Bis[(3,4-epoxycyclohexyl)methyl] adipate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 March 1988 to 10 March 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: BRRC Standard Operating Procedures 7.4.1B, 7.4.2B, 7.4.4C, 7.4.5B, 7.4.6B, 7.4.7C, 7.4.12C, 7.4.13 and 7.4.15

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis[(3,4-epoxycyclohexyl)methyl] adipate

EC Number:

221-518-5

EC Name:

Bis[(3,4-epoxycyclohexyl)methyl] adipate

Cas Number:

3130-19-6

Molecular formula:

C20H30O6

IUPAC Name:

1,6-bis({7-oxabicyclo[4.1.0]heptan-3-yl}methyl) hexanedioate

Test material form:

liquid: viscous

Method

Target gene:

- Histidine requirement in Salmonella typhimurium strains (Histidine operon).

Species / strain

Species / strain / cell type:

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

Remarks:

And TA 1538

Details on mammalian cell type (if applicable):

- Indicator organisms are stored at -80 °C. Working cultures are prepared monthly by inoculating nutrient broth from the frozen cultures and incubating with agitation overnight. Bacteria are then plated onto Vogel-Bonner Medium E agar plates (master plates) with an excess of histidine and biotin (required because of the lipopolysaccharide deficiency). After incubation for 24 hours, the strains are checked for their genetic markers to verify their identity and purity.
- For testing, the broth cultures are prepared by inoculating from the master plates or directly from frozen permanent stock cultures into nutrient broth and incubated overnight (8 to 10 hours) with agitation. The broth cultures are kept on ice during the day of testing. Fresh cultures are made each day of testing.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254 Induced, rat-liver homogenate (S9 Mix)

Test concentrations with justification for top dose:

- Preliminary cytotoxicity test: 0.01, 0.03, 0.1, 0.3, 1, 3, 5, 10, 30 and 115 mg/plate
- Main assay (without activation): 0.1, 0.3, 1, 3 and 5 mg/plate
- Main assay (with activation): 0.3, 1, 3, 5 and 10 mg/plate
- A preliminary toxicity test was performed with and without an S9 metabolic activation system using strain TA100 to determine the level of toxicity of the test material. Ten doses were tested for toxicity with a plate assay performed in the manner used for mutagenicity determinations. Toxicity was assessed at 48 hours after treatment by observing growth inhibition of the background lawn and/or a reduction in the number of spontaneous mutants.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- The sample was determined to be soluble in dimethylsulfoxide (DMSO) up to a concentration of 100 g/mL. Thus, DMSO was used as the solvent for dilution of the test agent. Dilutions were prepared fresh on the morning of each test day and the accuracy of dilution was verified by gravimetric analysis.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine 0.01mg without metabolic activation and 2-aminoanthracene 2.5 µg with metabolic activation

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST
- A preliminary toxicity test was performed with and without an S9 metabolic activation system using strain TA100 to determine the level of toxicity of the test material. Ten doses (0.01, 0.03, 0.1, 0.3, 1, 3, 5, 10, 30 and 115 mg/plate) were tested for toxicity with a plate assay performed in the manner used for mutagenicity determinations. Toxicity was assessed at 48 hours after treatment by observing growth inhibition of the background lawn and/or a reduction in the number of spontaneous mutants.
- Method: Sterile tubes were prepared containing 2 mL soft agar (6 g/L agar and 5 g/L NaCl) with a final concentration of 0.05 mM L-histidine and 0.05 mM D-biotin (the entire mixture was called top agar). Dilutions of the test material were made in an appropriate solvent so that the correct amount of test material can be added to each tube in 100 µL amounts. If less than 100 µL amounts of the test material are used, phosphate-buffered saline is used to adjust the total volume to 100 µL.
A 100 µL aliquot of an overnight broth culture of strain TA100 was added followed by the appropriate amount of test material. The mixture was vortexed and poured onto the surface of a Vogel-Bonner Medium E agar plate (VB-E plate). The top agar was allowed to harden and the plates were incubated at 37 °C for 48 hours. The plates were examined for the condition of their background lawns and growth is recorded as either confluent, sparse, or absent. Confluency is considered an indication of nontoxicity, sparse growth indicates moderate toxicity, and lack of growth is recorded as toxic. Numbers of revertant colonies were counted using an Artek® electronic colony counter or manually counted if the chemical produces a heavy precipitate.

