Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential of the target substance t-butyl-glycidyl ether to induce genotoxic effects was assessed in a weight-of-evidence approach by using data from several in vitro and in vivo studies conducted with the target substance as well as with the source substance n-butyl glycidyl ether. The target substance was tested positive in several Ames tests as reported by Dabney, 1976 and Canter et al., 1986. Positive results were also obtained from an E. coli SOS Chromotest.

In one in vitro sister chromatid assay (von der Hude, 1991) and in two in vitro Unscheduled DNA Synthesis test the target substance was tested positive for genotoxic effects.

Data from the source substance n-butyl glycidyl ether were used to assess the mutagenicity. In the publication by Thompson et al., 1981 the results from several genotoxicity tests (Ames, Mouse Lymphoma Assay, UDS) conducted with n-butyl glycidyl ether were presented. The UDS was negative, but the Ames and the Mouse Lymphoma assay showed a positive potential to induce mutagenicity.

Furthermore, n-butyl glycidyl ether is classified as Muta. 2 (H314) in the harmonized classification according to Annex VI of Regulation (EC) No 1272/2008.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- Short description of test conditions:
Chemicals were tested in Salmonella strains TA98, TA100, TA1535, and TA1537 and/or TA97 without metabolic activation and with liver S9 preparations from Aroclor 1254-induced male, Sprague-Dawley rats and Syrian hamsters, in a liquid incubation protocol with the tubes covered to retard loss of the volatile chemicals. The test doses used were determined by the solubility or toxicity of the individual chemicals but did not exceed 10 mg/plate. Testing was performed at 5 doses, using triplicate plates. Tests were repeated at least once; a chemical was not designated positive or negative unless the results were reproducible. A positive response was defined as a reproducible, dose-related increase in his + revertants over the solvent control level; it was not necessary for the increase to equal 2-fold over background. A chemical was considered mutagenic if at least one strain/activation combination yielded a reproducible positive response.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- CAS No.: 7665-72-7
- Supplier: Howard Hall International
Target gene:
Histidine locus
Species / strain / cell type:
other: TA97, TA98, TA100, TA1535 and TA1537
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 metabolic activation system
Test concentrations with justification for top dose:
- The test doses used were determined by the solubility or toxicity of the individual chemicals, but did not exceed 10 mg/plate.
Vehicle / solvent:
Not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
Not specified
Rationale for test conditions:
Not specified
Evaluation criteria:
- A positive response was defined as a reproducible, dose-related increase in his + revertants over the solvent control level; it was not necessary for the increase to equal 2-fold over background.
Statistics:
Fold increase was measured.
Key result
Species / strain:
other: TA97, TA98 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
other: TA1535 and TA100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
In this study, the mutagenicity of the test substance was tested in Salmonella strains TA98, TA100, TA1535, TA1537 and TA97 with and without S9 metabolic activation system. The test doses used were determined by the solubility or toxicity of the individual chemicals, but did not exceed 10 mg/plate. The test substance was found to be active in TA100 and TA1535 with and without activation and showed varying responses in other strains.
Executive summary:

In this study, the mutagenicity of the test substance was tested in Salmonella strains TA98, TA100, TA1535, TA1537 and TA97 with and without S9 metabolic activation system. The test doses used were determined by the solubility or toxicity of the individual chemicals, but did not exceed 10 mg/plate.The test substance was found to be active in TA100 and TA1535 with and without activation and showed varying responses in other strains.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1979-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
DNA repair in human lymphocytes (UDS).
GLP compliance:
not specified
Type of assay:
other: Induction of DNA repair
Specific details on test material used for the study:
Name: tertiary-butyl glycidyl ether
CAS No.: 7665-72-7
Lot No.: 21311-34GWS
Supplier: Shell
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: human peripheral blood lymphocytes were collected from 2 donors
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle's MEM
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
The test will be performed with various concentrations ranging up to the level at which 50% of cultured lymphocytes are found to be non-viable after 4.5 hours exposure to test item. The dosages will range between 100% cell viability and the toxic dose determined on cultured cells following an in vitro exposure of 4.5 hours.

The following doses were used:
10, 100, 1000 µg/mL (Donor A)
12.4, 37, 111, 333, 1000, 3000 mg/mL (Donor B)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
Saline
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1-Methyl-3-nitro-1-nitrosoguanidine
Untreated negative controls:
yes
Remarks:
Saline
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: n-Butyl glycidyl ether
Details on test system and experimental conditions:
The ability of the test item to induce excision repair in isolated human peripheral blood lymphocytes will be examined in short term in vitro assays. These assays will be performed with various concentrations of the test item ranging up to the level at which 50% of cultured lymphocytes are found to be non-viable after 4.5 hours exposure to test item under conditions to be employed in the repair induction assays. Control compounds will also be examined at concentrations extending to the TCD50. All viability assays will be determined by trypan blue exclusion in both initial determination of TCD50 and repair induction tests after 4.5 hours exposure.
Approximately 50 mL whole blood was taken from each healthy human donor employed in this study. The resulting heparinized whole blood was diluted to 80 mL with Eagles Minimal Medium (MEM) and distributed into two 50 mL sterile plastic centrifuge tubes, and underlayered with 10 mL Ficoll-Hypaque (density 1.077) per tube. After centrifugation at 400 x g for 40 min the mononuclear cell layers were removed, diluted into arginine-free MEM supplemented with 4% (v/v) heat-inactivated fetal calf serum and pelleted at 400 x g for 10 min. This wash procedure was repeated twice to minimize platelet contamination and to effectively deplete exogenous arginine from the cell suspension. The cells were resuspended at a final density of 2.0-3.0 x10^6 cells/mL and were incubated overnight in 50 µL polypropylene centrifuge tubes. The cells were then resuspended at the same cell density in fresh complete arginine-free medium and distributed as 1.4 mL aliquots in sterile, siliconized 12 x 75 mm borosilicate glass tubes. Into each culture tube was pipetted a standard 5.0 µL of agent diluted in DMSO, followed by immediate mixing on a vortex mixer. Each 1.4 mL treated culture was immediately redistributed into identical sterile siliconized culture tubes:
3 - 0.4 mL cultures for triplicate scintillation counting
1 - 0.1 mL culture for slide autoradiography
1 - 0.1 mL culture for viability assessment.
The triplicate cultures for scintillation counting were incubated at a final concentration of 5 µCi/mL 3H-thymidine (40-60 Ci/mM, NET-027 New England Nuclear); the culture for slide autoradiography were incubated at 10 µCi/mL. All cultures were incubated in 5% CO2 in air at 37 °C. At 4.5 hours viabilities of each sample series were determined; 20 µL trypan blue (0.4% in saline) was added to the 0.1 mL viability culture. After incubation for 10 min, each viability culture was vortexed and an aliquot leaded onto a hemacytometer for enumeration of trypan blue-stained and unstained cells. At the end of 5.0 hours, all remaining culture tubes were removed and chilled on ice. The triplicate cultures for scintillation counting were harvested on glass fiber filters (Reeve Angel 943AH) on a manifold harvester, and the filters were washed extensively in saline and distilled water. The filters were placed in 3.5 mL PCS (Amersham-Searle, diluted 0.5 volume xylol) in glass mini-vials, and were counted in a Beckman LS-8000 scintillation counter. The cultures for slide autoradiography were added 3.0 mL 0.075 MMK1 prior to centrifugation (400 x g, 15 min) aspiration of the supernatant. The cells were then resuspended in residual 0.75 M KCl by vortex and were diluted by addition of 3mL Carnoy’s solution (methanol and acetic acid, 4: 1); tubes were then refrigerated for 4-6 hours. The fixed cells were then pelleted by centrifugation and the supernatant was aspirated, after which the cells were resuspended in residual Carnoy's (approximately 50 µL). These suspensions were then pipetted onto clean glass slides and allowed to air dry. The slides were then cleaned of unincorporated 3H-thymidine by an acetone wash, followed by two washes in each distilled water in 5% trichloroacetic acid, distilled water. After air drying, the slides were dipped in Kodak NTB-2 emulsion diluted 1:1 with distilled water. In total darkness, the slides were placed into desiccated slide boxes and refrigerated at 4 °C in total darkness for 10 days. After developing in Kodak D-19 the slides were stained in 10% Giemsa for 2 min, rinsed in distilled water, air dried and mounted.
Rationale for test conditions:
The dilution scheme was designed to minimise the DMSO concentrations to which cells are exposed. The maximum DMSO concentration of 0.357% corresponds to the DMSO control dose.
Evaluation criteria:
not specified
Statistics:
Mean grain counts calculated using only cells with at least one nuclear grain.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
not applicable
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Table 1 in box "Any other information on results incl. tables" shows the results of assays for the induction of DNA repair, both by scintillation counting and by slide autoradiography. It may be noted that DNA repair induction occurs optimally at 333 µg/mL with the test compound. A dose-response relationship for both n- and t-BGE is apparent over the range of dose levels from 0 to 333 µg/mL. The viability assay employed may indicate little more than membrane integrity. Among the effects of these compounds on cellular metabolism at dose levels higher than 333 µg/mL appears to be an inhibitory of DNA repair synthesis. Any specifity for interference DNA repair function has not been examined, and may simply be a manifestation of overall cellular cytotoxicity.

