Reaction products of hexane-1,6-diol with 2-(chloromethyl)oxirane (1:2)

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

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

2-({[6-({1-chloro-3-[(oxiran-2-yl)methoxy]propan-2-yl}oxy)hexyl]oxy}methyl)oxirane; 2-[({6-[(oxiran-2-yl)methoxy]hexyl}oxy)methyl]oxirane; 3-chloro-2-({6-[(oxiran-2-yl)methoxy]hexyl}oxy)propan-1-ol; 6-[(oxiran-2-yl)methoxy]hexan-1-ol

EC Number:

618-939-5

Cas Number:

933999-84-9

Molecular formula:

C6H14O2 + C3H5ClO

IUPAC Name:

2-({[6-({1-chloro-3-[(oxiran-2-yl)methoxy]propan-2-yl}oxy)hexyl]oxy}methyl)oxirane; 2-[({6-[(oxiran-2-yl)methoxy]hexyl}oxy)methyl]oxirane; 3-chloro-2-({6-[(oxiran-2-yl)methoxy]hexyl}oxy)propan-1-ol; 6-[(oxiran-2-yl)methoxy]hexan-1-ol

Details on test material:

As per IUCLID5 Sections 1.1 - 1.4.

Method

Target gene:

histidine locus

Species / strain

Species / strain 1

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 2

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.