MUTAGENICITY ASSAY
- The test material was tested in triplicate at five doses chosen to span a range which included moderately toxic to relatively nontoxic concentrations. Testing was performed both with and without metabolic activation. Concurrent solvent and positive controls were tested in each experiment.
- Method: To a sterile tube containing 2 mL of top agar, a 100 µL aliquot of the appropriate bacterial culture was added followed by the addition of 100 µL of the appropriate solvent, control, or test material solution. Either 0.5 mL of S9 mix or 0.5 mL of phosphate-buffered saline (PBS) was added for tests with and without metabolic activation, respectively. The top agar mixture is then poured onto a VB-E plate. Each dose was tested in triplicate and with all five bacterial strains. Sterility checks were done on the PBS and/or S9 mix, all solvents, and the highest concentration of each test material. The plates were transferred to a darkened 37 °C incubator after hardening and incubated for 48 to 72 hours.

SCORING
- Bacterial colonies were counted manually or by an Artek Model #880 Colony Counter. The counter is calibrated for each test to check the counting accuracy.
- The numbers of colonies per plate were counted and recorded. An examination is also made of the background lawn on each plate. If toxicity is observed as an inhibition of growth of the background lawn, the plate was not counted, and was recorded as toxic. If the background lawn is sparse and the colony count was still recorded, that number is not used in the calculation of the mean number of colonies and its standard deviation. A reduction in the number of spontaneous revertant colonies is also an indication of toxicity, and these were labelled "toxic" when the average number of colonies was less than one half of the average number for the solvent control.

Evaluation criteria:

CRITERIA FOR TEST VALIDITY AND INTERPRETATION OF RESULTS
- The spontaneous reversion for the solvent controls should be within the laboratory's historical range.
- The positive controls should demonstrate that the test systems are responsive with known mutagens.
- A test material is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies.
- If a test material produces a marginal or weak response that cannot be reproduced in a second test, the test result will be considered negative.
- If there is no evidence of a dose-related increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test material is not considered to be a bacterial mutagen.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TEST
- In preliminary tests to determine cytotoxicity, ten concentrations of the test material ranging from 0.01 to 115 mg/plate were tested with and without the presence of an S9 metabolic activation system with strain TA100 only. Cytotoxicity was defined as either a reduction in the number of revertant colonies or an inhibition of growth of the background lawn.
- A dose level of 30 mg/plate was not soluble in the overlay agar in the test performed without S9. Thus, cytotoxicity was observed as absence of bacterial growth in zones around the precipitate while confluent growth was observed in the areas that were not exposed to the test material. A lower dose level of 5 mg/plate was also cytotoxic, allowing only sparse growth of the background lawn.
- In the preliminary test performed with activation, the two highest dose levels of 30 and 115 were not soluble in the overlay agar and thus allowed confluent growth of the background lawn with zones of growth inhibition around the precipitated test material. A dose of 10 mg/plate produced a moderate degree of cytotoxicity evident by sparse growth of the background lawn with no reduction in relative numbers of revertant colonies. When possible, the highest dose is selected to produce a moderate degree of cytotoxicity. Thus, based on the results of these preliminary toxicity tests, 5 doses ranging from 0.1 to 5 mg/plate were tested without S9 and 5 doses ranging from 0.3 to 10 mg/plate were tested in the presence of S9 in definitive mutagenicity tests.

MUTAGENICITY TEST
- Results of the mutagenicity test are shown in Table 1.
- In tests performed without S9, no indication of mutagenicity was observed with any of the strains, either by evidence of a dose-response relationship or a doubling of the average number of colonies over the average solvent control value. All five strains showed signs of treatment-related inhibition of growth of the background lawn with at least the highest dose level of test material tested (5 mg/plate).
- In the presence of S9, highly positive (> 10-fold) and dose-related increases in relative numbers of revertant colonies were observed with TA1535. Positive and dose-related mutagenic activity was also observed in the preliminary test performed with TA100 in the presence of S9. However, only weak mutagenic activity was evident in the definitive test performed with S9 using strain TA100. Thus, the test material was considered to be mutagenic in the presence of S9 activation in this in vitro bacterial assay.
- All five bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without S9 metabolic activation. Negative (solvent) controls were also tested with each strain, and in general, the average number of spontaneous revertants was within the historical ranges at the laboratory. All positive and negative controls were tested concurrently with the test material. Concurrent sterility testing showed that the S9 mix, PBS, the test material and the solvent control agent were sterile.