Table 1: DNA Repair Induction

Scintillation Autoradiography
Agent [µg/mL] % Viability CPM± SD % Cells with grains Grains per cell grains per labelled cell©
Donor A ≥2 ≥3 ≥6
tBGE 10.0 - 603 ± 25 22.8 8.9 0.7 0.84 1.9
100.0 88.4 720 ± 46 49.3 28.1 2.1 1.8 2.5
1000.0 90.0 731 ± 32 79.1 71.1 45.8 5.7 6.7
nBGE 10.0 85.9 586 ± 34 44.0 24.4 3.0 1.6 2.4
100.0 86.6 780 ± 24 65.9 52.8 13.8 2.9 3.7
500.0 85.5 814 ± 43 80.4 75.2 53.5 6.9 7.9
1000.0 72.1 429 ± 17 70.5 62.1 33.5 4.8 6.0
MNNG 0.36 91.2 913 ± 40 80.0 69.0 39.2 5.3 6.1
Saline (3.5 µL/mL) 91.2 552 ± 11 33.7 16.9 2.3 1.2 2.2
DMSO(b) (3.5 µL/mL) 93.4 546 ± 80 25.8 10.7 1.2 0.98 2.0
Machine background - 34 ± 2 - - - -
Donor B tBGE 12.4 94.1 709 ± 35 20.8 7.8 0.6 0.77 1.31
37.0 93.7 761 ± 34 33.1 13.8 3.1 1.19 2.21
111.0 92.5 957 ± 30 46.6 23.9 4.0 1.66 2.44
333.0 95.2 991 ± 80 81.6 71.8 39.4 4.78 5.42
1000.0 89.4 742 ± 31 70.7 55.7 25.1 3.89 4.86
3000(a) 72.2 257 ± 15 38.9 23.1 7.2 1.58 3.01
nBGE 4.1 - 709 ± 48 25.5 10.1 0.4 0.84 1.93
12.4 90.7 739 ± 50 29.2 15.4 1.0 1.05 2.16
37.0 95.0 867 ± 84 37.4 19.2 2.7 1.35 2.29
111.0 91.7 910 ± 44 56.8 38.6 8.7 2.27 3.17
333.0 86.0 1000 ± 95 81.3 67.9 27.3 4.25 4.89
1000.0 68.1 354 ± 15 53.3 39.2 18.5 3.06 4.53
MNNG 0.36 93.9 1144 ± 83 67.2 56.1 29.7 4.06 5.28
Saline (3.5 µL/mL) 93.2 654 ± 36 8.87 3.35 0.24 0.39 1.58
DMSO (3.5 µL/mL) 92.1 796 ± 18 17.6 7.3 0.75 0.64 1.92

(a): 4.6 µL tBGE added directly to culture (no DMSO)

(b): Dilution scheme employed in these assays was designed to minimise the DMSO concentrations to which cells are exposed. The maximum DMSO concentration of 0.357% corresponds to the DMSO control dose

(c): Meangrain counts calculated using only cells with at least one nuclear grain

Conclusions:
In this DNA repair induction study, tertiary-butyl glycidyl ether demonstrates genetic activity in human lymphocytes.
Executive summary:

In an in vitro DNA repair study, the DNA repair induction in human lymphocytes was tested with 1-tert-Butoxy-2-,3-expoxypropane at doses of 10.0, 100.0 and 1000.0 µg/mL (Donor 1) as well as 12.4, 37,0, 111.0, 333.0, 1000.0 and 3000.0 µg/mL (Donor 2). DNA repair induction occured optimally at 333 µg/mL with the test item in a dose-response relationship from 0 to 333 µg/mL. The viability assay employed may indicate little more than membrane integrity. Among the effects of these compounds on cellular metabolism at dose levels higher than 333 µg/mL appears to be an inhibitory effect on DNA repair synthesis. Any specificity for interference DNA repair function has not been examined, and may simply be a manifestation of overall cellular cytotoxicity.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1979-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- Principle of test: The test was conducted after the method described by Ames et al.
- Short description of test conditions:
- The test item was tested directly against strains TA1535, TA1537, TA1538, TA100, and TA98 of Salmonella typhimurium
- S-9 liver homogenate fraction, prepared from phenobarbital-induced or Aroclor-induced rats were employed in the liver homogenate mixes
- Petri plates containing 20 mL minimal glucose agar medium supplemented with 0.5 µg/mL biotin and 4 µg/mL histidine were used for the test . Two mL of molten top agar were mixed with 0.1 mL of nutrient broth cultures of the tester strain that had been grown overnight. This was then mixed with the test compound.
- The petri plates were incubated at 37 °C for 48 hours. Results were expressed as the mean colony forming units (CFU) of two plates.
- All strains were initially assayed with t-BGE at 2 µmoles/plate. Following the results of the initial assay, additional microbial assays of concentrations 0.5, 1.0, 2.0 and 4.0 µmoles/plate of the test item were used with and without metabolic activation to determine possible dose-responses and toxicity.
- Parameters analysed / observed: mean colony forming units (CFU)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name: tertiary-butyl glycidyl ether
- CAS No.: 7665-72-7
- Lot No.: 21311-34GWS
- Supplier: Shell
Target gene:
Histidine locus
Species / strain / cell type:
other: S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538
Details on mammalian cell type (if applicable):
Medium Used:
Spizzizens minimal medium supplemented with biotin and histidine, nutirent broth, bacto-agar, standard methods agar
The tests were carried out according to the procedure of Ames et al.
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate fraction
Test concentrations with justification for top dose:
2 µM/plate or all strains
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Hycanthone. 5 µg/plate TA1538, TA98, without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: MMNG, 5 µg/plate. TA1535, without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Epichlorohydrin
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The method described by Ames et al. were followed:
- Petri plates containing 20 mL minimal glucose agar medium (Spizzizen minimal medium) supplemented with 0.5 µg/mL biotin and 4 µg/mL histidine were used for the test. Two mL of molten top agar were mixed with 0.1 mL of nutrient broth cultures of the tester strain that had been grown overnight. This was then mixed with the test compound. The petri plates were incubated at 37 °C for 48 hours. Results were expressed as the mean colony forming units (CFU) of two plates.

