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Toxicological information

Genetic toxicity: in vitro

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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:
11 July 1989 to 25 August 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to a well reported method equivalent to a standard test guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Source: Shell Nederland Raffinaderij BV Rotterdam
Prepared as solution in DMSO
Batch No: 1986; 88009
Toxicology Ref No. ST88/2523
Date received: 18 Oct 1988
Appearance: clear colourless liquid
Purity: cis isomer 94.51-97.51%; trans isomer 1.5%; 1.2-dichloropropane 0.25%; 2.3 dichloropropane 0.01%, Water 0.03%; edenol D81 1%
Density: 1.224 g/mnL at 15./2 dec C
Storage: dark under nitrogen
Stability: no differences between 21 Nov 88 and 25 Sept 1989; stable during duration of study

Method

Target gene:
NDA
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver microsomes (S9 fraction)
Test concentrations with justification for top dose:
Test substance:
100 µl volumes of solutions of cis-l,3-dichloropropene in DMSO (2.3, 4.7,9.4, 18.8, 37.5, 75, 150 or 300 mg/ml) were added to the pre-incubation
mix to give final concentrations of 78.1, 156.3, 312.5, 625, 1250, 2500, 5000, or 10000 µg per ml, both in the presence and in the absence of rat liverS9 fraction. Subsequent assays with glutathione were conducted by adding 1250 µg/ml cis-1,3-dichloropropene (100 µl of 38.75 mg/ml) or solvent
(100 µl) to an overnight bacterial culture (0.5 ml) and either phosphate buffer pH 7.4 (2.4 ml) or 59 fraction (2.4 ml). After the addition of glutathione
(100 µl of 2.98, 5.95, 11.91, 23.82, 47.63,95.26 or 190.52 mg/ml to give final concentrations of 0.3125, 0.625, 1.25, 2.5, 5, 10 or 20 mM
respectively),

Positive controls:
E. coli -S9 Potassium dichromate (10 ug) ; +S9 Benzo(a)pyrene (5ug)
TA 1537 -S9 9-aminoacridine (12.5 ug); +S9 Neutral red (10ug)
TA 1535 -S9 sodium azide (1 ug); +S9 2-aminoanthracene (2.5 ug)
TA 100 -S9 sodium azide (1 ug) +S9 benzo(a)pyrene (5ug)
TA1538 -S9 2-nitrofluorene (2.5 ug); +S9 benzo(a)pyrene (5ug)
TA 98 -S9 2-nitrofluorene (2.5 ug); +S9 benzo(a)pyrene (5ug)
Vehicle / solvent:
dimethylsulphoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see compounds listed above
Details on test system and experimental conditions:
S9 fraction used in this study from male Fischer 344 rats pretreated with aroclor 1254 (prepared 3 June.1989). Preliminary cytotoxicity assay first carried out to assess the cytotoxicity of the test material, its solubility in the top agar and for any effect on the pH of the system; concentrations ranged up to 10000 ug per mL. After initial assays with each strain the top dose was reduced to 2500 ug/mL.
In each of the bacterial mutation assays control plates were set up with the solvent alone and with an appropriate known positive control compound. All tests were carried out in triplicate. Two replicate assays were carried out on different days in order to confirm reproducibility. Because of the volatile nature of the test material, assays were performed by the pre incubation method in sealed containers.
Evaluation criteria:
Reproducible dose-related increases or values of 2.5x control vlaues or greater are considered to indicate a mutagenic response.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Test material was miscible in the aqueous test system at all amounts testsed. The addition of 2400 ug/mL caused the pH to change from 7.37 to 7.42. Cytotoxicity was evident at the highest amounts 2500 ug/mL and above in the presence and absence of S9. The test material increased reverse gene mutation in both the presence and absence of the liver S9 activation in culutures of E coli WP2 uvrA pkm101, and S. typhimurium TA1535, TA100, and a lesser extent TA98. No increase was observed with S. Typhimurium TA1537 or /ta1538. A dose of 1250 ug/mL test material showed thegreatest increase of control cultures and was used to incorporate glutathion into the test; glutathione reduced mutagenicity of test material in presence of S9 and to a lesser extent without S9.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

The addition of cis-1,3-dichloropropene to cultures of Escherichia coli WP2 uvrA pkm101, Salmonella typhimurium TA 1535 and TA 100 showed reproducible, dose-related increases in reverse gene mutation in the presence and in the absence of rat liver S9 fraction. Smaller dose-related increases were also observed in S, typhimurium TA98 both in the presence and in the absence of rat liver S9 fraction. No increases were seen in S. typhimurium TA1537 or TA1538. The substance is considered to be potentially mutagenic under the conditions of the test.
Executive summary:

A GLP -compliant study has been conducted to a test method similar to OECD Guideline 471.The mutagenic activity of cis-1,3 -dichloropropene was investigated in agar layer cultures of selected bacterial tester strains of Salmonella typhimurium and Escherichia coli. Assays were performed in both the presence and absence of S9 microsomal fraction obtained from a liver homogenate of rat pretreated with Aroclor 1254. It was concluded that cis-1,3 -dichloropropene was direct-acting bacterial mutagen under the experimental conditions employed. A mutgenic dose of the test material was tested in the presence of a range of conentrations of glutathione. Glutathione reduced the mutagenicity of the test material in the presence and absence of S9 fraction.