m-bis(2,3-epoxypropoxy)benzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test

The test item was positive in Salmonella typhimurium strains TA1535 and TA100 (with and without metabolic activation) in out of 4 tested Salmonella typhimurium strains: TA1535, TA1537, TA98 and TA100 (Canter and Zeiger, 1984). The test item showed also a positive result in the Salmonella typhimurium strain TA100 of another paper (Seiler, 1984), however, there the metabolic activation was not specified.

in vitro chromosomal aberration test

Treatment of cultured Chinese hamster ovary CHO cells with the test item produced highly significant increases in chromosomal aberrations and sister chromatid exchanges both with and without S9 (Gulati et al, 1989).

The mutagenic and toxicity potential of the test item has been demonstrated in Chinese hamster CHO cells. The metabolic activation was not specified (Seiler, 1984).

A weight of evidence of the analyzed results suggest that the test item is positive for the genetic toxicity in vitro endpoint.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

Haworth et al (1983) with minor modifications

Principles of method if other than guideline:

- Principle of test: assess the mutagenicity in Salmonella of the test item using a standardized protocol.

- Short description of test conditions: The test item was tested in Salmonella strains TA98, TA100, TA1535 and TA1537 without metabolic activation and with liver S-9 preparations from Aroclor 1254-induced male, Sorague-Dawley rats and
Syrian hamsters, in a liquid incubation protocol with the tubes covered to retard loss of the volatile chemicals. The test item was tested using 10% S-9 in the S-9 mix up the limit of solubility or toxicity, or 10 mg/plate, at 5 doses, in triplicate plates. Tests were repeated at least once; a chemical was not called 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 two-fold over background.
A response was considered equivocal if a test was not reproducible; when a low-level, non-dose response was obtained; or when an increased response was seen at only one dose.
A chemical was considered mutagenic if at least one strain/activation combination yielded a reproducible positive response.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity test date: 87.9% (analyzed)

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

with liver S-9 preparations from Aroclor 1254-induced male Sprague-Dawley rats and Syrian hamsters

Test concentrations with justification for top dose:

0.5 µmol/plate (no justification for top dose provided)

Vehicle / solvent:

not reported

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

Not specified

Rationale for test conditions:

performed according to: Haworth ST, Lawlor´T, Mortelmans K, Speck W, Zeiger E. (1983) Salmonella mutagenicity results for 250 chemicals. Environmental Mutagenesis 5 (Suppl.): 3–142.

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 two-fold over background.
A response was considered equivocal if a test was not reproducible; when a low-level, non-dose response was obtained; or when an increased response was seen at only one dose.
A chemical was considered mutagenic if at least one strain/activation combination yielded a reproducible positive response.

Statistics:

Not specified

Species / strain:

S. typhimurium TA 98

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:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Conclusions:

Diglycidyl resorcinol ether was mutagenic in Salmonella strains TA100 and TA1535 with and withour metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

This study was accepted for publication on 17 October 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Reliability 2 was assigned as the study was conducted to a recognised procedure.

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

Performed according to Ames et al. (1975),

Principles of method if other than guideline:

The bacteria were incubated in Oxoid No. 2 broth for 6 h under continuous shaking until the desired cell density was reached. The number of living cells plated was checked in the following manner: 0.1 mL of the appropriate dilution of the bacteria in isotonic saline was added to 2 mL of top agar containing an amount of histidine sufficient for the unlimited growth of all bacteria. Other growth conditions, e.g. biotin content, composition of base agar, and incubation temperature, were equal to those of the mutagenicity test, and colony counts were also performed after 72 h incubation on a Biotran II Colony Counter. The number of spontaneous revertants (116 colonies per plate) has been subtracted. 5.8*10E07 cells were seeded per plate.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Resorcinoi diglycidyl ether (RDGE) was synthesized according to pub- lished methods (Ross et al., 1964); briefly, the parent compound resorcinol was first reacted with an approximately 10-fold excess of epichlorohydrin at slightly elevated temperatures. Upon completion of the reaction, a 50% sodium hydroxide solution was added slowly over a period of 1-3h in an amount equivalent to the number of reactive sites. The product was finally dissolved in toluene, washed with water and dried over anhydrous sodium sulfate. The solvent was then removed in vacuo and the product was purified by vacuum distillation. The purified product was analyzed by HPLC (on a silica column, with the same solvent as above) and was estimated to contain less than 2% impurities. The alkylating potency of the epoxide was measured in the 4-(4-nitro- benzyl)pyridine (NBP) assay according to Friedman and Boger (1961).

Target gene:

Not Specified

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

TA100: 50, 100, 200, 500 and 1000 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (Oxoid No.2 broth) for 6 h under continuous shaking until the desired cell density was reached

DURATION
- Exposure duration: 72 h

NUMBER OF CELLS EVALUATED:
The number of living cells plated was checked in the following manner: 0.1 ml of the appropriate dilution of the bacteria in isotonic saline was added to 2 ml of top agar containing an amount of histidine sufficient for the unlimited growth of all bacteria.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

DETERMINATION OF CYTOTOXICITY
- median number of revertant colonies per plate for strain TA100

Evaluation criteria:

For the test substance to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose-levels or at the highest practicable dose-level only. In addition there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

The median numbers of revertant colonies per plate for strain TA100 together with the median deviatin were calculated according to Vollmar (1981).