Evaluation criteria:
A compound was considered mutagenic in this assay if :
(1) control values of spontaneous revertants were within normal limits
(2) all sterility controls were negative
(3) positive controls gave acceptable results (a minimum of a doubling over the controls was demonstrated)
(4) a dose-response can be determined.
Statistics:
n.a.
Key result
Species / strain:
other: S. typhimurium TA1535, TA100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
other: TA98, TA100 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
T-BGE reverted the base-pair substitution type strains (TA1535 and TA100), but produced no response in the frame-shift type strains (TA1537, TA1538 and TA98).

Table 1: In vitro mutagenicity of test item using Salmonella typhimurium TA1535

Compound

µmoles / plate

No additions

Aroclor-induced Liver

Phenoarbital-induced Liver

 

 

CFU/plate

Rt/Rc

CFU/plate

Rt/Rc

CFU/plate

Rt/Rc

t-BGE

0.5

49

1.5

41

1.8

37

1.4

 

1.0

63

1.9

57

2.5

56

2.2

 

2.0

89

2.7

106

4.6

143

5.5

 

4.0

150

4.6

204

8.9

251

9.7

Cyclophosphamide

100 µg / plate

46

1.4

174

7.5

426

16.4

MNNG

5 µg / plate

~3000

 

~3000

 

18

 

Conclusions:
In conclusion, in an bacterial reverse gene mutation assay, the target substance t-BGE reverted the base-pair substitution type strains (TA1535 and TA100), but produced no response in the frame-shift type strains (TA1537, TA1538 and TA98).
Executive summary:

In conclusion, in an bacterial reverse gene mutation assay, the target substance t-BGE reverted the base-pair substitution type strains (TA1535 and TA100), but produced no response in the frame-shift type strains (TA1537, TA1538 and TA98).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1979-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Short description of test conditions:
Groups of 10 B6D2F1 female mice were given an oral dose of t-BGE in corn oil once a day for 4 days (0, 100, 200, and 400 mg/kg/day). Following the fourth treatment, each group of mice was placed in a separate metabolism cage, and urine was collected in a chilled container for 24 hours. The urine was tested for mutagenicity properties directly against TA1535 and TA98 with and without the addition of B-glucuronidase.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Name: tertiary-butyl glycidyl ether
CAS No. : 7665-72-7
Lot No. : 21311-34GWS
Supplier: Shell
Target gene:
Histidine locus
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
beta-glucuronidase
Test concentrations with justification for top dose:
0, 100, 200, 400 mg/kg/day; all strains in the microbial assay portion were initially assayed at 2.0 µmoles/plate since n-BGE (a similar substance) produced a mutagenic response at 2.0 µmoles/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: corn oil
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
corn oil
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: hycanthone, 50 mg/kg bw; Cytoxan, 50 mg/kg bw
Details on test system and experimental conditions:
METHOD OF APPLICATION: Groups of ten B6D2F1 female mice were given oral doses of t-BGE in corn oil once a day for four days. The mice were housed 5/cage in polypropylene cages with stainless steel wire-bar containment lids. Following treatment, the group of mice was placed in a metabolism cage, and urine was collected in a chilled container for 24 hours. Samples from the different dosage groups were frozen at -20 °C until all samples were collected. Frozen samples were then allowed to thaw at room temperature and were filtered into sterile glass tubes. 0.2 ml of urine was then tested directly against TA1535 and TA98 with and without the addition of 200 units/plate beta-glucuronidase.
- Petri plates containing 20 mL minimal glucose agar medium supplemented with 0.5 µg/mL biotin and 4 µg/mL histidine were used for the test. 2 mL of molten top agar were mixed with 0.1 mL of nutrient broth cultures of the tester strain that had been grown overnight. This was then mixed with the test compound. The petri plates were incubated at 37 °C for 48 hours. Results were expressed as the mean colony forming units of 2 plates.

DURATION
- Exposure duration: 37 °C for 48 hours





Evaluation criteria:
- A compound was considered mutagenic in this assay if:
1) control values of spontaneous revertants were within normal limits
2) all sterility controls were negative
3) positive controls gave acceptable results (a minimum of a doubling over the controls was demonstrated)
4) a dose-response can be determined
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
For detailed results please refer to table 1 in box "Any other information on results incl. tables".
Remarks on result:
other:
Remarks:
increase in mutagenicity is dose-dependent

Table 1: Mutagenic assay of the urine of mice treated with t-BGE using Salmonella typhimurium TA1535 and TA98

 Dose (mg/kg/day x 4)  beta-glucuronidase (200 units/plate)  TA1535 (CFU/plate)  TA98 (CFU/plate)
 - (negative control)  -  31  27
   +  31  28
 corn oil (0.01 ml/g; 0.2 ml sterile urine added/plate)  -  39  37
   +  45  36
 100 t-BGE  -  39  34
   +  75  34
 200 t-BGE  -  42  33
   +  132  41
 400 t-BGE  -  45  39
   +  126  32
 Cyclophosphamide (50 mg/kg/day x 2 in 5 mice)  -  580  n.t.
   +  460  n.t.
 Hycanthone (50 mg/kg/day x 2 in 5 mice)  -  n.t.  49
   +  n.t.  425
Conclusions:
In a body fluid analysis using a reverse bacterial mutation assay, Salmonella strains TA1535 and TA98 were tested directly with urine with and without the presence of beta-glucurondiase. The urine came from mice dosed with 0, 100, 200, and 400 mg/kg bw/day t-BGE on four consecutive days. The mean colony forming units were counted. A somewhat dose-dependent mutagenicity was seen in strain TA1535 with the addition of beta-glucuronidase; no mutagenicity was seen in TA98 with or without beta-glucuronidase.
Executive summary:

In a body fluid analysis using a reverse bacterial mutation assay, Salmonella strains TA1535 and TA98 were tested directly with urine with and without the presence of beta-glucurondiase. The urine came from a group of ten mice dosed with 0, 100, 200, and 400 mg/kg bw/day t-BGE on four consecutive days. The mean colony forming units were counted. A somewhat dose-dependent mutagenicity was seen in strain TA1535 in the presence of beta-glucuronidase. No mutagenicity was seen in tester strain TA98 with or without beta-glucuronidase.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
not specified
Type of assay:
other: Unscheduled DNA synthesis
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Dow Chemical Company supplied t-BGE, lot 21311-34GWS
Species / strain / cell type:
lymphocytes: normal human peripheral blood lymphocytes (HPBL)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: blood donors
- Sex, age and number of blood donors if applicable: two normal healthy women, 20 and 21 years of age, who were taking no medications.
- Whether whole blood or separated lymphocytes were used if applicable: approx. 50 ml whole blood was collected into an heparinized syringe. This volume of heparinized whole blood was diluted to 80 ml with Eagle's Minimal Medium (MEM, Grand Island Biologicals, NY) and a modification of the procedure of Boyum (1968) was used for the isolation of mononuclear leukocytes. After centrifugation at 400 × g for 40 min the mononuclear cell layer was removed, diluted into arginine-free MEM supplemented with 4% (v/v) heat-inactivated fetal calf serum (Microbiological Associates, Bethesda, MD), and the cells were pelleted again. This wash procedure was repeated twice to minimize platelet contamination and to effectively deplete exogenous arginine from the cell suspension.
- Methods for maintenance in cell culture if applicable:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle's Minimal Medmm (MEM, Grand Island Blologicals, NY), arginine-free MEM supplemented with 4% (v/v) heat-inactivated fetal calf serum (Microbiological Associates, Bethesda, MD), and complete arginine-free medium.
Test concentrations with justification for top dose:
Donor A: 10, 100, and 1000 ug/ml.
Donor B: 12.4, 37, 111, 333, 1000, and 3000 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
saline
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: MNNG
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): The HPBL were resuspended in the same medium at a final density of 2.0-3.0 × 10E6 cells/ml and were incubated overnight m 50-ml polypropylene centrifuge tubes at 37°C in 5% CO 2 in air. The HPBL were then centrifuged and resuspended at the same cell density m fresh, complete arginine-free medium and distributed as 1.4-ml aliquots m sterile, siliconized 12 X 75 mm borosilicate glass tubes.

DURATION
- Exposure duration: 5 hours

NUMBER OF REPLICATIONS: triplicate cultures for liquid scintillation counting (LSC), one for slide autoradiography, one for viability assessment.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 3 ml 0.075 M KC1 were added to each culture for shde autoradlography. The cultures were then centrifuged (400 × g, 10 min) and the supernatant was aspirated. The cells were then resuspended in residual 0.075 M KCI by vortexing and were diluted by addition of 3.0 ml Carnoy's solution (methanol and acetic acid, 4: 1). Tubes were then refrigerated for 4-6 h. The fixed cells were then pelleted by centrifugation and the supernatant was aspirated. The cells, resuspended in residual Carnoy's (approx. 50 ul), were then pipetted onto slides and allowed to air dry. The slides were then cleaned of unincorporated [3H]thymidlne by an acetone wash, followed by 2 washes in each: distilled water, 5% trichloroacetic acid (TCA), distilled water. After air-drying, the slides were dipped in Kodak NTB-2 emulsion diluted 1:2 with distilled water. In total darkness, the slides were placed into dessicated slide boxes and refrigerated at 4°C for 10 days. The slides were developed in Kodak D-19, stained in 10% Giemsa for 2 min, rinsed in distilled water, air-dried, and mounted. The slides were scored for cells exhibiting grains over nuclei, and the grain count of each nucleus was recorded. A minimum of 400 cells were scored at each dose level of chemical agent.

DETERMINATION OF CYTOTOXICITY
- Method: cell viability, determined by trypan blue exclusion.

OTHER EXAMINATIONS:
-Assessed induction of DNA repair, both by LSC and by slide autoradiography.

OTHER:
Methods for LSC and viability assessment - The triplicate cultures for LSC were incubated at a final concentration of 5 uCi/ml [3H]thymidine (40-60 CI/mM, NET-027Z, New England Nuclear); the cultures for slide autoradiography received 10 uCl/ml [3H]thymidine. All cultures were incubated at 37°C in 5% CO 2 in air. After 4.5 h of incubation, the viabilities of each sample series were determined. 20 ul trypan blue (0.4% in saline) was added to each 0.1-ml viability culture. After incubation for 5 min, each viability culture was vortexed and an aliquot loaded onto an hemacytometer for enumeration of trypan blue-stained and unstained cells. At the end of 5 h, all remaining culture tubes were removed and chilled on ice. The triplicate cultures for LSC were harvested on glass fiber filters (Reeve Angel 934AH) on a manifold harvester, and the filters were washed extensively in saline and distilled water. The filters were placed in 3.5-ml liquid scintillation cocktail (PCS, Amersham-Searle, diluted with 0.5 vol. xylol) m glass minivials, and were counted in a Beckman LS-8000 scintillation counter.
Rationale for test conditions:
not specified
Evaluation criteria:
not specifed
Statistics:
The Chi-square test was performed on data obtained from slide autoradlography in order to establish statistical significance levels. Additionally, mean grain count was calculated.
Species / strain:
lymphocytes: normal human peripheral blood lymphocytes (HPBL)
Metabolic activation:
not specified
Genotoxicity:
positive
Remarks:
In cells from donor A, elevated repair activity occurred in (Tert-butoxymethyl) oxirane treated cultures at both 100 and 1000 ug/ml doses. In cells from donor B, DNA-repair induction occurs optimally at 333 ug/ml.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Exposure to increasing doses of (Tert-butoxymethyl) oxirane led to a reduction in cell viability in a dose-response manner.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
A dose-related increase in percentage of nuclei with elevated gram counts was observed over the range of (Tert-butoxymethyl) oxirane concentration from 0 to 333 ug/ml.
Conclusions:
An unscheduled DNA synthesis assay was performed using normal human peripheral blood lymphocytes to determine DNA repair induction (via liquid scintillation counting (LSC), and slide autoradiography) and cell viability after a 5-hour exposure to different concentrations of (Tert-butoxymethyl) oxirane. Negative controls included saline and DMSO (vehicle), and MNNG was used a positive control. Exposure to increasing doses of (Tert-butoxymethyl) oxirane led to a reduction in cell viability in a dose-response manner. In cells from donor A, elevated repair activity occurred in (Tert-butoxymethyl) oxirane treated cultures at both 100 and 1000 µg/ml doses. In cells from donor B, DNA-repair induction occurs optimally at 333 µg/ml. A dose-related increase in percentage of nuclei with elevated gram counts was observed over the range of (Tert-butoxymethyl) oxirane concentration from 0 to 333 µg/ml. Under the described test conditions, (Tert-butoxymethyl) oxirane was seen to have genotoxic and cytotoxic effects.
Executive summary:

An unscheduled DNA synthesis assay was performed using normal human peripheral blood lymphocytes to determine DNA repair induction (via liquid scintillation counting (LSC), and slide autoradiography) and cell viability after a 5-hour exposure to different concentrations of (Tert-butoxymethyl) oxirane. Negative controls included saline and DMSO (vehicle), and MNNG was used a positive control. Exposure to increasing doses of (Tert-butoxymethyl) oxirane led to a reduction in cell viability in a dose-response manner. In cells from donor A, elevated repair activity occurred in (Tert-butoxymethyl) oxirane treated cultures at both 100 and 1000 µg/ml doses. In cells from donor B, DNA-repair induction occurs optimally at 333 µg/ml. A dose-related increase in percentage of nuclei with elevated gram counts was observed over the range of (Tert-butoxymethyl) oxirane concentration from 0 to 333 µg/ml. Under the described test conditions, (Tert-butoxymethyl) oxirane was seen to have genotoxic and cytotoxic effects.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: SOS-Chromotest using E. coli PQ37 as tester strain and the bacterial reverse mutation assay conducted according to Ames.
- Short description of test conditions:
a) SOS-Chromotest was performed with and without S9 mix (10%) S9 fraction. Test was carried out in 2 series of glass tubes containing 10 µl of dissolved test substance and 250 µl of bacterial suspension. After 2 hour incubation, b-galactosidase (b-GAL) and alkaline phosphatase (AP) were measured. b-GAL assay terminated 30 min after addition of o-nitrophenyl-b-galactopyranoside. AP assay was terminated 10 min after p-nitrophenyl phosphte had been added.
b) Salmonella/microsome test: the test was performed according to Ames et al., 1975.
GLP compliance:
no
Type of assay:
other: Ames test and SOS-Chromotest
Specific details on test material used for the study:
- Name of test material: 2,3-Epoxypropyl-tert-butylether
- Source: Aldrich
- Purity: 99%
- CAS number: 7665-72-7


