Reaction mass of (Z)-1-chloro-1-propene and 2-chloropropane and 2-chloropropene

Genetic toxicity: in vitro

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:

September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD guideline n° 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-chloropropene

EC Number:

209-187-5

EC Name:

2-chloropropene

Cas Number:

557-98-2

Molecular formula:

C3H5Cl

IUPAC Name:

2-chloroprop-1-ene

Constituent 2

Reference substance name:

2-chloropropane

EC Number:

200-858-8

EC Name:

2-chloropropane

Cas Number:

75-29-6

Molecular formula:

C3H7Cl

IUPAC Name:

2-chloropropane

Constituent 3

Reference substance name:

1-chloropropene

EC Number:

209-675-8

EC Name:

1-chloropropene

Cas Number:

590-21-6

IUPAC Name:

1-chloroprop-1-ene

Details on test material:

Identification Reaction mass containing mainly 2-chloropropene
Molecular formula C3H5Cl
Molecular weight 76.53
CAS Number 557-98-2
Description Clear light yellow to brown liquid (determined at NOTOX)
Batch RBA100301A
Purity Minimum 60.0%
Test substance storage In refrigerator (2-8°C) in the dark
Stability under storage conditions Stable
Expiry date 08 March 2011 (allocated by NOTOX, 1 year after receipt of the test substance)

Study specific test substance information:
General information Extremely flammable
Stability in vehicle Dimethyl sulfoxide: Not indicated
Solubility in vehicle Dimethyl sulfoxide: Not indicated

Method

Target gene:

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains, and in a gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with tester strain TA100 in the absence and presence of 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of Reaction mass containing mainly 2-chloropropene used in the subsequent mutation assay was 5000 μg/plate or the level at which the test substance inhibited bacterial growth.

Vehicle / solvent:

dimethyl sulfoxide - DMSO (SeccoSolv, Merck, Darmstadt, Germany)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Relevant positive controls (sodium azide, 2-nitrofluorene,methylmethanesulfonate,4-nitroquinoline N-oxide, 2-aminoanthracene) were concurrently tested in each strain.

Details on test system and experimental conditions:

TEST SYSTEM
Test System Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale Recommended test system in international guidelines (e.g. OECD, EC).
Source Salmonella typhimurium strains:
Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames):
TA100 received on 14-06-2006, used batch: TA100.241109
TA98 received on 14-06-2006, used batch: TA98.241109
TA1537 received on 14-06-2006, used batch: TA1537.041209
TA1535 received on 14-06-2006, used batch: TA1535.241109
Escherichia coli strain:
(Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK)
WP2uvrA received on 06-02-2008, used batch: EC.090210
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment (Ref.1). The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

CELL CULTURE
Preparation of bacterial cultures:
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.05 ± 0.05 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

Agar plates:
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi). The agar plates for the test with the Salmonella typhimurium strains also contained
12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Merck) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan (Acros Organics).

Top agar:
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) Sodium Chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions:
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 34.8 – 39.0°C) in the dark. Temporary deviations of maximally 1 hour (in the range of 34.8 – 36.0°C) occurred due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity. The temporary deviations from the temperature (with maxima of 38.7 and 39.0°C) are explained in protocol deviation 1.

METABOLIC ACTIVATION SYSTEM
S9-fraction:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany.

The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the Standard Operating Procedures. The rats were orally dosed at three consecutive days with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight) in corn oil (they were denied access to food for 3 to 4 hours preceding each dosing). One day after the final exposure (24 h), the rats were sedated using oxygen/carbon dioxide and then killed by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C) for a maximum of 1 year.

Before use, all S9 batches were characterised with the mutagens Benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA98 at concentrations of 5 µg/plate and 1 µg/plate, respectively.

Preparation of S9-mix:
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KCl solution. The above solution was filter (0.22 µm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment. The S9-batch used was
no. 10-5.