Reference: Vollmar J (1981) Fundamental microbiological aspects of the Ames Test, Panel Discussion: Statistical Problems in the Ames test, in : A. KAppas (Ed) Progress in Mutation Research, Vol 2, Elsevier, Amsterdam: 149-193.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not specified

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The in vitro alkylating potency of Resorcinol diglycidyl ether compared to NBP is given in Table 1. The Salmonella mutagenicity test data are given in Table 2.

Table 1: Alkylating Potency of Resorcinol Diglycidyl Ether Measured by the Formation of Coloured NBP Adducts

	Optical density at 540nm (measured against negative control)
	12.5 µg	25 µg	50 µg	100 µg
Resorcinol Diglycidyl Ether	0.23	0.55	1.17	2.18

Table 2: Salmonella Mutanegenicity Test Data

	Amount per Plate
	50 µg	100 µg	200 µg	500 µg	1000 µg
Resorcinol Diglycidyl Ether	322 (25)	493 (22)	656 (38)	1 (32)	Toxic

Table indicates the median numbers of revertant colonies per plate for TA 100, together with the median deviation in brackets. The number of spontaneous revertants (116 colonies per plate) has been subtracted. 5.8*10E07 cells were seeded per plate.

Conclusions:

The substance possesses mutagenic activity related to its epoxy groups.

Executive summary:

The mutagenic potential of resorcinol diglycidyl ether has been demonstrated in Salmonella TA100 bacterial cells. There was concentration-related increase over the range tested: 50, 100 and 200 µg/plate. At 500 µg/plate the test item showed already cytotoxicity. There was no information on used or not used metabolic activation system. In addition, there are no data on positive or negative control used. According to the current OECD 471 guideline requirements there only one (S. typhimurium TA100) out of five minimum required strains of bacteria was used.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

chromosomal aberrations

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

This study was accepted for publication on 17 October 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Reliability2 was assigned as the study was conducted to a recognised procedure it is a comparative study with no clear endpoint data.

Qualifier:

no guideline available

Principles of method if other than guideline:

For the in vitro chromosomal aberration tests, Chinese hamster cells were chosen. The CHO-K1 cells were obtained from Flow Laboratories AG (Baar, Switzerland); they were cultured in F-12 medium, containing 10% foetal calf serum, at 37°C and in an atmosphere of 5% CO2. For the test, 2x10E05 cells were seeded into 25-cm2 culture flasks and incubated for 24 h. After this period, the test compounds were added in DMSO solution, and the cells were incubated for a further period of 6-24 h. Two hours prior to harvesting, the cells were arrested in C-mitosis by the addition of 10 µg/ml Colcemid. Harvesting of mitotic cells was accomplished by the mitotic shake-off method of Terasima and Tolmach (1961). The suspended cells were collected by centrifugation at 400 g and submitted to 161 hypotonic treatment (75 mM KCI, 37°C, 20 min). After further centrifugation, the cells were fixed by dropwise addition of ice-cold, freshly prepared methanol/acetic acid fixative (3:1, v/v) under continuous shaking. The fixed cells were left in the refrigerator for at least 30 min, but preferably overnight; they were then spun down and fresh fixative was added; after further centrifugation. the cells were taken up in a few drops of fresh fixative and dropped onto ice-cold, wet slides, where the metaphases were spread by vigorous blowing and cautious flaming. Chromosomes were stained in orcein (2% in 60% acetic acid) for 10 min. The slides were cleared by dipping them twice briefly into absolute ethanol and by treating them in two changes of xylene before mounting. Although the slides were not coded, every precaution was taken to reduce possible bias. These measures included the strict adherence to predefined damage types (Gebhart, 1970; Schoeller and Wolf, 1970; Evans and O'Riordan, 1975). Also, only well spread and complete metaphases, deviating not more than 2 from the modal number of chromosomes characteristic for this cell line, were considered for analysis, i.e. only metaphases with 18-22 chromosomes were analyzed.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Resorcinoi diglycidyl ether (RDGE) was synthesized according to pub- lished methods (Ross et al., 1964); briefly, the parent compounds, N-methylaniline, aniline, and resorcinol, resp., were first reacted with an approximately 10-fold excess of epichloro- hydrin at slightly elevated temperatures. Upon completion of the reaction, a 50% sodium hydrox- ide solution was added slowly over a period of 1-3h in an amount equivalent to the number of reactive sites. The product was finally dissolved in toluene, washed with water and dried over anhydrous sodium sulfate. The solvent was then removed in vacuo and the product was purified by vacuum distillation. The purified product was analyzed by HPLC (on a silica column, with the same solvent as above) and was estimated to contain less than 2% impurities. The alkylating potency of the epoxide was measured in the 4-(4-nitro- benzyl)pyridine (NBP) assay according to Friedman and Boger (1961).

Target gene:

Not Specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster cells were obtained the CHO-KI cells from Flow Laboratories AG (Baar, Switzerland); they were cultured in F-12 medium, containing 10% foetal calf serum, at 37°C and in an atmo- sphere of 5% CO 2.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

2.5, 8 and 25 µg/mL

Vehicle / solvent:

CHO Cells: Solvent used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

Chinese hamster CHO-KI cells were obtained from Flow Laboratories AG (Baar, Switzerland); they were cultured in F-12 medium, containing 10% foetal calf serum, at 37°C and in an atmosphere of 5% CO 2.