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: diluted in half-log intervals and each dose was tested in triplicate.
Species / strain / cell type:
other: PQ37
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: M. Hofnung (Paris, France)
Metabolic activation:
with and without
Metabolic activation system:
10% S9 fraction
Test concentrations with justification for top dose:
not specified
Vehicle / solvent:
not specified
Details on test system and experimental conditions:
SALMONELLA/MICROSOME TEST
Performed according to Ames et al. (1975). as a plate incorporation assay with and without S9 mix (4% S9 fraction from Aroclor 1254-pretreated male Wistar rats, 180-200 g, in the mix). All chemicals were generally tested without an external metabolization system up to concentrations giving a cytotoxic effect (indicated by a clear background lawn) or up to the highest possible solubility of the test compound. Additional tests were performed with S9 mix only when the test substances did not show a direct mutagenic effect.

SOS-CHROMOTEST
The test was performed with and without S9 mix (10% S9 fraction). The test was carried out in 2 series of glass tubes containing 10 ul of dissolved test substance and 250 ul of bacterial suspension. After incubating for 2 hours, b-galactosidase (b-GAL) was measured in one series of glass tubes and alkaline phosphatase (AP) in the other. The b-GAL assay was terminated 30 min after the addition of o-nitrophenyl-b-galactopyranoside. The AP assay was terminated 10 min after p-nitrophenyl phosphate had been added. SOS induction factor represents the ratio of the optical density measured at 420 nm of b-GAL and AP divded by the ratio of the solvent control. Positive test result was determined if the b-GAL activity was increased and the induction factor raised to more than 1.5.
Rationale for test conditions:
not specified
Evaluation criteria:
not specified
Statistics:
not specified
Species / strain:
other: PQ37
Metabolic activation:
not specified
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Using the SOS -Chromotest, the test substance tert-butyl glycidyl ether showed a dose-related increase of the b-GAL activity and the induction factor.

RESULTS OF THE SOS-CHROMOTEST

 Concentration (mmole/L)  b-GAL (OD 560)  AP (OD 560)  SOS induction factor
 DMSO  0.18  0.47  1.0
 0.3  0.18  0.45  1.1
 1.0  0.22  0.42  1.4
 3.3  0.23  0.40  1.5
 10.0  0.33  0.31  2.8
Conclusions:
Using the SOS-Chromotest, the test substance tert-butyl glycidyl ether showed a dose-related increase of the b-GAL activity and the induction factor. It also had a positive result during the Ames test. This indicates that the test substance has genotoxic effects.
Executive summary:

Using the SOS -Chromotest, the test substance tert-butyl glycidyl ether showed a dose-related increase of the b-GAL activity and the induction factor. It also had a positive result during the Ames test. This indicates that the test substance has genotoxic effects.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990-12-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Principles of method if other than guideline:
- Principle of test: Sister-chromatid exchange (SCE)-inducing potency is a measure of genotoxicity via observing the structure-activity relationships of the test substances in mammalian cells
- Short description of test conditions:
Chinese hamster V79 cell line grown in MEM supplemented with 10% heat-inactivated fetal calf serum and penicillin, streptomycin at 37 °C and 5% CO2. After 18 hours the test substances were added in varying concentrations. 2 hours after exposure, the medium was discarded and washed 2 times, replaced with medium containing 10E-5 M BrdU. Mitotic cells were harvested by shake-off after 28 hours of incubation with Colcemid (2x10E-7 M) for last 4 hours. Cells fixed on slides and stained. 25 metaphases with harlequin-stained chromosomes were scored for SCE per experimental point. Mean values of 2 experiments (total 50 metaphases) analyzed by pair-wise comparison of the solvent control.
-
GLP compliance:
no
Type of assay:
sister chromatid exchange assay in mammalian cells
Specific details on test material used for the study:
- Name of test material: 2,3-Expoxypropyl-tert.-butylether
- Source: Aldrich
- Purity: 99% purity
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Aldrich

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimal essential medium with Earle's salts supplemented with 10% heat-inactivated fetal calf serum and penicillin (100 U/mL), streptomycin (0.1 mg/mL) at 37 °C and 5% CO2
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Not reported
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Epichlorohydrin
Details on test system and experimental conditions:
The Chinese Hamster V79 cell line was grown in minimal essential medium with Earle´s salts (MEM) supplemented with 10% heat-inactivated fetal calf serum and penicillin (100 U/mL), streptomycin (0.1 mg/mL) at 37 °C and 5% Co2. V79 cells ( 5 x 10^5) were seeded into 25 cm² flasks and after 18 h the test substances were added in appropriate concentrations. The mdeium with the test substance was discarded 2 h later, the cells were washed 2 times with fresh medium containing 5-bromo-2´-deoxyuridine (10^-5 M). Mitotic cells were harvested by shakeoff after 28 h of incubation with Colcemid (2 x 10^-7 M) for the last 4 h. The cells were fixed on slides and stained according to standard protocols (Latt et al., 1981).
Statistics:
All results were confrmed in independent experiments and mean values of 2 experiments with a total of 50 metaphases scored per experimental point were analyzed by pair-wise comparison of the solvent control, using one-tailed Student's t test. The distribution of first, second, and third mitoses was determined by counting 100 metaphases per experimental point to calculate the replication index (RI).
To calculate the SCE-inducing potency (SCEIP) the slopes of the dose-response relationships were calculated by a computed linear regression analysis of the pooled results of the 2 independent experiments.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
SCE
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All tested compounds were tested up tp cytotoxic concentrations which were detected by delay in the replication indey (RI). The calculated SCE-Inducing potency for the test item was calculated with 1.9.

Below is Table 1 showing the results of two independent experiments; each experimental point is the mean value of 25 metaphases±SD and the replication index (RI) determined from 100 cells.

Slope of dose-response curve calculated as the mean of 2 experiments.

Table 1. V79/SCE test with 2,3 -epoxypropyl-tert.-butylether *denotes statistically significant from solvent at p<0.0005

   Experiment 1     Experiment 2     r  slope
   Mean (SD)  RI  Mean (SD)  RI    
 DMSO  5.4 (1.9)  2.13  4.7(2.2)  2.00  0.98  1.9
 0.31  6.1 (2.9)  2.7  2.14  4.8(2.0)  2.01  
 0.625  6.6 (2.4)  2.11  5.7 (2.8)  2.00    
 1.25  8.0 (2.2)  2.02*  6.5 (3.3)  2.00    
 2.5  9.4 (4.5)  2.00*  9.1 (3.5)  2.00*    
 5.0  15.1 (6.1)  1.60*  13.6 (5.2)  1.96*    

* SCE value different from the solvent, p< 0.005

Conclusions:
The mutagenic action of t-BGE in mammalian cells was investigated using the Chinese hamster V79 cell line. After exposing the cells to DMSO as a control and test substances concentrations ranging from 0.31 to 5.0 mmol/L, the sister chromatid exchange (SCE)-inducing potency was calculated by determining the slopes of the dose-response relationships via computed linear regression analysis of the pooled results of 2 independent experiments. The SCE-inducing potency (SCEIP) was 1.9 for test substance 2,3-epoxypropyl-tert.-butylether according to experiments with V79 cells in vitro without an external metabolizing system.
Executive summary:

The mutagenic action of t-BGE in mammalian cells was investigated using the Chinese hamster V79 cell line. After exposing the cells to DMSO as a control and test substances concentrations ranging from 0.31 to 5.0 mmol/L, the sister chromatid exchange (SCE)-inducing potency was calculated by determining the slopes of the dose-response relationships via computed linear regression analysis of the pooled results of 2 independent experiments. The SCE-inducing potency (SCEIP) was 1.9 for test substance 2,3-epoxypropyl-tert.-butylether according to experiments with V79 cells in vitro without an external metabolizing system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The source compound n-butyl glycidyl ether (EC 219-376-4, CAS 2426-08-6) is considered a suitable read across partner for the target substance tert-butyl glycidyl ether (EC 231-640-0, CAS 7665-72-7). This read-across is based on the hypothesis that source and target substances have similar toxicological properties because:
- structural similarity of the target and the source substances (the presence or absence of additional functional groups or substituents that could influence the behaviour of a chemical),
- similarity in physico-chemical profile of the source and target substances (water solubility, partitioning behaviour (log Kow value))
Both substances are structural isomers regarding the alkyl chain structure with identical molecular weight. Both ethers have similar physico-chemical properties. Both are highly water soluble (20 g/L vs 90 g/L), exhibit similar log Kow values (0.63 vs 0.97), have similar boiling points (169 °C vs 152 °C) and vapour pressure (3.5 hPA vs 6.26 hPa).
Reason / purpose:
read-across source
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
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:
positive
Remarks:
enhancement of the response was seen (especially noticeable with TA1535) in the presence of the S9 fraction.
Cytotoxicity / choice of top concentrations:
not specified
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
Remarks:
enhancement of the response was seen in the presence of the S9 fraction.
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The results of the assays for strain TA1537, TA1538 and TA98 were negative. Moreover, in all cases the positive controls exhibited at least a 10-fold increase in the number of revertant colonies (mutation index >10). In strain TA1535 the mutation index at 2000 µg/plate was calculated to be 27.7 (-S9) and 69.2 (+S9). In strain TA100 the mutation index at 2000 µg/plate was 9.1 (-S9) and 9.6 (+S9). Thus, in these both strains the test showed positive results.
Conclusions:
A Salmonella/mammalian microsome plate assay was performed as described by Ames et al., 1975 with butyl glycidyl ether. Butyl glycidyl ether was tested positive with and without metabolic activation in Salmonella typhimurium strains TA1535 and TA100.
Executive summary:

A Salmonella/mammalian microsome plate assay was performed as described by Ames et al., 1975 with butyl glycidyl ether. The concentrations chosen for the main test were based on the results from a preliminary toxicity test. Concentrations of 2000, 666.7, 222.2, 74, 24.7, 8.2 and 0 µg/plate were tested in the presence and absence rat liver microsome activation system (S9) using Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, and TA98. Both solvent (0.1 ml DMSO) and water negative controls were prepared. Three plates were prepared for each dose level and controls. In all cases the positive controls exhibited at least a 10-fold increase in the number of revertant colonies (mutation index >10). Butyl glycidyl ether was positive with and without metabolic activation in strains TA1535 and TA100, although enhancement of the response was seen (especially noticeable with TA1535) in the presence of the S9 fraction. The results of the assay with strains TA1537, TA1538 and TA98 were negative. Butyl glycidyl ether was considered mutagenetic in strains TA1535 and TA100 under the test conditions described.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The source compound n-butyl glycidyl ether (EC 219-376-4, CAS 2426-08-6) is considered a suitable read across partner for the target substance tert-butyl glycidyl ether (EC 231-640-0, CAS 7665-72-7). This read-across is based on the hypothesis that source and target substances have similar toxicological properties because:
- structural similarity of the target and the source substances (the presence or absence of additional functional groups or substituents that could influence the behaviour of a chemical),
- similarity in physico-chemical profile of the source and target substances (water solubility, partitioning behaviour (log Kow value))
Both substances are structural isomers regarding the alkyl chain structure with identical molecular weight. Both ethers have similar physico-chemical properties. Both are highly water soluble (20 g/L vs 90 g/L), exhibit similar log Kow values (0.63 vs 0.97), have similar boiling points (169 °C vs 152 °C) and vapour pressure (3.5 hPA vs 6.26 hPa).
Reason / purpose:
read-across source
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
In general, the highest mutagenic responses were obtained in the assays without metabolic activation. Responses obtained were slightly reduced in the assays that used the non-induced S9 preparations, whereas much lower responses were obtained with the Aroclor-induced S9 preparations. Cell toxicity was reduced proportionally, i.e. the same dose of chemical produced a more toxic response in the absence of any S9 and a less toxic response in the presence of the S9.
Conclusions:
The L5178Y mouse lymphoma assay was conducted as described by Clive and Spector, 1975 and later modified by Clive et al., 1979. Twelve concentrations of butyl glycidyl ether were tested in the presence and absence rat liver microsome activation system (S9) using L5178Y mouse lymphoma cells. In general, the highest mutagenic responses were obtained in the assays without metabolic activation. Responses obtained were slightly reduced in the assays that used the non-induced S9 preparations, whereas much lower responses were obtained with the Aroclor-induced S9 preparations. Cell toxicity was reduced proportionally, i.e. the same dose of chemical produced a more toxic response in the absence of any S9 and a less toxic response in the presence of the S9. Butyl glycidyl ether produced a positive response either with one of the S9 preparations or without activation. Under the described test conditions, butyl glycidyl ether was considered mutagenic.
Executive summary:

The L5178Y mouse lymphoma assay was conducted as described by Clive and Spector, 1975 and later modified by Clive et al., 1979. Twelve concentrations of butyl glycidyl ether were tested in the presence and absence rat liver microsome activation system (S9) using L5178Y mouse lymphoma cells. In general, the highest mutagenic responses were obtained in the assays without metabolic activation. Responses obtained were slightly reduced in the assays that used the non-induced S9 preparations, whereas much lower responses were obtained with the Aroclor-induced S9 preparations. Cell toxicity was reduced proportionally, i.e. the same dose of chemical produced a more toxic response in the absence of any S9 and a less toxic response in the presence of the S9. Butyl glycidyl ether produced a positive response either with one of the S9 preparations or without activation. Under the described test conditions, butyl glycidyl ether was considered mutagenic.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The source compound n-butyl glycidyl ether (EC 219-376-4, CAS 2426-08-6) is considered a suitable read across partner for the target substance tert-butyl glycidyl ether (EC 231-640-0, CAS 7665-72-7). This read-across is based on the hypothesis that source and target substances have similar toxicological properties because:
- structural similarity of the target and the source substances (the presence or absence of additional functional groups or substituents that could influence the behaviour of a chemical),
- similarity in physico-chemical profile of the source and target substances (water solubility, partitioning behaviour (log Kow value))
Both substances are structural isomers regarding the alkyl chain structure with identical molecular weight. Both ethers have similar physico-chemical properties. Both are highly water soluble (20 g/L vs 90 g/L), exhibit similar log Kow values (0.63 vs 0.97), have similar boiling points (169 °C vs 152 °C) and vapour pressure (3.5 hPA vs 6.26 hPa).
Reason / purpose:
read-across source
Species / strain:
other: WI38 cells
Metabolic activation:
with and without
Genotoxicity:
other: Demonstrable, although not considered positive, responses were obtained for butyl glycidyl ether in the presence of S9 and ethyl glycidyl ether in the non-activated assay.
Cytotoxicity / choice of top concentrations:
other: Demonstrable, although not considered positive, responses were obtained for butyl glycidyl ether in the presence of S9 and ethyl glycidyl ether in the non-activated assay.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
An unscheduled DNA synthesis assay was performed using WI38 cells to determine DNA repair after exposure to five different concentrations of butyl glycidyl ether in the presence and absence rat liver microsome activation system (S9). The positive controls were 4-nitroquinoline-N-oxide (4NQO), a compound that induces UDS in the absence of metabolic activation, and dimethylnitrosamine (DMN), a compound that induces UDS in vitro only with metabolic activation. The negative control was DMSO diluted in culture medium. The response index was calculated by dividing the amount of thymidine incorporation in the test results by the thymidine incorporation in the solvent control. A compound was considered positive in this assay if a dose-related increased in the amount of [3H]thymidine incorporated into DNA over at least 3 concentrations with the highest response equal to at least twice the solvent control was attained. Demonstrable, although not considered positive, responses were obtained for butyl glycidyl ether in the presence of S9 and ethyl glycidyl ether in the non-activated assay.
Executive summary:

An unscheduled DNA synthesis assay was performed using WI38 cells to determine DNA repair after exposure to five different concentrations of butyl glycidyl ether in the presence and absence rat liver microsome activation system (S9). The positive controls were 4-nitroquinoline-N-oxide (4NQO), a compound that induces UDS in the absence of metabolic activation, and dimethylnitrosamine (DMN), a compound that induces UDS in vitro only with metabolic activation. The negative control was DMSO diluted in culture medium. The response index was calculated by dividing the amount of thymidine incorporation in the test results by the thymidine incorporation in the solvent control. A compound was considered positive in this assay if a dose-related increased in the amount of [3H]thymidine incorporated into DNA over at least 3 concentrations with the highest response equal to at least twice the solvent control was attained. Demonstrable, although not considered positive, responses were obtained for butyl glycidyl ether in the presence of S9 and ethyl glycidyl ether in the non-activated assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In contrast to the in vitro data, the target substance was tested negative in several in vivo studies (in vivo chromosome aberration test, in vivo micronucleus test, dominant lethal test).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1976-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Short description of test conditions:
Metaphase cells from bone marrow preparations of the animals were scored for structural chromosomal aberrations. A total of 100 metaphases from each animal were scored.
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Specific details on test material used for the study:
- Name: tertiary-butyl glycidyl ether
- CAS No. : 7665-72-7
- Lot No. : 21311-34GWS
- Supplier: Shell
Species:
mouse
Strain:
other: B6D2F1
Sex:
female
Details on test animals and environmental conditions:
- Weight: 20 - 25 g
Route of administration:
oral: gavage
Vehicle:
Vehicle used: Corn oil
Details on exposure:
- Dilutions of t-BGE in corn oil were administered orally once a day for five days to separate groups of ten B6D2F1 female mice
- The positive control compounds were administered only twice.
- All animals were sacrificed 4 hours after the last treatment.
Duration of treatment / exposure:
5 days
Frequency of treatment:
once a day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
400 mg/kg bw/day
No. of animals per sex per dose:
10 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cytoxan and hycanthone were used as positive controls
Tissues and cell types examined:
Metaphase cells from bone marrow preparations were used for scoring of structural DNA damage.
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The frequency of gaps was essentially the same for both treated and control groups. The known positive controls Cytoxan and hycanthone demonstrated frequencies of damaged cells in the same range as prvious studies conducted in this laboratory. This test showed no differences between the control and the t-BGE dosage group oin the percentage of metaphases with chromosomal damage.
Conclusions:
In an in-vivo cytogenicity toxicity test, t-BGE was orally exposed to ten female B6D2F1mice weighing 20 - 25 g at concentrations of 100 - 400 mg/kg once per day for five consecutive days. All animals were sacrificed 4 hours after the last treatment. Extraction of bone marrow, preparation of smears, staining and analysis was conducted and the metaphase cells from bone marrow preparations were scored for structural chromosomal aberrations. A total of 100 metaphases from each animal were scored. The frequency of gaps was essentially the same for both treated and control groups. The known positive controls Cytoxan and hycanthone demonstrated frequencies of damaged cells in the same range as prvious studies conducted in this laboratory. This test showed no differences between the control and the t-BGE dosage group oin the percentage of metaphases with chromosomal damage.
Executive summary:

In an in-vivo cytogenicity toxicity test, t-BGE was orally exposed to ten female B6D2F1mice weighing 20 - 25 g at concentrations of 100 - 400 mg/kg  once per day for five consecutive days. All animals were sacrificed 4 hours after the last treatment. Extraction of bone marrow, preparation of smears, staining and analysis was conducted and the metaphase cells from bone marrow preparations were scored for structural chromosomal aberrations. A total of 100 metaphases from each animal were scored. The frequency of gaps was essentially the same for both treated and control groups. The known positive controls Cytoxan and hycanthone demonstrated frequencies of damaged cells in the same range as prvious studies conducted in this laboratory. This test showed no differences between the control and the t-BGE dosage group oin the percentage of metaphases with chromosomal damage.

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1979-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Principles of method if other than guideline:
- Principle of test: The dominant lethal test assesses the potential mutagenicity of test substance t-BGE by observing fetal deaths, reduced pregnancy rates and implants over different dosage levels.
- Short description of test conditions: Undiluted t-BGE was applied topically at dosages of 0, 0.375, 0.75, and 1.5 g/kg. At least 10 males of proven fertility were randomly selected for each dosage group and the negative control group (topically applied 0.9% saline), and were treated with the dosage three times per week for eight weeks. Following the treatment period, three untreated virgin females were randomly caged per treated male for 1 week. At the end of the 1st week the females were replaced with three other untreated virgin females for the second week and the same occurred the third week. Females were sacrified 13-14 days from the midweek of caging and presumptive mating and were scored for pregnancy, total number of implants and fetal deaths.

- Parameters analysed / observed:
1) the mean number of implants per pregnant female
2) the percent of fertile matings: the number of pregnant females / number of females used
3) the number of dead implantations per total implants: number of dead implantations / total number of implantations
4) the mean number of dead implants per pregnant female
GLP compliance:
not specified
Type of assay:
rodent dominant lethal assay
Specific details on test material used for the study:
- Name: tertiary-butyl glycidyl ether
- CAS No. : 7665-72-7
- Lot No. : 21311-34GWS
- Supplier: Shell
Species:
mouse
Strain:
other: B6D2F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
- Age at study initiation: males were 8-10 weeks at experiment start; females 8-10 weeks when mated
- Weight at study initiation: not reported
- Assigned to test groups randomly: yes
- Fasting period before study: not reported
- Housing: not reported
Route of administration:
dermal
Vehicle:
No vehicle; test substance is undiluted
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Approximately 15-20% of the surface dorsal area of each mouse was clipped by electric shears and the hair remaining on the clipped area was chemically depilated so that the absorption of the chemical would be unimpeded. (Chemical depilation was used only as needed following the initial removal of hair and did not exceed one depilation per week. A minimum of 24 hours was allowed between chemical depilation and application os the test chemical.

MATING:
- Following the treatment period, three untreated virgin females were randomly caged per treated male for 1 week. At the end of the first week, the females were replaced with three other untreated virgin females for the second week and likewise for the third week.
Duration of treatment / exposure:
Each male was treated with the dosage three times per week for eight weeks. Each male was weighed weekly, and the approximate dosage was determiend weekly from the mean weight of each dosage group.
Frequency of treatment:
Treated 3 times per week for 8 weeks
Post exposure period:
Not specified
Dose / conc.:
0 other: g/kg
Dose / conc.:
0.375 other: g/kg
Dose / conc.:
0.75 other: g/kg
Dose / conc.:
1.5 other: g/kg
No. of animals per sex per dose:
10 males
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Positive control(s):
None
Tissues and cell types examined:
total number of implants and fetal deaths
Statistics:
- The percent of fertile matings or percent pregnancy and the number of dead implants per total implants were also transformed using the arc-sine transformation with Bartlett's modification for values of 0 and 100% pregnancy rates.
- The transformed and non-transformed parameters were analyzed by a two-factor analysis of variance with unequal replications. In the case where non-pregnancies occurred from an individual male mouse, the values for the other three parameters (number of deaths/total implants, number of implants/pregnant female and mean number of deaths/pregnant female) were treated as missing, and that individual male was eliminated from the analysis.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not specified
Remarks on result:
not determinable
Remarks:
no classification for germ cell mutagenicity
Additional information on results:
PREGNANCY RATIOS
- No significant difference between the t-BGE dosage groups and negative control in the pregnancy ratios
- No significant trend in the pregnancy ratios with time and no significant interaction was detected.

MEAN NUMBER OF IMPLANTS
- No significant difference between the dosage groups in the mean number implants/pregnant females.
- There was a significant increase (p<0.001) in mean number implants/pregnant female at all dosage groups across time.
- There was no significant interaction.
- There was no evidence to indicate that the dosage level decreased the number of implants/pregnant female.

MEAN NUMBER DEAD IMPLANTS/TOTAL IMPLANTS
- No significant difference between the dosage groups; no significant interaction detected.

MEAN NUMBER DEAD IMPLANTS/PREGNANT FEMALE
- No significant difference between the dosage groups; no significant interaction detected.
- Significant increases across time in mean number dead implants/pregnant female.

VARIATIONS AND STATISTICAL SIGNIFICANCE
- Variations from dose level to dose level and time period to time period can be explained by the random variation observed.

Conclusions:
In an in vivo genetic toxicity test, conducted similar to guideline OECD 478, t-BGE was dermally exposed to 10 B6D2F1 male mice (per dosage group) at the concentrations 0, 0.375, 0.75, 1.5 g/kg bw. The male mice were exposed for 8 weeks and then mated with untreated females. The dominant lethal assay results indicated that increased dosage levels of t-BGE play no role in modifying either pregnancy rates, number of dead implants, number implants or the dead implant rates.
Executive summary:

In an in vivo genetic toxicity test, conducted similar to guideline OECD 478, t-BGE was dermally exposed to 10 B6D2F1 male mice (per dosage group) at the concentrations 0, 0.375, 0.75, 1.5 g/kg bw. The male mice were exposed for 3 times per week for a total of 8 weeks and then mated with untreated females. The dominant lethal assay results indicated that increased dosage levels of t-BGE play no role in modifying either pregnancy rates, number of dead implants, number implants or the dead implant rates.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1979-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
- Name: tertiary-butyl glycidyl ether
- CAS No. : 7665-72-7
- Lot No. : 21311-34GWS
- Supplier: Shell
Species:
mouse
Strain:
other: B6D2F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 20-25 g
Route of administration:
oral: gavage
Vehicle:
- Vehicle used:Corn oil
Details on exposure:
- Dilutions of t-BGE in corn oil were administered orally once a day for five days to separate groups of ten B6D2F1 female mice
- The positive control compounds were administered only twice.
- All animals were sacrificed 4 hours after the last treatment.
Duration of treatment / exposure:
5 days
Frequency of treatment:
Once a day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
400 mg/kg bw/day
No. of animals per sex per dose:
10 females
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive controls: Cytoxan and Hycanthone
- Route of administration: Oral
- Doses / concentrations: 50 mg / kg Cytoxan and 50 mg / kg Hycanthone
Evaluation criteria:
- The test substance is mutagenic if the frequency of structural aberrations in metaphase cells or micronuclei in the polychromatic erythrocytes of mouse bone marrow is comparable to the positive controls
Statistics:
- The data are expressed as percent cells containing micronuclei
Key result
Sex:
female
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
In an in-vivo genetic toxicity test, conducted similar to guideline OECD 474, t-BGE was orally exposed to ten female B6D2F1 mice weighing 20 - 25 g at concentrations 100 - 400 mg / kg once per day for five consecutive days. A total of 1000 polychromatic red cells from bone marrow preparations from each animal in control and t-BGE tested groups were scored for micronucleus. The test substance did not induce any increase in the frequency of the micronuclei in the polychromatic erythrocytes of the bone marrow. Thus, in this study, the test item is considered to be non-mutagenic.
Executive summary:

In an in-vivo genetic toxicity test, conducted similar to guideline OECD 474, t-BGE was orally exposed to ten female B6D2F1 mice weighing 20 - 25 g at concentrations 100 - 400 mg / kg once per day for five consecutive days. A total of 1000 polychromatic red cells from bone marrow preparations from each animal in control and t-BGE tested groups were scored for micronucleus. The test substance did not induce any increase in the frequency of the micronuclei in the polychromatic erythrocytes of the bone marrow. Thus, in this study, the test item is considered to be non-mutagenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The potential of the target substance t-butyl-glycidyl ether to induce genotoxic effects was assessed in a weight-of-evidence approach by using data from several in vitro and in vivo studies conducted with the target substance as well as with the source substance n-butyl glycidyl ether. The target substance was tested positive in several Ames tests as reported by Dabney, 1976 and Canter et al., 1986. Positive results were also obtained from an E. coli SOS Chromotest.

In one in vitro sister chromatid assay (von der Hude, 1991) and in two in vitro unscheduled DNA synthesis tests the target substance was tested positive for genotoxic effects.

Data from the source substance n-butyl glycidyl ether were used to assess the in vitro mutagenicity. In the publication by Thompson et al., 1981 the results from several genotoxicity tests (Ames, Mouse Lymphoma Assay, UDS) conducted with n-butyl glycidyl ether were presented. The UDS was negative, but the Ames and the Mouse Lymphoma assay showed a positive potential to induce mutagenicity.

In contrast to the in vitro data, the target substance was tested negative in several in vivo studies (in vivo chromosome aberration test, in vivo micronucleus test, dominant lethal test).

Furthermore, the structural analogue n-butyl glycidyl ether (source substance) is harmonized classified as Muta. 2 (H341) according to Annex VI of Regulation (EC) No 1272/2008.

Based on the results of the available data, and the fact that n-butyl glycidyl ether is harmonized classified as Muta. 2, H341 the target substance is considered as a Category 2 mutagen (Muta 2, H341).

Justification for classification or non-classification

Based on the results of the available data, and the fact that the structural analogue n-butyl glycidyl ether (source substance) is harmonized classified as Muta. 2, H341 the target substance is considered as a Category 2 mutagen (Muta 2, H341).