STUDY DESIGN
Dose range finding test:
Selection of an adequate range of doses was based on a dose range finding test with tester strain TA100 in the absence and presence of 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of Reaction mass containing mainly 2-chloropropene used in the subsequent mutation assay was 5000 μg/plate or the level at which the test substance inhibited bacterial growth.

Mutation assay:
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.

In the first experiment Reaction mass containing mainly 2-chloropropene was tested both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and WP2uvrA. In an independent repeat of the assay with additional parameters, the test substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.

The vehicle control and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Top agar in top agar tubes was melted by heating to 45°C. The following solutions were preincubated for 30 minutes by 70 rpm at 20°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in dimethyl sulfoxide. After the preincubation period the solutions were added to 3 ml molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted.

Colony counting:
The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate are present. If more than 40 colonies were present, these could be counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually and evidence of test substance precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

Electronic data capture:
Observations/measurements in the study were recorded electronically using the following programme: REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): temperature.

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight to moderate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight to moderate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight to moderate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST
Reaction mass containing mainly 2-chloropropene was tested in tester strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. This dose range finding test is reported as a part of the first experiment of the mutation test (Table 2).

Precipitate
Precipitation of Reaction mass containing mainly 2-chloropropene on the plates was not observed at the start or at the end of the incubation period.

Toxicity
To determine the toxicity of Reaction mass containing mainly 2-chloropropene, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

A slight to moderate reduction of the bacterial background lawn was observed at test substance concentrations of 3330 and 5000 µg/plate in the absence and presence of S9-mix.

Mutagenicity
Reaction mass containing mainly 2-chloropropene induced an up to 3.6-fold dose related increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix.

MUTATION ASSAY
Experiment 1:
Based on the results of the dose range finding test, Reaction mass containing mainly 2-chloropropene was tested up to the dose level of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix with the tester strains, TA1535, TA1537, TA98 and WP2uvrA. The results are shown in Table 2.

Precipitate
Precipitation of Reaction mass containing mainly 2-chloropropene on the plates was not observed at the start or at the end of the incubation period.

Toxicity
There was no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. The reduction of the bacterial background lawn is presented in Table 1.

Mutagenicity
In tester strain TA1535, Reaction mass containing mainly 2-chloropropene induced an up to 8.5-fold dose related increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix. In the other tester strains, no increase in the number of revertants was observed upon treatment with Reaction mass containing mainly 2-chloropropene.

Experiment 2:
To obtain more information about the possible mutagenicity of Reaction mass containing mainly 2-chloropropene, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the following dose ranges were selected for the second mutation experiment:
TA1535 and TA1537
Without S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
With S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
TA98, TA100 and WP2uvrA
With and without S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
The results are shown in Table 3.

Precipitate
Precipitation of Reaction mass containing mainly 2-chloropropene on the plates was not observed at the start or at the end of the incubation period.

Toxicity
There was no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. A slight to moderate reduction of the bacterial background lawn was observed at test substance concentrations of 3330 and 5000 µg/plate in the tester strains TA1535 and TA1537 in the presence of S9-mix. A slight reduction of the bacterial background lawn was observed at the test substance concentration of 3330 µg/plate in the tester strains TA98, TA100 and WP2uvrA in the absence and presence S9-mix and at the tester strains TA1535 and TA1537 in the absence S9-mix.

Mutagenicity
Reaction mass containing mainly 2-chloropropene induced up to 13- and 3.5-fold dose related increases in the number of revertant colonies compared to the solvent control in tester strain TA1535 and TA100, respectively in the presence of S9-mix. In the other tester strains, no increase in the number of revertants was observed upon treatment with Reaction mass containing mainly 2-chloropropene.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1           Toxicity of Reaction mass containing mainly 2-chloropropene in the first experiment

              (Reduction of the bacterial background lawn )

