

90

443

1000T

5

3

10

28

301

2000T

0

0

0

0

148

Positive Controls

 

 

 

 

 

2-AA       4

186

179

1726

1349

-

2-AA     10

-

-

-

-

1150

P: Precipitation T: Toxicity -: Not tested Underlined values are regarded as increased

Historical Range

5 - 22

2 - 29

12 - 68

54 - 197

252 - 531

Mutagenic activity of Chloroacetic anhydride in S. typhimurium Preincubation (Summary)

Without metabolic activation

μg/plate

Mean Revertants/Plate

S. typhimurium

TA 98

TA 100

Negative Control

DMSO

25

60

Chloroacetic anhydride

 

 

50

29

60

100

24

55

250

30

65

500        

33

73

1000

39

67

Positive Controls

 

 

NaN3          5

-

1346

2-NF         10

1531

-

With metabolic activation

μg/plate

Mean Revertants/Plate

S. typhimurium

TA 98

TA 100

Negative Control

DMSO

23

79

Chloroacetic anhydride

 

 

50

25

84

100

34

85

250

31

80

500        

27

86

1000

32

91

Positive Controls

 

 

2-AA             4

1614

817

P: Precipitation T: Toxicity -: Not tested Underlined values are regarded as increased 

Historical Range

12 - 68

54 - 197

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Chloroacetic anhydride caused neither base-pair substitutions nor frameshift mutations in different S. typhimurium strains in the presence and
absence of metabolic activation when tested up to the bacteriotoxic concentration in the plate test. In addition, the purer batch of chloroacetic
anhydride was also negative in S. typhimurium strains TA 98 and TA 100 under conditions that previously induced slight increases.
Based on these results it was concluded, that chloroacetic anhydride itself is "Ames negative" under the conditions of this study.
Executive summary:

For worker safety, Chloroacetic anhydride was investigated under GLP conditions to see whether it induces mutations in the Salmonella typhimurium strains TA 1535, TA 100, TA 102 (sensitive to base-pair substitution), TA 1537 and TA 98 (sensitive to frameshift mutagens) both in the presence and absence of a metabolic activation system (S9 mix: Aroclor 1254-induced rat liver microsomal fraction and co-factors). Chloroacetic anhydride was dissolved in DMSO and added to the bacterial cultures in triplicates to give the final concentrations of 100 to 2000 μg/plate. A repeat experiment was performed in strains TA 98 and TA 100 at final concentrations of 50 to1000 μg/plate using the preincubation method for comparison purposes, since a previous screening Ames has shown a positive result.

Solubility and Toxicity

Chloroacetic anhydride did not precipitate up to 2000 μg/plate but it was bacteriotoxic primarily at 500 μg/plate in absence and at 1000 μg/plate in presence of S9 mix in the plate test. There was no bacteriotoxicity up to 1000 μg/plate using the preincubation method.

Mutagenicity

Chloroacetic anhydride did not increase the number of revertant colonies in different tester strains of S. typhimurium in presence and absence of a metabolic activation system compared to the negative control when tested up to the bacteriotoxic concentrations. Furthermore, chloroacetic anhydride did not increase the number of revertant colonies in TA 98 and TA 100 in the preincubation test when tested up to 1000 μg/plate. It is possible that an impurity in the batch (purity 97.4%, darkbrown, solidified melt) used for the screening Ames study was responsible for the slight increase in TA 98 (1000 μg/plate with S9 mix) and TA 100 (500 μg/plate without S9 mix) observed in that study using the preincubation method. The validity of this study is given since the vehicle control plates showed spontaneous revertants in different tester strains of S. typhimurium at frequencies similar to those described in the literature and within the historical control range experienced in our laboratory. All of the positive control mutagens NaN3, 9-AA, 2-NF, MMC and 2-AA showed the expected strain specific responses with and without metabolic activation.

Conclusion

Chloroacetic anhydride caused neither base-pair substitutions nor frameshift mutations in different S. typhimurium strains in the presence and absence of metabolic activation when tested up to the bacteriotoxic concentration in the plate test. In addition, the purer batch of chloroacetic anhydride was also negative in S. typhimurium strains TA 98 and TA 100 under conditions that previously induced slight increases. Based on these results it was concluded, that chloroacetic anhydride itself is "Ames negative" under the conditions of this study.