Butyl 2,3-epoxypropyl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material induced mutations in Salmonella typhimurium strain TA 1535 and TA 100 with and without the presence of a metabolic activations system.

The test material induced unscheduled DNA synthesis, with a dose response relationship, in the presence of metabolic activation.

The test material induced mutagenicity in the mouse lymphoma assay in the absence of a metabolic activation system and in the presence of a non-induced metabolic activation system, but not in the presence of an induced metabolic activation system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

To evaluate the in vitro genotoxicity of the test material an Ames test was performed on the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 with and without a metabolic activation system (S-9).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test material is indicated as R0065 in the study report.

Target gene:

his revertants

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver Homogenate (S-9)

Test concentrations with justification for top dose:

8.2, 24.7, 74.0, 222.2, 666.7, and 2000 µg/plate.
Based on preliminary toxicity testing.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: Methylnitronitrosoguanidine (MNNG) for strain TA1535 and TA100 without metabolic activation and, 2-aminoanthracene for all strains with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

NUMBER OF REPLICATIONS: 3
To each of 2 mL of complete top agar, 0.1 mL of an overnight broth culture of each tester strain, 0.1 ml of the test material or diluent and 0.5 mL of the S-9 mix for the activated test will be added. The contents of the tube will be mixed thoroughly and poured onto VBE minimal agar plates. Plates will be gently rotated and tilted to assure uniform distribution of the top agar, allowed to harden on an even surface for 1 hour, inverted and put in a dark 37±0.5 °C incubator. After two days, the colonies in both test plates and controls were counted.

Evaluation criteria:

The following criteria were established in the testing laboratory:
- Demonstration of toxicity of the chemical for the S. typhimurium strains
- The solvents control are within normal range, and
- Confirmation of sensitivity and responsiveness of the tester strains to mutagenic action.
If above mentioned criteria are met, a chemical that exhibits a positive, dose-related response over 3 concentrations with the baseline increase equal to twice the solvent control is considered to be mutagenic.
A chemical will be considered negative if the maximum non-inhibitory level exhibits less than a twofold increase in the number of induced revertants when compared to the solvent control. A non-linear dose response over three concentrations of the test substance will indicate mutagenic potential, but a dose-related response provided more persuasive evidence of mutagenesis.

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 1537, TA 1538, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The sensitivity of TA1535 to the action of the test material was demonstrated over 6 concentrations and activation improved this response at the 666.7 and 2000 µg/plate levels. In contrast, increased revertant numbers of TA100 were recorded only at the 3 highest concentrations of the test material and no substantial enhancement was obtained with the addition of a metabolic activation system (S-9).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

To evaluate the potential in vitro genetic toxicity of the test substance the mouse lymphoma assay was performed. Concentrations tested were 100, 200, 300, 400 and 500 µg/mL.

GLP compliance:

no

Type of assay:

other: L5178Y mouse lymphoma assay

Specific details on test material used for the study:

Test material is indicated as R0065 in study report.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclar 1254 induced rat liver homogenates (S-9), non-induced S9 mix

Test concentrations with justification for top dose:

100, 200, 300, 400 and 500 µg/mL
Preliminary toxicity screening was performed to determine the dose ranges to be used in the subsequent mutagenicity assays. The toxicity of the test substance was determined by testing a range of chemical concentrations for relative inhibition of the growth of L5178Y TK+/- cells in suspension culture in F10p following a four hour exposure, and two day incubation period.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

Five concentrations of the test substance were selected for the mutagenicity assays. The cells were exposed for four hours to the test and control chemicals with and without metabolic activation systems, washed free of the chemical, and incubated for a two day expression time. The cell concentration in each tube was adjusted to 300,000 cells per mL if necessary after the first expression day. Plating in selective and non-selective cloning media was accomplished after the second day. For plating, a sufficient sample from each culture was centrifuged and re-suspended at 10^-6 viable cells/mL in F10p. One-half mL of this concentrated cell suspension was added to each of three selective medium plates, and one mL of a 10^-4 dilution was added to each of three non-selective medium plates.