Strain	Without S9-mix		With S9-mix
Dose          Bacterial background lawn                   (μg/plate)			Dose       Bacterial background lawn   (µg/plate)
TA1535		3330          slight 5000          moderate	5000          normal
TA1537		3330          slight 5000          moderate	5000          slight
TA98		3330          slight 5000          moderate	3330          slight 5000          moderate
WP2uvrA		3330          slight 5000          moderate	3330          slight 5000          moderate

Table2           Experiment 1: Mutagenic response of Reaction mass containing mainly 2-chloropropene in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

 

Dose                    Mean number of revertant colonies/3 replicate plates (± S.D.) with

(µg/plate)              different strains of Salmonella typhimurium and one Escherichia coli strain

 

 

                                   TA1535           TA1537           TA98            TA100           WP₂uvrA

                                               Without S9-mix

 

   positive control       248 ± 22       181 ± 47      1153 ± 90       799 ± 51        489 ± 17

    solvent control        12 ±  2          5 ±  2         15 ±  4        109 ± 18         22 ±  1

 

                  3                                                            95 ±  9

                 10                                                            91 ±  9

                 33                                                           111 ± 21

                100         10 ±  5          4 ±  2         15 ±  2        114 ± 12         14 ±  2

                333          9 ±  4          5 ±  2         16 ±  3        150 ± 15         15 ±  1

               1000          9 ±  2          4 ±  1         21 ±  3        105 ± 15         20 ±  6

               3330         12 ±  1 s       3 ±  0 s       12 ±  1 s       85 ±  4 s       15 ±  5 s

               5000          8 ±  2 m       3 ±  3 m      13 ±  1 m       88 ±  6 m       15 ±  1 m

------------------------------------------------------------------------------------------------------------

                                                With S9-mix¹

 

   positive control       891 ± 14       423 ± 11       913 ± 59      1193 ± 10        318 ± 18

    solvent control        17 ±  5         4 ±  1         23 ±  1         93 ± 10         25 ±  6

 

                  3                                                            89 ± 18

                 10                                                            90 ±  3

                 33                                                           109 ± 11

                100         17 ±  4         5 ±  1         22 ±  1       102 ±  3         23 ±  3

                333         31 ±  5         4 ±  1         25 ±  4        99 ± 16         19 ±  3

               1000         81 ± 16         6 ±  3         22 ±  5       299 ± 24         18 ±  4

               3330        145 ± 45        4 ±  1         25 ±  1 s     336 ± 34 s       17 ±  4 s

               5000         89 ± 21          3 ±  1 s       29 ±  6 m     337 ± 65 m      15 ±  2 m

 

Solvent control: 0.1 ml dimethyl sulfoxide

1    The S9-mix contained 5% (v/v) S9 fraction

s    Bacterial background lawn slightly reduced

m    Bacterial background lawn moderately reduced

Table3           Experiment 2: Mutagenic response of Reaction mass containing mainly 2-chloropropene in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

 

Dose                    Mean number of revertant colonies/3 replicate plates (± S.D.) with

(µg/plate)              different strains of Salmonella typhimurium and one Escherichia coli strain

 

                                   TA1535           TA1537           TA98            TA100           WP₂uvrA

                                               Without S9-mix

 

   positive control       872 ± 35       366 ± 81      1025 ± 14       758 ± 111       466 ± 59

    solvent control        10 ±  2         4 ±  1         16 ±  3         86 ±  9         13 ±  3

 

                 33          9 ±  3          5 ±  2         13 ±  1         87 ±  7         18 ±  4

                100         12 ±  2         4 ±  1         16 ±  2         99 ±  8         12 ±  3

                333         10 ±  3         4 ±  0         18 ±  5         90 ±  7         18 ±  4

               1000         11 ±  5         6 ±  3         14 ±  2         93 ± 11         17 ±  1

               3330         13 ±  3 s      5 ±  1 s       14 ±  3 s       77 ±  9 s       10 ±  2 s

------------------------------------------------------------------------------------------------------------

