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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From July 07 to August 25, 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to internationally accepted testing procedures and GLP procedures. The OECD recommended combination of strains was tested. The test was performed on the analogous hydroxyethyl derivative (CAS 17958-73-5). Justification for Read Across is detailed in the endpoint summary and in the Category Justification Report attached to the section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
deviation had no impact on the outcome of the study; details below
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
deviation had no impact on the outcome of the study; details below
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his (trp) locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.
Metabolic activation:
with and without
Metabolic activation system:
S9 post mitochondrial fraction
Test concentrations with justification for top dose:
First experiment: 50, 150, 500, 1500 and 5000 µg per plate.
As neither precipitation nor toxicity was observed in the first experiments, the second experiments were done with the same dose-range. Experiments with metabolic activation differ from the first by different amount of S9 (30 µl – 5.7 %, 50 µl - 9.5 %, 100 µl – 19.0 %).
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Remarks:
untreated controls contain no solvent
Negative solvent / vehicle controls:
yes
Remarks:
0.1 ml of DMSO
Positive controls:
yes
Remarks:
reference mutagens
Positive control substance:
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
other: 4-nitro-o-phenylenediamine (NPD), 2-aminofluorene (2-AF), 2-aminoanthracene (2-AA), 9-aminoacridine hydrochloride monohydrate (9-AAc)
Remarks:
Without S9: AS 1.5 µg/plate TA100 and TA1535; NPD 20 µg/plate TA98; 9 AAc 100 µg/plate TA1537; ENNG 20 µg/plate E. coli. With S9: 2-AA 1.0 µg/plate TA 1535, 2.5 µg/plate TA1537, 25 µg/plate E. coli; 2-AF 10 µg/plate TA100 and TA98.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar, plate incorporation. 100 µl of test substance of required concentration, 100 µl of 16-18 h culture of tester strain, 0.5 ml relevant buffer and 30 µl of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 ml of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45 ± 3 °C. After shaking the mixture was poured into a minimal glucose agar plate.
Fresh solutions of test substance were prepared before each experiment. All concentrations of the test substance solution were dosed in the volume of 0.1 ml per plate.

PREPARATION and USING OF S9: the metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/ml, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 ml/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 °C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer. Each plate in all experiments with metabolic activation contained 0.5 ml of buffer with NADP and glucoso-6-phosphate and 30 µl S9 (the concentration of S9 in the S9mix was 5.7 %). In experiments without metabolic activation only buffer was added to the top agar.

DURATION
- Incubation period: 48 - 72 h at 37 ± 1 °C.

TOXICITY ASSAY: the starting solution (5000 µg/0.1 ml) was diluted to concentration series (10-5000 µg per plate), which was tested for toxicity in strain TA 100 without metabolic activation. No toxicity was observed in any dose.

COUNTER SYSTEM: after incubation the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000.

NUMBER OF REPLICATIONS: triplicate plating was used at each dose level except in the toxicity test with strain TA 98, where test substance was tested in duplo. Each experiment was repeated.

GENOTYPES: genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).
Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as "biologically relevant":
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no evidence of a rising trend in the number of revertants with increasing dose
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Current historical ranges of revertant numbers in bacterial strains used in the study and live bacteria count used in the first experiments

Control Spont.rev. DMSO PC -S9 PC +S9 Number of CFU/ml
S.t. TA 100 78 - 182 70 - 174 317 - 829 541 - 1573 1.06*109
S.t. TA 1535 10 - 30 9 - 29 254 - 626 69 - 257 7.87*108
S.t. TA 98 12 - 48 12 - 44 271 - 1559 523 - 2175 2.13*108
S.t. TA 1537 5 - 21 4 - 20 450 - 1398 60 - 280 7.43*108
E.c. WP2 uvrA 11 - 47 8 - 40 375 - 1095 0 - 387 9.40*108

Toxicity testing in strain TA 100

Test substance (mg per plate) Revertants per plate Rt/Rc Bacteriotoxic effect
Plates x ± sd
0 128 107 114 116 ± 9 - not evaluated
DMSO 106 113 95 105 ± 7 - not evaluated
10 97 96 NT 97 ± 1 0.9 bacterial background normal
100 110 109 NT 110 ± 1 1.0 bacterial background normal
500 106 109 NT 108 ± 2 1.0 bacterial background normal 
1000 98 101 NT 100 ± 2 1.0 brighter colonies, bacterial background normal background normal
2500 96 98 NT 97 ± 1 0.9 colonies are unlikely coloured (brighter, more solid), bacterial background normal
5000 103 93 NT 98 ± 5 0.9 colonies are unlikely coloured (brighter, more solid), background coloured yellow, bacterial background normal
AS 554 603 NT 579 ± 25 5.5 not evaluated

The results obtained in most experiments did not show substantial/biologically significant increases in the number of revertants versus solvent controls (Rt/Rc< 2), and no one of the experiments gave evidence of a rising trend in the number of revertants with increasing dose.

Moderate increase of number of revertants was observed in the first experiment in E. coli with metabolic activation Rt/Rc = 1.5 in the two highest doses. Practically no increase is observed in comparison with negative control and no such increase was observed in the second experiment. This result was not considered as biologically relevant (see criteria reported above).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance was non-mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains tested without metabolic activation.
Executive summary:

Method

The test substance was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was dissolved in dimethylsulfoxide and assayed in doses of (10) 50-5000 µg per plate, which were applied to plates in volume of 0.1 ml.

Experiments were performed without as well as with metabolic activation with rat liver and a mixture of cofactors.

Results

The test substance was non-mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains tested without metabolic activation.