Evaluation criteria:

An acceptable assay will have:
1) plating efficiencies of at least 70% in the negative controls,
2) spontaneous mutation frequencies in the negative controls of 10 or less per 10^5 surviving cells,
3) responses to the positive control compounds within acceptable ranges as determined by previous testing. Assuming these criteria are met within the experiment, a positive result will be indicated by a dose-related response to the test substance which there are significantly greater (p≤ 0.05) numbers of mutants at the higher test concentrations as compared to the negative controls. The experiments will be repeated using an appropriate optimized dose range for each chemical.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

In the precence of induced S-9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

non-induced S-9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The two highest concentrations (500 and 400 µg/mL) did not yield enough cells for cloning following the two-day expression time in the absence of rat liver preparations. Significant mutagenicity was obtained with the test substance at the three succeeding dose levels. The toxicity and mutagenic activity was reduced considerably in the presence of induced S-9 such that the three lower doses no longer gave appreciable mutagenic activity. In the presence of non-induced S-9 a dose related mutagenic was obtained over the 200 to 500 µg/mL concentrations of the test substance.
induce

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

To evaluate the in vitro genetic toxicity of the test substance the unscheduled DNA synthesis assay was performed with and without a metabolic activation system. A preliminary test and 3 assays were performed in order to precisely define the response within the concentration range between the observed cytotoxicity and a no-effect level.

GLP compliance:

no

Type of assay:

other: unscheduled DNA synthesis assay

Specific details on test material used for the study:

Test material is indicated as R0065 in the study report.

Species / strain / cell type:

mammalian cell line, other: WI-38 cells

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from adult male Swiss-Webster mice

Test concentrations with justification for top dose:

Preliminary assay: 0.002, 0.02, 0.2, and 2.0 µL/mL without metabolic activation; 0.004, 0.04, 0.4, and 4.0 µL/mL with metabolic activation.
First assay: 1.778, 2.667, 4.0, 6.0, and 9.0 µL/mL without metabolic activation; 0.5, 1.0, 2.0, 4.0, and 8.0 µL/mL with metabolic activation.
Second assay: 1.6, 2.0, 2.6, 3.2, 4.0 µL/mL without metabolic activation; 2.56, 3.2, 4.0, 5.0, and 6.25 µL/mL with metabolic activation.
Third assay: 0.237, 0.356, 0.533, 0.8, 1.2 µL/mL without metabolic activation; Assay was not performed with metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (0.5% for preliminary tests, second and third assay without metabolic activation and 1% for first assay with and without metabolic acitivation and second assay with metabolic activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

N-dimethylnitrosamine

Details on test system and experimental conditions:

Preliminary tests:
Performed according the described UDS procedure, except that:
- A broader dose range was tested.
- Fewer replicate samples tested at each concentration (4 replicates)
- The end point indicator was either an apparent elevation in 3H-TdR incorporation into DNA or a cytotoxic effect, as indicated by either a reduction in 3H-TdR incorporation or a loss of cells from the culture, observed as a reduction in the DNA content of the culture. The preliminary assays were performed to establish the appropriate ranges of concentrations to be tested in the actual UDS assays.

UDS Assays:
The contact-inhibited WI-38 cells were incubated at 37 °C with dilutions of the test substance and with 1 µCi/mL of 3H-TdR (sp act, 6.7 Ci//mmole). For testing in the absence of metabolic activation, the cells were exposed simultaneously to the compounds and to 3H-TdR for 3 hours. For testing with metabolic activation, the cells were incubated with the compounds, 3H-TdR, and the metabolic activation preparation for 1 hour and then with only 3H-TdR in culture medium for an additional 3 hours. (The shorter exposure time for metabolic activation testing prevents cytotoxic effects to the WI-38 cells by the liver homogenate preparation.) To extract DNA from the cells, a modification of the PCA-hydrolysis procedure was used; one aliquot of the DNA solution was used to measure the DNA content, after reaction with diphenylamine, and a second aliquot was used for scintillation counting measurements of the extent of incorporation of 3H-TdR. Six replicates per test concentration were tested. Results were expressed as disintegrations per minute (dpm) or incorporated 3H- TdR per unit of DNA and were compared with the rate of incorporation of 3H-TdR into cells exposed to solvent only (negative control).