                                                With S9-mix¹

 

   positive control       189 ± 30       275 ± 27       329 ± 16       709 ± 64        212 ±  2

    solvent control        10 ±  2         7 ±  3         24 ±  3         68 ± 11        15 ±  5

 

                 33                                           25 ±  7         65 ±  5        14 ±  5

                100         13 ±  3         5 ±  3         22 ±  6         80 ± 12        14 ±  3

                333         21 ±  5         5 ±  2         26 ±  2         94 ± 15        18 ±  2

               1000         52 ± 11        4 ±  2         25 ±  6        146 ±  6       18 ±  6

               3330        125 ±  9 s     4 ±  1 s      21 ±  2 s      235 ±  7 s    16 ±  4 s

               5000        110 ± 11 m    3 ±  1 m

 

Solvent control: 0.1 ml dimethyl sulfoxide

1    The S9-mix contained 5% (v/v) S9 fraction

s    Bacterial background lawn slightly reduced

m    Bacterial background lawn moderately reduced

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation in Salmonella typhimurium
negative in Escherichia coli

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In the absence of S9-mix, all five bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, increase in the number of revertants in two independently repeated experiments.

In the presence of S9-mix, Reaction mass containing mainly 2-chloropropene induced up to 3.5- to 13-fold, dose-related increases in the number of revertant colonies compared to the solvent control in tester strain TA1535 and TA100. In the other tester strains TA1537, TA98 and WP2uvrA, Reaction mass containing mainly 2-chloropropene did not induce a dose-related, two-fold, increase in the number of revertant colonies. Since the increases in the number of revertant colonies were outside the laboratory historical control data range, in two independently repeated experiments, in two strains and were dose-related, these increases are considered to be biologically relevant and Reaction mass containing mainly 2-chloropropene is considered to be mutagenic.

Based on the results of this study it is concluded that Reaction mass containing mainly 2-chloropropene is mutagenic in the Salmonella typhimurium reverse mutation assay and is not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation.

Executive summary:

Reaction mass containing mainly 2-chloropropene was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP₂uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone) with the inclusion of a preincubation step. 

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch RBA100301A of Reaction mass containing mainly 2-chloropropene was a clear light yellow to brown liquid with a purity of minimum 60.0%. The test substance was dissolved in dimethyl sulfoxide.

 

In the dose range finding test, Reaction mass containing mainly 2-chloropropene was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in tester strain TA100. Reaction mass containing mainly 2-chloropropene did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed both in the absence and presence of S9-mix at test substance concentrations of 3330 and 5000 µg/plate. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

 

In the first mutation assay, Reaction mass containing mainly 2-chloropropene was tested up to concentrations of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98 and WP₂uvrA. Toxicity was observed in all tester strains, except in tester strain TA1535 in the presence of S9-mix.

 

In the second mutation assay, Reaction mass containing mainly 2-chloropropene was tested up to concentrations of 3330 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains, TA98, TA100 and WP₂uvrA and up to 3330 and 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix, respectively in the tester strains TA1535 and TA1537. Toxicity was observed in all tester strains.

 

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

In the absence of S9-mix, all five bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, increase in the number of revertants in two independently repeated experiments.

 

In the presence of S9-mix, Reaction mass containing mainly 2-chloropropene induced up to 3.5- to 13-fold, dose-related increases in the number of revertant colonies compared to the solvent control in tester strain TA1535 and TA100. In the other tester strains TA1537, TA98 and WP₂uvrA, Reaction mass containing mainly 2-chloropropene did not induce a dose-related, two-fold, increase in the number of revertant colonies.Since theincreases in the number of revertant colonies were observed outside the laboratory historical control data range, in two independently repeated experiments, in two strains and were dose-related, these increases are considered to be biologically relevant and Reaction mass containing mainly 2-chloropropene is considered to be mutagenic.

 

Based on the results of this study it is concluded that Reaction mass containing mainly 2-chloropropene is mutagenic in the Salmonella typhimurium reverse mutation assay and is not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation.