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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to O.E.C.D. Testing Guideline 471 with GLP compliance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
As per IUCLID5 Sections 1.1 - 1.4.

Method

Target gene:
histidine locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Strain TA 1538 was also used.
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rrat liver S9 fraction.
Test concentrations with justification for top dose:
TA 98 and TA 100 (ug/plate): 1, 3, 10, 33, 100, 333, 1000, 5000
TA 1535, TA 1537, and TA 1538 (ug/plate): 1, 3, 10, 33, 100, 333, 1000, 5000
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
sodium azide, 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Positive control substance:
other: 2-Aminoantracene was the positive control for S9 metabolic activation.
Details on test system and experimental conditions:
The bacterial tester strains were stored frozen in nutrient broth plus 8% v/v DMSO in liquid nitrogen. Liqiud bacterial cultures were initiated from the frozen stock in 20 mL Nutrient broth in 250 mL Erlenmeyer flasks. Cultures were incubated with shaking at approximately 37 degrees C for 6 hr. The plating medium was 1.5% Vogel-Bonner minimal medium supplemented agar. The sofy overlay medium was minimal medium supplemented with 8.0 gram Difco Bacto and 10.5 mg L-histidine / liter.

The S9 liver fraction was prepared from the livers of 8 - 12 week old male Wistar rats that had been induced with 500 mg/kg Aroclor 1254 by i.p. injection. The livers were harvested and washed in ice cold 150 mM KCL and homogenized. The liver homogenated was diluted 1:3 with ice cold KCL and centrifuged in the cold at 9,000 g for 10 minutes. The resulting S9 fraction was stored frozen at approximately -70 C. The protein content of the S9 fraction was standardized to 20 to 45 mg/mL. The S9 mix was prepared by diluting the S9 fraction 3:7 with S9 cofactors and storing on ice for the study.

For treatment 0.1 mL of test substance solution or control, 0.5 mL of S9 metabolic activation mix or 0.5 mL buffer (no metabolic activation) 0.1 mL bacterial culture and 2 mL overlay soft agar was added and mixed rapidly in pre=warmed test tubes. The contents of the test tubes were then poured over and distributed on the minimal medium plates. All plating were made in triplicate. The plates were incubated for approximately 72 hr at 37 C before scoring. The bacterial colonies were counted by means of a BIOTRAN III counter (BIOTRO-NIK, D-6000 Frankfurt, F.R.G.). The counter ws intrfaced to an Apple II minicomputer for data acquision and calculation.
Evaluation criteria:
Solvent control background mutant frequcies are within the laboratory accepted range. A test substance is considered to induce a positev mutational response if in strain TA 100 an increse over the concurent control background value is at least 2-fold. In tester strains TA 1535, 1537 1538 and TA 98 the increase over the control background value must be 3-fold. Also, a dose-related increase of the mutant frequency is desirable.
Statistics:
No data.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
1,6-Hexanediol Diglycidylether (HDDGE) was not cytotoxic in a preliminary assay conducted upto a high dose level of 5.0 mg/plate. Treatment of tester strains TA 1535 and TA 100 with HDDGE resulted in dose-related increase of the mutant frequency in both the presence and absence of rat liver derived S9 metabolic activation perperation. Treatment with HDDGE also induced a dose-related increase of the mutant frequency in strain TA 1538. without S9 metabolic activation. In strain ta 1535 at 5.0 mg/plate the mutant frequency was 26-fold the concurrent control value with S9 metabolic activation and 15-fold without S9 metabolic activation. In tester strain TA 100 the mutant frequency was 12-fold the vehicle control value at 1.0 mg/plate with S9 metabolic avtivation and 9-fold the control background value without S9 metabolic avtivation.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Test Substance: Grilonit RV 1812

S9 mix from: rat liver (Batch R 270886)

Without S9 mix

Revertants/plate

Mean from three plates

Dose

ug/plate

TA 1535

TA 1537

TA 1538

TA 98

TA 100

0.0

10.0

33.3

100.0

333.3

1000.0

5000.0

30

40

45

64

108

210

460

14

12

13

20

18

18

28

33

31

44

45

61

84

87

33

50

54

41

57

14

0

129

200

225

497

922

1176

722

Positive controls

Sodium azide (10 ug/plate)

4-nitro-o-phenylene-diamine (50 ug/plate)

584

 

 

257

 

 

1735

 

 

1318

975

With S9 mix

Revertants/plate

Mean from three plates

Dose

ug/plate

TA 1535

TA 1537

TA 1538

TA 98

TA 100

0.0

10.0

33.3

100.0

333.3

1000.0

5000.0

36

39

40

96

260

424

942

24

17

20

28

23

23

25

35

38

33

39

40

48

50

54

50

51

38

47

95

36

142

157

193

233

701

1737

1031

Positive controls

2-amino-anthracene (10ug/plate)

451

464

1283

1370

859

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

It can be stated that during the described mutagenicity test and under the experimental conditions reported, 1,6-Hexanediol Diglycidylether (HDDGE) did induce point mutations by base pair changes (or frameshifts in strain TA 1538) in the genome of the strains used. Therefore, HDDGE is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

1,6-Hexanediol Diglycidylether (HDDGE) was evaluated for mutagenic potential in an O.E.C.D. bacterial mutation 471 Testing Guideline study with GLP compliance. Dose-related increases of the mutant frequency were observed in tester strains TA 1535, TA 1538 and TA 100. HDDGE was mutagenic to strains TA 1535 and TA 100 with and without rat liver derived S9 metabolic activation preperation. Therefore, under the experimental conditions reported, 1,6-Hexanediol Diglycidylether did induce point mutations by base pair changes (or frameshifts in strain TA 1538) in the genome of the strains used and HDDGE is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.