Evaluation criteria:

We have defined as an acceptable assay one in which the response of the positive control compound is predicted, within the 95% confidence limits, by least-squares regressions of average dpm/µg DNA versus average dpm/µg for background.

Statistics:

least-squares regressions of average dpm/µg DNA versus average dpm/µg for background.

Key result

Species / strain:

mammalian cell line, other: WI-38

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: WI-38

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary assay:
Results suggested neither a positive response nor cytotoxic effects at any of the test concentrations without metabolic activation. With metabolic activation the results of the assay suggested an elevation in 3H-TdR incorporation at a test concentration of 4.0 µL/mL.

First assay:
This assay of the test substance without metabolic activation was conducted at concentrations predominantly in excess of the 2.0 µL/mL maximum used in the preliminary assay. The results of this assay suggested cytotoxic effects at concentrations of 4.0 µL/mL and greater; the effects at lower test concentrations were neither clearly cytotoxic nor indicative of an absence of effects. With metabolic activation, a statistically significant elevation at a test concentration of 4.0 µL/mL was apparent. However, a dose response relationship wat not established, presumably due to the spacing of the test concentrations.

Second assay:
This assay was performed to further define the response within the concentration range between the observed cytotoxicity and a no-effect level. The results clearly indicated depressions in 3H-TdR incorporation at all test concentrations. Thus, the concentration range initially selected for testing apparently was too high to permit adequate evaluation of the potential of the test material to induce UDS. With metabolic activation, the results showed a statistically significant elevation in 3H-TdR incorporation and also indicated a dose response relationship for the elevation observed. Thus, UDS was observed in response to the test substance in the presence of metabolic activation.

Third assay:
The results of this assay, performed only in the absence of metabolic activation, did not suggest an elevation in 3H-TdR incorporation. Therefore, it was concluded that no UDS response occurred in the absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test material causes structural chromosomal aberrations in the rat bone marrow cells when tested in the Bone Marrow Cytogenetics study.

No significant micronuclei increases were observed in mice treated orally (gavage).

Upon single and 2 -time intraperitoneal administration of the test substance increases in micronuclei were seen at doses of 675 and 900 mg/kg.

Results obtained from 3 dominant lethal assays were ambiguous.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

To evaluate the in vivo genetic toxicity of the test substance a Bone Marrow Cytogenetics study was performed in the rat at levels of 31.3, 104.2 and 312.5 mg/kg bw per day.

GLP compliance:

no

Type of assay:

other: Mammalian Bone Marrow Chromosome Aberration Test

Specific details on test material used for the study:

Test material is indicated as R0065 in the study report.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

CR-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Wilmington, Massachusetts
- Age at study initiation: sexually mature
- Weight at study initiation: 150-200 gram
- Assigned to test groups randomly: yes
- Housing: individually in hanging wire mesh cages
- Diet: Purina Laboratory Chow ad libitum
- Water: via water bottles ad libitum
- Acclimation period: one week

Route of administration:

intraperitoneal

Vehicle:

distilled water

Details on exposure:

The test material was dissolved in an appropriate vehicle (distilled water) and was made up fresh each dose day. The entire study was performed using a single batch of the chemical.

Duration of treatment / exposure:

5 days

Frequency of treatment:

daily

Post exposure period:

On Day 6, all animals received an intraperitoneal injection of colchicine (2.0 mg/kg body weight) to inhibit mitosis in dividing cells. Approximately two to four hours after injection of colchicine, the animals were sacrificed using carbon-dioxide (C02) anaesthesia followed by cervical dislocation.

Dose / conc.:

31.3 mg/kg bw/day (actual dose received)

Dose / conc.:

104.2 mg/kg bw/day (actual dose received)

Dose / conc.:

312.5 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylene melamine (TEM)
- Route of administration: intraperitoneal injection
- Doses / concentrations: Single injection at Day 5, 0.04 mg/kg (Dose volume 5 mL/kg)

Tissues and cell types examined:

Bone marrow cells collected from both femurs of each animal.
Fifty cells in the metaphase state of mitosis were examined from each rat that provided analysable cells. The slides were scanned with a low power objective (10x) to find metaphases and, once found, the metaphases were analysed via high power oil immersion lens (100x).

Details of tissue and slide preparation:

Immediately following sacrifice, bone marrow cells were collected from both femurs of each animal by aspiration into a 12 mL syringe filled with 5 mL of pre-warmed (37 °C) Hank's balanced salt solution at pH 7.4. The aspirate was transferred to a centrifuge tube and centrifuged for five minutes at 100 x g. The supernatant was decanted and 3.0 mL of 0.075M KCl was added to each tube. After standing at room temperature for 25 minutes, each tube was centrifuged for five minutes at 100 x g. The supernatant was decanted and 5 mL of freshly prepared Carnoy's Fixative (3:1, methanol : glacial acetic acid v:v) was added to each tube. The tubes stood at room temperature for 20 minutes and were centrifuged for five minutes at 100 x g. The supernatant was discarded and the cells were re-suspended in 5 mL of fresh fixative. Each tube was sealed and refrigerated overnight (4 °C). The next day each tube was centrifuged at 100 x g for five minutes. The supernatant was decanted and the cells were suspended in 2-3 mL fresh Carnoy's Fixative. Three drops of this final cell suspension were dropped onto pre-cleaned glass microscope slides which were then gently blown dry. Three slides were made for each animal. Each slide was identified with the animal's number.

Evaluation criteria:

The metaphases were observed for the cytogenetic abnormalities, mitotic index (number of cells undergoing mitosis per 100 cells counted), and modal number (number of chromosomes in each metaphase). Chromosomal aberrations were classified into one of four basic groups: (1) chromatid and (2) chromosome breaks; (3) markers which involve dicentrics, exchanges, rings and other miscellaneous configurations and (4) severely damaged cells.

Statistics:

The mean changes in body weights, the mean modal number (number of chromosomes in each metaphase), and the mean mitotic index (number of cells undergoing mitosis per 100 cells counted) were analysed using Bartlett's test for equality of variance (Bartlett, 1937) and the one-way classification of analysis of variance (ANOVA) (Snedecor and Cochran, 1967). If significant results were obtained from both analyses, group mean values were compared using the multiple comparison procedure of Games and Howell (Games and Howell, 1976). If only the ANOVA were significant, Scheffe's multiple comparison procedure (Scheffe, 1953) was used to compare group means. The aberrant cells and the categories of chromatid breaks, chromosome breaks, markers, and severely damaged cells were statistically analysed either by Wilcoxon`s nonparametric comparison of group means (Snedecor and Cochran, 1957) or by chi-square analysis (Snedecor and Cochran, 1967). Regression analyses (Draper and Smith, 1966) were performed on all data when significant responses were observed in the treated groups.

Key result

Sex:

male/female

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Clinical observations and mortality:
One animal from the high-dose group died. There were no observable gross pathological findings. Statistical analysis of the mean body weight increases revealed no significant differences among any of the groups.
Analysis of marker aberrations resulted in a significant elevated mid-dose group. The low- and high-dose groups were elevated with respect to the incidence of severely damaged cells. The percent of aberrant cells (total number of aberrant cells per animal) was significantly increased among all treated groups when compared to the negative control. Therefore, under the conditions of this study, the test substance administered intraperitoneally for five consecutive days at doses as low as 31.3 mg/kg bw/day resulted in structural chromosomal aberrations in the rat bone marrow cells at all levels.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Genetic toxicity in vitro:

Regarding the in vitro genetic toxicity of the test material, 7 Ames tests, 3 unscheduled DNA synthesis assays and 2 mouse lymphoma assays are relevant.