Any other information on results incl. tables

Table 1: Summary of Mutagenicity Experiment

± S9 Mix	Concentration (mg/plate)	Mean number of colonies/plate
		TA100	TA1535	TA1538	TA98	TA1537
-	Solvent 0.1 0.3 1 3 5	98 103 120 116 56 T	18 19 21 29 16 (S) T	8 5 12 5 4 (T) T	19 23 30 17 13 T/S	4 6 5 7 1 (T) T
+	Solvent 0.3 1 3 5 10	100 113 133 134 127 (S) 103 (S)	13 34 95 155 66 (S) 127	19 21 16 12 6 4	28 29 25 26 21 12	5 4 7 5 4 (T) 3
Positive Controls
-	Name	SA	SA	4NPD	4NPD	9AA
	Concentration (mg/plate)	0.01	0.01	0.01	0.01	0.06
	Mean no. colonies/plate	1915	1811	908	604	251
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2.5	2	2.5	2.5	2.5
	Mean no. colonies/plate	610	59	785	1500	66

4NPD = 4-nitro-0-phenylenediamine

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

S= Sparse growth of background lawn; counts not included in calculation of mean and standard deviation.

T = TOXIC: Clearing of background lawn, or average number of colonies < ½ solvent control value.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study the test material was considered to be mutagenic in the presence of metabolic activation with Salmonella typhimurium strain TA1535 and marginally mutagenic with Salmonella typhimurium TA100.

Executive summary:

The potential of the test material to cause mutagenic activity was investigated in a study similar to OECD 471, under GLP conditions.

Test doses for the Ames test were chosen from data obtained in a preliminary study using Salmonella typhimurium strain TA100. Preliminary cytotoxicity results of tests performed without the presence of a rat-liver S9 metabolic activation mixture showed that a concentration of 10 mg/plate produced a complete absence of growth of the background lawn of bacteria while a lower dose of 5 mg/plate allowed sparse growth of the bacterial lawn. A higher dose of 30 mg/plate was not soluble in the overlay agar which resulted in confluent growth of the background lawn. In the preliminary test with S9, a dose of 10 mg/plate produced sparse growth of the background lawn. Higher doses were not soluble which permitted confluent growth of the background lawn except around the areas where the chemical formed precipitate. Based on these results, the test material was tested with five concentrations ranging from 0.1 to 5 mg/plate in the absence of S9, and from 0.3 to 10 mg/plate in the presence of S9, using triplicate cultures at each dose level.

For the main mutagenicity tests, a sterile tube containing 2 mL of top agar, a 100 µL aliquot of the appropriate bacterial culture was added followed by the addition of 100 µL of the appropriate solvent, control, or test material solution. Either 0.5 mL of S9 mix or 0.5 mL of phosphate-buffered saline (PBS) was added for tests with and without metabolic activation, respectively. The top agar mixture was then poured onto a VB-E plate. Each dose was tested in triplicate and with all five bacterial strains. Sterility checks were done on the PBS and/or S9 mix, all solvents, and the highest concentration of each test material. The plates were transferred to a darkened 37 °C incubator after hardening and incubated for 48 to 72 hours. Concurrent solvent and positive controls were tested in each experiment.

In tests performed without S9, no indication of mutagenicity was observed with any of the strains, either by evidence of a dose-response relationship or a doubling of the average number of colonies over the average solvent control value. All five strains showed signs of treatment-related inhibition of growth of the background lawn with at least the highest dose level of test material tested (5 mg/plate). In the presence of S9, highly positive (> 10-fold) and dose-related increases in relative numbers of revertant colonies were observed with TA1535. Positive and dose-related mutagenic activity was also observed in the preliminary test performed with TA100 in the presence of S9. However, only weak mutagenic activity was evident in the definitive test performed with S9 using strain TA100.

All five bacterial strains exhibited a positive mutagenic response with the positive controls tested both with and without S9 metabolic activation. Negative (solvent) controls were also tested with each strain, and in general, the average number of spontaneous revertants was within the historical ranges at the laboratory. All positive and negative controls were tested concurrently with the test material. Concurrent sterility testing showed that the S9 mix, PBS, the test material and the solvent control agent were sterile.

Under the conditions of this study the test material was considered to be mutagenic in the presence of metabolic activation with Salmonella typhimurium strain TA1535 and marginally mutagenic with Salmonella typhimurium TA100.