For the test, 2x10E05 cells were seeded into 25-cm2 culture flasks and incubated for 24 h. After this period, the test compounds were added in DMSO solution, and the cells were incubated for a further period of 6-24 h. Two hours prior to harvesting, the cells were arrested in C-mitosis by the addition of 10 µg/ml Colcemid. Harvesting of mitotic cells was accomplished by the mitotic shake-off method of Terasima and Tolmach (1961). The suspended cells were col- lected by centrifugation at 400 g and submitted to hypotonic treatment (75 mM KCI, 37°C, 20 min). After further centrifugation, the cells were fixed by dropwise addition of ice-cold, freshly prepared methanol/acetic acid fixative (3:1, v/v) under continuous shaking, The fixed cells were left in the refrigerator for at least 30 min, but preferably overnight; they were then spun down and fresh fixative was added; after further centrifugation. the cells were taken up in a few drops of fresh fixative and dropped onto ice-cold, wet slides, where the metaphases were spread by vigorous blowing and cautious flaming. Chromosomes were stained in orcein (2% in 60% acetic acid) for 10 min. The slides were cleared by dipping them twice briefly into absolute ethanol and by treating them in two changes of xylene before mounting. Although the slides were not coded, every precaution was taken to reduce possible bias. These measures included the strict adherence to predefined damage types (Gebhart, 1970; Schoeller and Wolf, 1970; Evans and O'Riordan, 1975): thus, according to these definitions, some damage although possibly representing an interstitial deletion, was classified as "gap" and therefore excluded from the aberration frequency calculations. Also, only well spread and complete metaphases, deviating not more than 2 from the modal number of chromosomes characteristic for this cell line, were considered for analysis, i.e. only metaphases with 18-22 chromosomes were analyzed.

Statistics:

Statistical significance of the aberration frequencies was estimated with the aid of the tables of Kastenbaum and Bowman (1970).

Reference:
Kastenbaum MA and Bowman KO (1970) Tables for determining the statistical significance of mutation frequencies. Mutation Res 9; 527-549.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

not specified

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 8 and 25 µg/mL

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The analysis of chromosomal damage caused by these substances in Chinese hamster cells in vitro, showed that resorcinol diglycidyl ether was very active (Table 1).
The compound was also observed to be toxic to CHO cells as seen by the reduction in the number of mitotic cells obtained at the early harvesting time of 6h and as a reduction in the number of metaphases obtained at both times at the highest test substance concentration. At the highest concentration of 25 µg/mL, resorcinol diglycidyl ether left virtually no cell undamaged.

Table 1: In Vitro Chromosomal Aberrations in CHO Cells induced by Resorcinol Diglycidyl Ether

	Treatment time (h)	Conc (µg/mL)	Metaphases scored	No. aberrant metaphases (%)	No. Breaks	No. transformations	Remarks
RDGE	6	2.5	100	8 (8)	11
		8	33	8 (24)	10		p<0.01
		25	25	11 (44)	16		p<0.01
Control		0	100	1 (1)		1
RDGE	24	2.5	100	9 (9)	9	4
		8	50	24 (48)*	18	7	p<0.01
		25	15	14 (93)*	14	1	p<0.01
Control		0	100	2 (2)	3

* Metaphases with multiple aberrations

Conclusions:

The test material demonstrates potential to cause chromosomal damage and cytotoxicity.

Executive summary:

The mutagenic and toxicity potential of resorcinol diglycidyl ether has been demonstrated in Chinese hamster CHO cells. The metabolic activation was not specified.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

chromosomal aberrations (ABS) and sister chromatid exchanges (SCE)

Type of information:

other: Publication

Adequacy of study:

key study

Study period:

This study was published in 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

no guideline followed

Principles of method if other than guideline:

Principle of test:
Short-term tests that detect the induction of chromosomal effects in cultured mammalian cells are an integral part of most testing schemes proposed to identify potential chemical carcinogens acting through genotoxic mechanisms. Cultured Chinese hamster ovary (CHO) cells are widely used to test chemicals for their ability to induce chromosomal aberrations (ABS) and sister chromatid exchanges (SCE).

GLP compliance:

no

Type of assay:

other: chromosomal aberrations and sister chromatid exchanges tests

Specific details on test material used for the study:

Chemical: DIGLYCIDYL RESORCINOL ETHER (EC 101-90-6)
Supplier: Ciba-Geigy
Purity: 87.69%
Solvent: DMSO (precipitated at 2500 µg/ml)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells, up to 15 passages since cloning, were used for all testing. Stock cells were obtained from Litton Bionetics (Kensington, MD) and stored in liquid nitrogen.
At least once per year representative cells were sent to Flow Laboratories (McLean, VA) for mycoplasma testing using the Hoechst stain test followed by the Agar and Hyorhinis test. Results from all tests for mycoplasma contamination were negative. CHO cells were maintained in McCoy’s 5A medium (modified) supplemented with L-glutamine (2 mM), antibiotics, and 10% fetal bovine serum (FBS). Pre- mixed culture medium and FBS were purchased from GIBCO Laboratories (Grand Island, NY). All stock and experimental cultures were maintained at 37°C in an atmo- sphere of 5% COz in air and 95% relative humidity

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

A liver fraction (S9) prepared from Aroclor 1254-induced male Sprague Dawley rats (Microbiological Associates, Bethesda, MD). The final concs of the S9 fraction, NADP, and isocitric acid were 0.02 ml, 2.4 mg, and 4.5 mg/mL culture medium respectively.