Ames tests:

To evaluate the in vitro genotoxicity of the test material an Ames test was performed (plate incorporation) on the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 with and without a metabolic activation system (S-9) (US EPA, 1997). Test concentrations were 8.2, 24.7, 74.0, 222.2, 666.7, and 2000 µg/plate (based on preliminary testing). The test material induced gene mutations in TA 1535 and TA 100 with and without metabolic activation. The sensitivity of TA1535 to the action of the test material was demonstrated over 6 concentrations and activation improved this response at the 666.7 and 2000 µg/plate levels. In contrast, increased revertant numbers of TA100 were recorded only at the 3 highest concentrations of the test material and no substantial enhancement was obtained with the addition of a metabolic activation system (S-9).

Six other Ames tests were identified and are briefly described below.

Connor et al (1980) tested the test substance in S typhimurium strains TA 1535, TA 1537, TA 1538, TA 100 and TA 98 at 2.0 µmoles (260 µg) per plate according to the Ames methods with and without metabolic activation (S9). Similar as in the previous study the test material induced gene mutations in TA 1535 and TA 100 with and without metabolic activation. 

Canter et al (1986), tested 28 chemicals (among which the test substance) in Salmonella strains TA98, TA100, TA1535, and TA1537 with and without metabolic activation (S9) at 0, 33, 100, 333, 1000 and 3333 µg/plate. Again, results showed that the test substance induced gene mutations in TA 1535 and TA 100 with and without metabolic activation. Ambiguous results were obtained for TA 98 and TA 1537 with and without metabolic activation.

Thompsom et al (1981), tested 10-fold dilutions according to the standard plate assay procedure of Ames (100, 10 and 1 mg/mL, 100, 10 and 1 µg/m). Further testing was performed at 0, 8.2, 24.7, 74, 222.2, 666.7 and 2000 µg/plate (The concentration of chemical which reduced the background lawn by 50-75% compared to the solvent control was selected as the highest testable dose.). The substance was tested with S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 1538 with and without metabolic activation (S9). Results showed that the test substance induced gene mutations in TA 1535 and TA 100 with and without metabolic activation.

 Wade et al. (1979), spot tested the test substance in S typhimurium strains TA 100 and TA 98 with and without the addition of a metabolic activation system. Doses tested were 0.05 and 10 mg/plate. At 10 mg/plate, cytotoxicity was observed in the TA 98 strain. Results showed that the test substance induced gene mutations in TA 100 without metabolic activation.

In another study (US EPA, 1982) the test substance was investigated in agar layer cultures of Salmonella typhimurium TA 1535 and TA 1538 both with and without the incorporation of rat liver micrsomal enzymes (S9). The addition of the test substance to agar layer cultures of S. typhimurium TA 1535 and TA 1538 induced an increase in reverse gene mutation at high concentrations both with and without the incorporation of a rat liver microsomal fraction.

Finally, in TSCATS document OTS0206138 (US EPA, 1978), the test substance was tested in S typhimurium strains TA 1535 and TA 98 (plate incorporation) with and without the addition of Aroclor or phenobarbital induced liver homogenate. A dose-related increase in mutagenic response was observed in strain TA1535 without metabolic activation. The addition of the S9 mix from either Aroclor or phenobarbital liver homogenate decreased the mutagenic Activity. No mutagenic effect was observed in strain TA 98.

UDS assay:

Besides the identified Ames tests 4 unscheduled DNA synthesis assays were identified.

