Chloroacetic anhydride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 January - 18 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

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

Test material information

Constituent 1

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

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 / 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:

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 results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Species / strain:

S. typhimurium TA 98

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

Test results 4

Species / strain:

S. typhimurium 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

Test results 5

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

Test results 6

Species / strain:

S. typhimurium TA 98

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

Test results 7

Species / strain:

S. typhimurium 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.

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:

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.