Test concentrations with justification for top dose:

Dose selection ten or eleven dose levels, at half-log intervals beginning at a high dose of 5 mg/ml (or as limited by solubility), were used for the first trial of the SCE study. The dose levels for ABS studies were chosen based on the toxicity of the test chemical observed in the SCE studies.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: MMC

Details on test system and experimental conditions:

Protocol for SCE Studies
Approximately 24 hr prior to cell treatment, 1 X lo6 cells were seeded per 75 cm2 flask. A culture was established for each dose both with and without metabolic activation. For assays without metabolic activation, the medium was replaced with fresh medium immediately before chemical treatment. Cells were treated with test or control substances for 2 hr to allow interaction with cells before the addition of bromodeoxyuridine (BrdUrd). BrdUrd was then added (final concentration 10 pM), and incubation was continued for an additional 24 hr. The medium was removed, and fresh medium containing 10 pM BrdUrd and colcemid was added and incubation was continued for 2-3 hr. For assays with metabolic activation, the cells were rinsed twice with phosphate buffered saline (PBS), after which culture medium without FBS was added. Cells were incubated for 2 hr in the presence of the test or control substances and the S9 reaction mixture. FBS was omitted to avoid the binding of serum proteins to short-lived, highly reactive intermediates. After the 2 hr exposure period, cells were washed twice with PBS, and then complete medium containing 10% FBS and 10 pM BrdUrd was added. Cells were incubated for an additional 26 hr, with colcemid present for the final 2-3 hr of incubation. Two to three hours after addition of colcemid, cells were harvested by mitotic shake-off. Prior to harvesting, the percent confluency in each flask was estimated using a widefield microscope. Harvested cells were treated for about 3 min at room temperature with hypotonic KCI (75 mM), washed with fixative (3: 1 methano1:glacial acetic acid, v/v), dropped onto slides, and air dried. Staining for the detection of SCE was accomplished by a modified fluorescence plus Giemsa (FPG) technique [Goto et al., 19781. Fifty second-division metaphase cells were scored per dose for the incidence of SCE. The number of chromosomes in each cell was also recorded. Any cell that had fewer than 19 or more than 23 chromosomes was excluded. All slides except for the high-dose positive controls were coded.

Protocol for ABS Assay
Approximately 24 hr prior to cell treatment, 1.2 x lo6 cells were seeded per 75 cm2 flask. A culture was established for each dose both with and without metabolic activation. For assays without metabolic activation, the testing approach was similar to the corresponding SCE studies except that cells were treated for about 10 hr and BrdUrd was omitted. Colcemid was added 2-3 hr prior to cell har- vest by mitotic shake-off. The test protocol for assays with metabolic activation was also similar to the corresponding SCE studies except that BrdUrd was omitted and cells were harvested approxi- mately 1 1 hr after removal of the S9 fraction. Colcemid was added 2 hr prior to harvest. Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored. The chromosome number was recorded for each cell and chromosome or chromatid type aberrations were classified into three categories: simple (breaks, fragments, double minutes), complex (interchanges, rearrangements), and other (pulverized, more than ten aberrationslcell).

Repeat Tests
Positive results in initial tests were confirmed by addi- tional tests. If both -S9 and +S9 studies gave a positive response and required confirmation, they were done se- quentially (-S9 first). If the -S9 repeat was positive, the repeat +S9 study was not always performed.

Cell Cycle Delay
The standard time for obtaining second-division meta- phase cells in SCE studies was 26 hr after adding BrdUrd. For chemicals that caused cell cycle delay, harvest times were extended, generally in 5 hr increments, with colcemid present for the last 2 hr. For ABS tests, harvest times were similarly extended based on the observation of cell cycle delay in the SCE trials.

pH During Chemical Treatment
In instances when a change in the pH of the culture me- dium was noticed after addition of the test chemical and the overall response was negative, the test was considered sufficient. If, however, the overall response was positive, the experiment was repeated with the pH adjusted to 7.4.

Precipitation of the Test Compound
If the test chemical was not soluble at 5 mg/ml, it was tested up to doses at which precipitate was visible (precipitation of diglycidyl resorcinol ether in DMSO above 500 µg/ml).

Statistics:

Statistical analyses were conducted on both the slopes of the dose-response curves and the individual dose points. An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occur- ring by chance at one dose point is cO.01; the probability for such a chance occurrence at two dose points is <0.001. Thus a trial with one dose showing an increase of 20% or greater was considered weak evidence of a positive re- sponse, and a trial with two doses showing an increase of 20% or greater was concluded to be positive. For the ABS data, the percentage of cells with aberrations was analyzed. As with SCE, both the dose-response curve and individual dose points were statistically analyzed. A statistically significant (P < 0.003) trend test or a significantly elevated dose point (P < 0.05) was sufficient to indicate a chemical effect. A detailed discussion of these statistical methods is presented in Margolin et al. [ 19861, and their application in determining test conclusions is further explained in Gallo- way et al. [1987]

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Remarks:

DMSO

Positive controls validity:

not specified

Additional information on results:

Treatment of cultured CHO cells with DGRE produced highly significant increases in ABS and SCE both with and without S9. These results agree with those presented by Seiler [1984] showing DGRE to be an extremely effective inducer of ABS in vitro: Treatment of CHO cells with 25 pg/ml caused chromosome damage in 93% of the cells. In contrast, oral administration of DGRE to mice in doses up to the LD50 did not elevate the frequency of micronucleated erythrocytes in the bone marrow [Seiler, 1984].

Conclusions:

Treatment of cultured CHO cells with DGRE (m-bis(23-epoxypropoxy)benzene) produced highly significant increases in ABS and SCE both with and without S9.