In TSCATS document OTS0200642 (US EPA, 1979) the unscheduled DNA synthesis assay was performed using WI-38 cells with and without a metabolic activation system (S9 liver homogenate from adult male Swiss-Webster mice). A preliminary test and 3 assays were performed in order to precisely define the response within the concentration range between the observed cytotoxicity and a no-effect level (ranging from 0.002 to 8.0 µl/mL). With metabolic activation, the results showed a statistically significant elevation in 3H-TdR incorporation and also indicated a dose response relationship for the elevation observed. Thus, UDS was observed in response to the test substance in the presence of metabolic activation. The results of the assay, performed only in the absence of metabolic activation, did not suggest an elevation in 3H-TdR incorporation. Therefore, it was concluded that no UDS response occurred in the absence of metabolic activation.

In TSCATS document OTS0206138 (US EPA, 1978) the test substance was examined for induction of excision repair measured as unscheduled DNA-synthesis in human mono-nucleated white cells (G-0 phase) in a short-term in vitro assay using liquid scintillation spectroscopy. Test concentrations were 1, 10, 100 or 500 ppm in DSMO. A linear dose-response relationship occurred in 3HTdR incorporation with the dosages of 1. 10. and 100 ppm. The values at 10 and 100 ppm were significantly different than the control (p<0.05). Cell viability was dramatically reduced over the range of 100 to 500 ppm, with an obvious toxic effect occurring at 500 ppm. This effect undoubtedly influenced incorporation of 3HTdR at 500 ppm as seen by a significant reduction at this dosage level. The results of the radio-autographic studies indicate a large increase of labelled cells from the treatment over the control.

Thompson et al. (1981) evaluated test substance for genetic toxicity in the unscheduled DNA synthesis assay using WI-38 cells with and without a metabolic activation system. For testing in the absence of metabolic activation, the cells were exposed simultaneously to the compounds and to 3H-TdR for 3 h. For testing with metabolic activation, the cells were incubated with the compound. The UDS assay demonstrated roughly a dose-related response, both in the presence and absence of metabolic activation. However, the response index (the ratio of test/control) was <1 at all test concentrations without metabolic activation. The test substance is not very soluble in aqueous solutions as evidenced by the presence of oil droplets on the surface of the medium at higher concentrations. The protein and lipids present in S9 potentially enhances the solubility of butyl glycidyl ether, thus increasing the exposure of the epoxides to the cells.

MLA:

In a study (US EPA, 1979) the potential in vitro genetic toxicity of the test substance was evaluated in the L5178Y mouse lymphoma assay. Based on preliminary toxicity screening doses of 100, 200, 300, 400 and 500 µg/mL in DMSO were selected. The cells were exposed for 4r hours to the test and control chemicals with and without metabolic activation systems, washed free of the chemical, and incubated for a two day expression time. The cell concentration in each tube was adjusted to 300,000 cells per mL if necessary after the first expression day. Plating in selective and non-selective cloning media was accomplished after the second day. For plating, a sufficient sample from each culture was centrifuged and re-suspended at 10-6 viable cells/mL in F10p. One-half mL of this concentrated cell suspension was added to each of three selective medium plates, and one mL of a 1/10-4 dilution was added to each of three non-selective medium plates. The two highest concentrations (500 and 400 µg/mL) did not yield enough cells for cloning following the two-day expression time in the absence of rat liver preparations. Significant mutagenicity was obtained with the test substance at the 3 succeeding dose levels. The toxicity and mutagenic activity was reduced considerably in the presence of induced S-9 such that the three lower doses no longer gave appreciable mutagenic activity. In the presence of non-induced S-9 a dose related mutagenic was obtained over the 200 to 500 µg/mL concentrations of the test substance.

In addition Thompson et al. (1981) performed the L5178Y mouse lymphoma assay to evaluate the genetic toxicity of the test substance both with and without the presence of a metabolic activation system (S9). Based on an earlier performed toxicity assay. A narrow range of concentrations around the dose which gave approx. 50% inhibition of growth was selected for further testing (0, 84, 100, 130, 164, 200, 250, 300, 320, 400, 500, 640 and 800 µg/mL). The mutation frequency was calculated by dividing the number of colonies appearing in the selective plates (TFT) by the number of surviving cells in non-selective plates. The mutation index was calculated by dividing the mutation frequency of the test results by the mutation frequency of the solvent control. A compound was considered positive if dose-related increases in the mutation index over at least 3 concentrations with the highest response at least equal to 3.0 were obtained. 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.

Genetic toxicity in vivo:

In a study (US EPA, 1979) the in vivo genetic toxicity of the test substance was assessed via a Bone Marrow Cytogenetics test in the rat at levels of 31.3, 104.2 and 312.5 mg/kg bw per day (ip) for 5 days. On Day 6, all animals received an intraperitoneal injection of colchicine (2.0 mg/kg body weight) to inhibit mitosis in dividing cells. Approximately two to four hours after injection of colchicine, the animals were sacrificed using carbon-dioxide (C02) anaesthesia followed by cervical dislocation. One animal from the high-dose group died. There were no observable gross pathological findings. Statistical analysis of the mean body weight increases revealed no significant differences among any of the groups. Analysis of marker aberrations resulted in a significant elevated mid-dose group. The low- and high-dose groups were elevated with respect to the incidence of severely damaged cells. The percent of aberrant cells (total number of aberrant cells per animal) was significantly increased among all treated groups when compared to the negative control. Therefore, under the conditions of this study, the test substance administered intraperitoneally for five consecutive days at doses as low as 31.3 mg/kg bw/day resulted in structural chromosomal aberrations in the rat bone marrow cells at all levels.

In another study (US EPA, 1978) the test substance was evaluated in an in vivo micronucleus test performed on B6D2F1 female mice (oral exposure (gavage), 200 mg/kg for 5 days). Of the 10 animals treated with the test substance 1 animal died Of the 10 animals in the solvent control group also 1 animal died. In the positive control group no animals died. The mean % micronuclei of the test group was 0.04 ± 016.The mean % micronuclei of the positive control group was 4.20 ± 1.31. The mean % micronuclei of the solvent control group was 0.14 ± 0.1. Based on these results no genotoxic effect was observed.

In another mouse micronucleus test, Connor et al (1980) found upon single intraperitoneal administration of the test substance increases in micronuclei at doses of 675 and 900 mg/kg. Although micronuclei increases upon 2-time administration were higher than the solvent controls at 450 and 225 mg/kg, their difference was not detected at P = 0.050. In contrast, treatment at 200 mg/kg orally on 5 consecutive days produced no increase in micronuclei over the controls.

In addition, three dominant lethal assays were identified. Whorton et al (1983) conducted two dominant lethal studies with BDF hybrid mice. Males were 8-10 weeks old at the commencement of experiments and females were 8-10 weeks old when mated. Each male was mated with 3 virgin females per week beginning 2 weeks prior to administration of the test compound in order to determine fertility, litter size and frequency of spontaneous fetal deaths. Males of proven fertility were randomly assigned to the 3 treatment groups (0.375, 0.75, 1.5 g/kg) and the negative control. Due to its high lipophilicity, application resulted in rapid penetration of the skin without the occurrence of excess or runoff. Males were treated 3 times per week for a total of 8 weeks. Each male was weighed weekly, the group mean weight determined and the dosage adjusted accordingly. Following the last week of treatment, each male was mated to 3 virgin females per week for a total of 3 weeks. At autopsy, females were checked for total number of implants and fetal deaths. Following the final mating period, each male was sacrificed, and the testes were removed and preserved in Bouin's solution at a volume ratio of at least 10:1. After fixation, these testes were embedded in paraffin and cut on a rotary microtome. The sections were stained with hematoxylin eosin and assessed in blind pathological studies. No significant dose-related changes either in pregnancy rates or in average number of implants per pregnant female were found. Although a significant increase in fetal death rates was observed by the end of the first week after the highest dosage was administered, the results were not altogether conclusive. Moreover, analysis of the frequency and distribution of abnormal testicular cells revealed that there were no significant dose-related testicular changes, and that the number of altered cells was so low in every dosage group that it is unlikely that sperm count viability was affected.

A similar performed test was described in TSCATS document OTS0200451 (US EPA, 1978). To evaluate the potential in vivo genetic toxicity of the test substance the dominant lethal assay was performed on 10 male B6D2F1 mice of proven fertility. Only 1 dose level was assessed; 1500 mg/kg bw dermally. Approximately 15-20% of the surface area were clipped by electric shears in the dorsal area. The remaining hair was chemically depilated. Chemical depilation was only used 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 of the test chemical and application of the test material. The group of males was weighed weekly, and the mean weight for each week was used to adjust the dosage. Following the treatment period, 3 untreated virgin females were randomly caged per treated male for one week. At the end of the first week the females were replaces with 3 other untreated virgin females for the duration of the second week. All females were sacrificed by cervical dislocation 13-14 days from the midweek of the caging and presumptive mating. In this study, there was a significant increase ( p=0.04) in the proportion deaths/pregnancy of the treated animals over the control animals. In addition there was a significant effects on the same variable due to time and a very time-treatment interaction, although not significant at the 0.05 level. Significant effects (p=0.01) were also detected in the number implants/pregnancy due to time and time-treatment dependency. The number of pregnant females in the BGE group was consistently lower than the control group and was significant with p=0.05.

In TSCATS documents OTS0206194 (US EPA, 1982) the same test was performed but, this time with three dose levels (37.5, 750, 1500 mg/kg bw. Pregnancy rate (PR) was calculated by first calculating the PR for each time period-dosage group males using the pregnancy status of three females per male. All males and females were included in the calculation and analysis of the variable. Statistical analysis was performed both for the PR as calculated for each male together with a re-expression of the PR as TPF=arc sin √PR. Average number of implants per pregnant female, IPF, was calculated for each time-does male by adding the number of implants for the pregnant females and dividing by the number of pregnant females. Proportion of dead implants (per pregnant female), DPMI, was calculated for each time-dose male by adding all deaths observed in all pregnant females and dividing by the total number of implants observed. Averaged Proportion of dead implants (per pregnant female), DPM2, was also calculated for each time-dose male by first calculating the proportion of dead implants per pregnant female and then averaging these resulting proportions. Averaged number of dead implants per pregnant female, DPF, was calculated for each time-dose male by adding the number of dead implants in all pregnant females and dividing by the resultant number of pregnant females. Results of the statistical analysis indicate that there is insufficient evidence to conclude that adverse effects due to increase dosage exist. While there was a weak suggestion of a highest dose effect at the post-treatment period 1 for the variable Averaged Proportion of dead implants (per pregnant female), this results could not be meaningfully distinguished from its corresponding saline control group. Also when tabulated in the form of percentage females in each does-time cell having one or more dead implant, data indicate that, although the 1500 mg/kg bw groups appears to result in a larger than expected number of females having one or more dead implant in the first post treatment period, the dead implant rate is not significantly elevated over the saline control group.

Justification for classification or non-classification

The substance was demonstrated to induce mutations in Salmonella typhimurium strain TA 1535 and TA 100 with and without the presence of a metabolic activations system. The substance was also shown to be positive in an unscheduled DNA synthesis test in the presence of metabolic activation and in a mouse lymphoma assay without metabolic activation and with a non-induced S-9 mix.

When tested in vivo in the Bone Marrow Cytogenetics study the test material caused structural chromosomal aberrations in the rat bone marrow cells when tested. However, no significant micronuclei increases were observed in mice treated orally with the test substance in the micronucleus test. Upon single and 2-time intraperitoneal administration of the test substance, increases in micronuclei were seen. However, intraperitoneal injection is generally not recommended since it is not an intended route of human exposure and, since only one dose was tested no dose related increase could be demonstrated.

 

Based upon the results obtained in the available in vitro and in vivo genetic toxicity testing, the test substance should be classified as Muta. 2, H341: Suspected of causing genetic defects in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.