Executive summary:

Short-term tests that detect the induction of chromosomal effects in cultured mammalian cells are an integral part of most testing schemes proposed to identify potential chemical carcinogens acting through genotoxic mechanisms. Cultured Chinese hamster ovary (CHO) cells are widely used to test chemicals for their ability to induce chromosomal aberrations (ABS) and sister chromatid exchanges (SCE).

Cell line and Culture Medium: CHO cells, up to 15 passages since cloning, were used for all testing. Stock cells were obtained from Litton Bionetics (Kensington, MD) and stored in liquid nitrogen. At least once per year representative cells were sent to Flow Laboratories (McLean, VA) for mycoplasma testing using the Hoechst stain test followed by the Agar and Hyorhinis test. Results from all tests for mycoplasma contamination were negative. CHO cells were maintained in McCoy’s 5A medium (modified) supplemented with L-glutamine (2 mM), antibiotics, and 10% fetal bovine serum (FBS). Premixed culture medium and FBS were purchased from GIBCO Laboratories (Grand Island, NY). All stock and experimental cultures were maintained at 37°C in an atmosphere of 5% CO2 in air and 95% relative humidity.

Metabolic Activation: A liver fraction (S9) prepared from Aroclor 1254-induced male Sprague Dawley rats (Microbiological Associates, Bethesda, MD) was used to provide exogenous metabolic activation. The final concentrations of the S9 fraction, NADP, and isocitric acid were 0.02 mL, 2.4 mg and 4.5 mg, respectively, per milliliter culture medium.

Dose Selection: Ten or eleven dose levels, at half-log intervals beginning at a high dose of 5 mg/ml (or as limited by solubility), were used for the first trial of the SCE study. The dose levels for ABS studies were chosen based on the toxicity of the test chemical observed in the SCE studies.

Protocol for SCE Studies Approximately 24 h prior to cell treatment, 1x10^6 cells were seeded per 75 cm2 flask. A culture was established for each dose both with and without metabolic activation. For assays without metabolic activation, the medium was replaced with fresh medium immediately before chemical treatment. Cells were treated with test or control substances for 2 h to allow interaction with cells before the addition of bromodeoxyuridine (BrdUrd). BrdUrd was then added (final concentration 10 µM), and incubation was continued for an additional 24 h. The medium was removed, and fresh medium containing 10 µM BrdUrd and colcemid was added and incubation was continued for 2-3 h. For assays with metabolic activation, the cells were rinsed twice with phosphate buffered saline (PBS), after which culture medium without FBS was added. Cells were incubated for 2 h in the presence of the test or control substances and the S9 reaction mixture. FBS was omitted to avoid the binding of serum proteins to short-lived, highly reactive intermediates.After the 2 h exposure period, cells were washed twice with PBS, and then complete medium containing 10% FBS and 10 µM BrdUrd was added. Cells were incubated for an additional 26 h, with colcemid present for the final 2-3 h of incubation.

Two to three hours after addition of colcemid, cells were harvested by mitotic shake-off. Prior to harvesting, the percent confluency in each flask was estimated using a widefield microscope. Harvested cells were treated for about 3 min at room temperature with hypotonic KCI (75 mM), washed with fixative (3:1 methano1:glacial acetic acid, v/v), dropped onto slides, and air dried. Staining for the detection of SCE was accomplished by a modified fluorescence plus Giemsa (FPG) technique [Goto et al., 19781]. Fifty seconddivision metaphase cells were scored per dose for the incidence of SCE. The number of chromosomes in each cell was also recorded. Any cell that had fewer than 19 or more than 23 chromosomes was excluded. All slides except for the high-dose positive controls were coded.

Protocol for ABS Assay: Approximately 24 h prior to cell treatment, 1.2 x 10^6 cells were seeded per 75 cm2 flask. A culture was established for each dose both with and without metabolic activation. For assays without metabolic activation, the testing approach was similar to the corresponding SCE studies except that cells were treated for about 10 hr and BrdUrd was omitted. Colcemid was added 2-3 h prior to cell harvest by mitotic shake-off.

The test protocol for assays with metabolic activation was also similar to the corresponding SCE studies except that BrdUrd was omitted and cells were harvested approximately 11 h after removal of the S9 fraction. Colcemid was added 2 h prior to harvest. Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored. The chromosome number was recorded for each cell and chromosome or chromatid type aberrations were classified into three categories: simple (breaks, fragments, double minutes), complex (interchanges, rearrangements), and other (pulverized, more than ten aberrations/cell).

Repeat Tests: Positive results in initial tests were confirmed by additional tests. If both -S9 and +S9 studies gave a positive response and required confirmation, they were done sequentially (-S9 first). If the -S9 repeat was positive, the repeat +S9 study was not always performed.

Cell Cycle Delay: The standard time for obtaining second-division metaphase cells in SCE studies was 26 h after adding BrdUrd. For chemicals that caused cell cycle delay, harvest times were extended, generally in 5 h increments, with colcemid present for the last 2 h. For ABS tests, harvest times were similarly extended based on the observation of cell cycle delay in the SCE trials.

pH During Chemical Treatment: In instances when a change in the pH of the culture medium was noticed after addition of the test chemical and the overall response was negative, the test was considered sufficient. If, however, the overall response was positive, the experiment was repeated with the pH adjusted to 7.4.

Statistical Analysis: Statistical analyses were conducted on both the slopes of the dose-response curves and the individual dose points. An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose point is cO.01; the probability for such a chance occurrence at two dose points is <0.001. Thus a trial with one dose showing an increase of 20% or greater was considered weak evidence of a positive response, and a trial with two doses showing an increase of 20% or greater was concluded to be positive. For the ABS data, the percentage of cells with aberrations was analyzed. As with SCE, both the dose-response curve and individual dose points were statistically analyzed. A statistically significant (P < 0.003) trend test or a significantly elevated dose point (P < 0.05) was sufficient to indicate a chemical effect. A detailed discussion of these statistical methods is presented in Margolin et al. [ 19861, and their application in determining test conclusions is further explained in Galloway et al. [1987].

Results: Treatment of cultured CHO cells with DGRE produced highly significant increases in ABS and SCE both with and without S9. These results agree with those presented by Seiler [1984] showing DGRE to be an extremely effective inducer of ABS in vitro: Treatment of CHO cells with 25 µg/ml caused chromosome damage in 93% of the cells. In contrast, oral administration of DGRE to mice in doses up to the LD50 did not elevate the frequency of micronucleated erythrocytes in the bone marrow [Seiler, 19841].

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

in vivo mouse: cytogenicity / bonne marrow chromosome aberration

Genetic toxicity tests in vivo in mouse were documented in two publications (Seiler, 1984 and Shelby et al, 1993). The study of Seiler (1984) has demonstrated that the test item was completely inactive in vivo micronucleus assay in mice after single oral dosing at 300 and 600 mg/kg. In the paper of Shelby et al (1993) was noted that due to the test item toxicity characteristics in a designed protocol was not possible to use of a sufficiently high exposure to induce observable genetic toxicity (the highest reported intraperitoneal IP dose 270 mg/kg). Since the paper is conducted not exactly according to guideline (e.g. missing historical control), it is difficult to conclude on reliability of outcome. However, based on in vivo micronucleus results, which were inconsistent depending on the testing protocol, the Dutch Expert Committee on Occupational Safety a Committee of the Health Council of the Netherlands considered that resorcinol diglycidyl ether may have genotoxic potential.

A weight of evidence of the analyzed results suggest that the test item is negative for the genetic toxicity in vivo endpoint.

Link to relevant study records

Reference

Reference 1

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:

This study was accepted for publication on 17 October 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Reliability 2 was assigned as the study was conducted to a recognised procedure it is a comparative study with no clear endpoint data.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The micronucleus test was performed in mice according to the established procedures (Schmid, 1976; Heddle and Salamone, 1981). Male and female mice were given the test compounds orally, dissolved in polyethyleneglycol (PEG 400), in doses up to acutely toxic levels. 24 h after this single dose, the mice were sacrificed and the bone marrow cells were flushed out into foetal calf serum. In the case of a negative outcome of the test, a second assay was performed with fixation times of 24, 48, and 72 h, resp. After centrifugation at 400 g, the cells were spread onto slides, air-dried and stained with May-Grünwald/Giemsa. In this case, the slides were coded and analysed by two individuals separately.

GLP compliance:

no

Type of assay:

other: Chromosomal aberration

Specific details on test material used for the study:

Resorcinoi diglycidyl ether (RDGE) was synthesized according to pub- lished methods (Ross et al., 1964); briefly, the parent compounds, N-methylaniline, aniline, and resorcinol, resp., were first reacted with an approximately 10-fold excess of epichloro- hydrin at slightly elevated temperatures. Upon completion of the reaction, a 50% sodium hydrox- ide solution was added slowly over a period of 1-3h in an amount equivalent to the number of reactive sites. The product was finally dissolved in toluene, washed with water and dried over anhydrous sodium sulfate. The solvent was then removed in vacuo and the product was purified by vacuum distillation. The purified product was analyzed by HPLC (on a silica column, with the same solvent as above) and was estimated to contain less than 2% impurities. The alkylating potency of the epoxide was measured in the 4-(4-nitro-benzyl)pyridine (NBP) assay according to Friedman and Boger (1961).

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Mice of the ICR-strain were obtained from the Institute of Animal Husbandry of the University of Zurich

Route of administration:

oral: gavage

Vehicle:

Polyethylene glycol (PEG 400)

Details on exposure:

Male and female mice of body weight circa 25 g, were given the test compounds orally, dissolved in polyethylene-glycol (PEG 400), in doses up to acutely toxic levels.

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

24h

Dose / conc.:

300 mg/kg bw (total dose)

Remarks:

first assay: 24 after single dose

Dose / conc.:

600 mg/kg bw (total dose)

Remarks:

second assay was performed with fixation times of 24, 48 and 72 h

No. of animals per sex per dose:

4 males and 4 females

Control animals:

no

Tissues and cell types examined:

polychromatic erythrocytes from the bone marrow

Details of tissue and slide preparation:

24 h after this single dose, the mice were sacrificed and the bone marrow cells were flushed out into foetal calf serum. In the case of a negative outcome of the test, a second assay was performed with fixation times of 24, 48, and 72 h, resp. After centrifugation at 400 g, the cells were spread onto slides, air-dried and stained with May-Grtinwald/ Giemsa.

Evaluation criteria:

Not specified

Statistics:

There the standard deviation of the frequency of micronucleated erythrocytes (per thousand polychromatic erythrocytes) was calculated. The spontaneous frequency of micronucleated polychromatic erythrocytes (3-5 per thousand polychromatic erythrocytes, standard deviation 1.8) has been subtracted.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

one out of four mice died within 48 h in each group

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Mice were given solutions of the test compound orally in dosages up to the LD50, and the polychromatic erythrocytes from the bone marrow of such treated animals were checked for the presence of micronuclei. The in vitro much more active RDGE, on the other hand, proved to be completely inactive. The inability of RDGE to induce micronuclei in the bone marrow of mice in vivo could not be traced to some influence on the length of the cell cycle, since the compound was also inactive at 48 and 72 h after dosage, nor was the dose given insufficient, since 1 out of 4 mice died within 48 h in each experimental group.

Table 1 INDUCTION OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES 1N THE BONE MARROW OF MICE TREATED WITH RESORCINOL DIGLYCIDYL ETHER

Fixation Time (h)	Frequency (and standard deviation) of micronucleated erythrocytes (per thousand polychromatic erythrocytes) at dose (mg/kg p.o.) of
	300	400	500	600	800	1000	1500
24	0.0 (1.5)			0.0 (1.9)
48				0.9 (1.3)
72				0.4 (0.6)

The spontaneous frequency of micronucleated polychromatic erythrocytes (3-5 per thousand polychromatic erythrocytes, standard deviation 1.8) has already been subtracted.

Conclusions:

The article has demonstrated that resorcinol diglycidyl ether (RDGE) was completely inactive in vivo micronucleus assay in mice.

Executive summary:

The micronucleus test was performed in male and female mice of the ICR-strain. The test item resorcinol diglycidyl ether (RDGE) dissolved in polyethylene-glycol (PEG 400) was given orally in doses up to acutely toxic level. 24 h after the single dose (300 mg/kg po), the mice were sacrificed and the bone marrow cells were flushed out into foetal clf serum. In the case of negative outcome of the test, a second assay was performed with fixation times of 24, 48 and 72 h, respectively. After centrifugation at 400 g, the cells were spread onto slides, air-dried and stained with May-Grünwald/Giemsa. The slides were coded and analysed by two individuals separately. The polychromatic erythrocytes from the bone marrow of treated animals were checked for the presence of micronuclei. RDGE proved to be completely inactive. The inability of RDGE to induce micronuclei in the bone marrow of mice in vivo could not be traced to some influence on the length of the cell cycle, since the compound was also inactive at 48 and 72 h after dosage, nor was the dose given insufficient, since 1 out of 4 mice died within 48 h.

Reference 2

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 5 male B6C3F1 mice were treated by intraperitoneal (IP) injection to the 3 doses of test item at a volume of 0.4 mL per mouse on three consecutive days. Animals were monitored twice daily and 24 h after the third treatment. The surviving mice were euthanized by CO2 asphyxiation. Bonne marrow smears (two slides/tissue/mouse) were prepared by a direct technique (Tice et al, 1990). Air-dried smears were fixed using absolute methanol and stained with acridine orange (Tice et al, 1990a). Bone marrow smears from each animal were evaluated at 1000 x magnification using epi-illuminated fluorescence microscopy (450-490 nm excitation, 520 nm emission) for determination of the percentage of PCE among 200 erythrocytes.
For the initial MN test, groups of 5 mice were injected IP on three consecutive days with either the test item (at 1x, ½ x and 1/ x ,where x is the maximum dose determined in the dose determination experiments), a weakly active dose of the positive control chemical (DMBA in corn oil) or the appropriate solvent. Mice were euthanized with CO2, 24 hr after the third treatment. Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol and stained with acridine orange. For each animal, slides were evaluated at 1,000 x magnification for the number of MN-PCE among 2,000 PCE and for the percentage of PCE among 200 erythrocytes.
Repeat tests were performed for the test item based on results of the initial micronucleus test since the results suggested a possible effect.
The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.3), which was designed specifically for in vivo micronucleus data.
The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE. The number of MN-PCE were pooled within each dose group
and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability. In the event that the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positives. The %PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each group and the concurrent solvent control group was by an unadjusted one-tailed Pearson chi squared test which incorporated the calculated variance inflation factor for the study.

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
NTP chemical repository (Radian Corporation, Austin, TX)

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: the National Toxicology Program production facility at Taconic Farms
- Age at study initiation: between 9 and 14 weeks
- Weight at study initiation: between 25 and 33 g

Route of administration:

intraperitoneal

Vehicle:

- Vehicle/solvent used: corn oil
- Justification for choice of solvent/vehicle: water-insoluble chemical
- Concentration of test material in vehicle: 0, 15.2, 30.4 and 60.8 mg/kg/day
- Amount of vehicle (IP):0.4 mL/mouse

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended using a Tek-Mar Tissumizer(R) and administered within 30 min of preparation

Duration of treatment / exposure:

Three consecutive days

Frequency of treatment:

Once per day

Post exposure period:

24 h after the third (last) treatment

Dose / conc.:

0 mg/kg bw/day

Remarks:

Initial test, 3-day treatment

Dose / conc.:

15.2 mg/kg bw/day

Remarks:

Initial test, 3-day treatment

Dose / conc.:

30.4 mg/kg bw/day

Remarks:

Initial test, 3-day treatment

Dose / conc.:

60.8 mg/kg bw/day

Remarks:

Initial test, 3-day treatment

Dose / conc.:

0 mg/kg bw/day

Remarks:

Single-exposure MN test

Dose / conc.:

90 mg/kg bw/day

Remarks:

Single-exposure MN test

Dose / conc.:

180 mg/kg bw/day

Remarks:

Single-exposure MN test

Dose / conc.:

270 mg/kg bw/day

Remarks:

Single-exposure MN test

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

7,12-dimethylbenzanthracene purchased from Eastman Kodak (Rochester, NY)
- Route of administration: IP
- Doses / concentrations: a weakly active dose

Tissues and cell types examined:

Bonne marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: doses were selected at 1, 0.5 and 0.25 of the maximum dose determined in the dose determination experiments

TREATMENT AND SAMPLING TIMES: for initial test 3-day treatment and sampling after 24 h of the last treatment, for single-exposure MN test sampling after 24 h after treatment

DETAILS OF SLIDE PREPARATION: 2 slides mouse fixed in absolute methanol and stained with acridine orange

METHOD OF ANALYSIS: For each animal, slides were evaluated at 1,000x magnification for the number of MN-PCE among 2,000 PCE and for percentage of PCE among 200 erythrocytes

Evaluation criteria:

To determine whether a specific treatment resulted in a significant increase in MN-PCE, the number of MN-PCE were pooled within each dose groupand analyzed by a one-tailed trend test. The level of significance was set at an alpha level of 0.05.

Statistics:

The %PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each group and the concurrent solvent control group was by an unadjusted one-tailed Pearson chi squared test which incorporated the calculated variance inflation factor for the study.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: Overall result

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

dose-related increase in micronuclei

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: concentrations: 90, 180 and 270 mg/kg

Conclusions:

The initial test was positive, but the two repeat tests were negative. The test was limited since the toxicity was too high in the repeated tests to induce observable genetic toxicity. Therefore a single high-dose test was performed and the results showed a statistically significant dose-related increase in cells with micronuclei.

Executive summary:

Groups of 5 male B6C3F1 mice were treated by intraperitoneal (IP) injection to the 3 doses of test item at a volume of 0.4 mL per mouse on three consecutive days. Animals were monitored twice daily and 24 h after the third treatment. The surviving mice were euthanized by CO2 asphyxiation. Bone marrow smears (two slides/tissue/mouse) were prepared by a direct technique (Tice et al, 1990). Air-dried smears were fixed using absolute methanol and stained with acridine orange (Tice et al, 1990a). Bone marrow smears from each animal were evaluated at 1000 x magnification using epi-illuminated fluorescence microscopy (450-490 nm excitation, 520 nm emission) for determination of the percentage of PCE among 200 erythrocytes.

For the initial MN test, groups of 5 mice were injected IP on three consecutive days with either the test item (at 1x, ½ x and 1/ x ,where x is the maximum dose determined in the dose determination experiments), a weakly active dose of the positive control chemical (DMBA in corn oil) or the appropriate solvent. Mice were euthanized with CO2, 24 hr after the third treatment. Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol and stained with acridine orange. For each animal, slides were evaluated at 1,000 x magnification for the number of MN-PCE among 2,000 PCE and for the percentage of PCE among 200 erythrocytes.

Repeat tests were performed for the test item based on results of the initial micronucleus test since the results suggested a possible effect.

The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.3), which was designed specifically for in vivo micronucleus data.

The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE. The number of MN-PCE were pooled within each dose group

and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability. In the event that the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positives. The %PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each group and the concurrent solvent control group was by an unadjusted one-tailed Pearson chi squared test which incorporated the calculated variance inflation factor for the study.

The initial test was positive to 60.8 mg/kg with trend P = 0.038. Repeal tests to 60.8 mg/kg and 91.2 mg/kg were both negative and the overall result was concluded to be negative. Because authors knew this chemical was highly effective at inducing chromosomal aberrations in mouse bone marrow cells following single exposures to doses up to 300 mg/kg (McFee personal communication; Tice, unpublished), a single-exposure micronucleus test was conducted. Bone marrow cells were harvested 24 h after treatment. A significant dose-related increase in MN-PCE was observed at doses of 90, 180 and 270 mg/kg (Table 2). It appears that due to the toxicity characteristics of DGRE, a three-exposure protocol does not permit use of a sufficiently high exposure to induce observable genetic toxicity.

With the test item, the range of daily doses used in the three-exposure protocol (15.2 to 91.2 mg/kg) was also lower than the range of doses used in the single exposure chromosome aberration studies (62.5 to 400 mg/kg). In a single exposure MN test, positive results were obtained using doses up to 270 mg/kg, indicating that the cumulative toxicity of multiple exposures restricted the daily doses to a level that did not induce a detectable increase in MN. The 91.5 mg/kg/day exposure added up to approximately 270 mg/kg, but there was apparently no substantial induction of MN, perhaps due to effective detoxification of the low exposures of efficient repair of induced damage during the intervals between exposures.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

Resorcinol diglycidyl ether showed some mutagenic potential in vivo and was mutagenic in vitro, therefore the mode of action may be described by a stochastic genotoxic mechanism.

Additional information

The conclusion of the results is supported by the report: Health Council of the Netherlands. Resorcinol diglycidyl ether. Evaluation of the carcinogenicity and genotoxicity. Subcommittee on the Classification of Carcinogenic Substances of the Dutch Expert Committee on Occupational Safety a Committee of the Health Council of the Netherlands (2016). No. 2016/03

Justification for classification or non-classification

Based on the available data, it is assumed that the substance acts by a stochastic genotoxic mechanism and may be classified as a germ cell mutagen category 2, H341 Suspected of causing genetic defects (state route of exposure if it is conclusively proven that no other routes of exposure cause the hazard) in accordance with Regulation (EC) No. 1272/2008 and its amendments.