3-methyl-1,3-butanediol-1-ethylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-08-23 to 2018-08-25 (experiment start-end)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1,3-butanediol-1-ethylate

EC Number:

816-856-0

Cas Number:

5205-01-6

Molecular formula:

C7H14O3

IUPAC Name:

3-methyl-1,3-butanediol-1-ethylate

Specific details on test material used for the study:

3-methyl-1,3-butanediol-1-acetate
Batch number: 161121
Purity: 86.5%
Correction factor: 1.156
Expiry date: May 2019
Appearance: Colourless liquid
Storage conditions: Room temperature (15-25°C)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Strains TA98, TA1535 and TA102 obtained from MOLTOX, INC., USA
Strains TA100 and TA1537 obtained from Xenometrix AG
All cell stocks stored in liquid nitrogen

Metabolic activation:

with and without

Metabolic activation system:

Male wistar rats treated orally with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three days

Test concentrations with justification for top dose:

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
5.0 µL is the maximum volume as recommended by OECD guideline 471 (1997)

Vehicle / solvent:

Purified water

Controls

Untreated negative controls:

yes

Remarks:

Purified water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

Samples of each tester strain were grown by culturing for 12 h at 37°C in Nutrient Broth to the late exponential or early stationary phase of growth (approximately 10^9 cells/mL). The nutrient medium consisted of 8g/L nutrient broth and 5 g/L sodium chloride.
A solution of 125 µL ampicillin (10 mg/mL) (TA98, TA100, TA102) was added in order to retain the phenotypic characteristics of the strain.
For the plate incorporation method 100 µL test item solution or negative or positive control, 500 µL S9 mix or S9 substitution buffer, 100 µL bacterial suspension, 2 mL overlay agar were mixed in a test tube and poured over the surface of a minimal agar plate:
For the pre-incubation method 100 µL of the test item solution or negative or positive control was pre-incubated with the tester strains (100 µL) and S9 substitution buffer or S9 mix (500 µL) for 60 minutes at 37°C prior to adding the overlay agar (2 mL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used. After solidification, the plates were inverted and incubated at 37 °C for at least 48 h in the dark.
The colonies were counted automatically. Tester strains with a low spontaneous mutation frequency i.e. strains TA1535 and TA1537 were counted manually.

Rationale for test conditions:

Plate incorporation methodology was employed in Experiment 1. Since there was no evidence of any mutagenicity, Experiment 2 comprised pre-incubation methodology.

Evaluation criteria:

The Mutation Factor was calculated by dividing the mean value of the revertant counts by the mean values of the negative control (unrounded values).
A test item was considered as mutagenic if:
1. a clear and dose-related increase in the number of revertants occurred and/or
2. a biologically relevant positive response for at least one of the dose groups occurred
in at least one tester strain with or without metabolic activation.
A biologically relevant increase was described as a concentration related increase in revertant numbers is at least 1.5-fold (in strain TA102), 2-fold (in strains TA98 or TA100), or 3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values in at least one tester strain with or without metabolic activation.

The test item is regarded as positive in this assay if all of the above criteria are met
The test item is regarded as negative in this assay if none of the above criteria are met.

Statistics:

None, the mutation factor was used.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

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:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

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:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test article was observed.

Any other information on results incl. tables

Experiment 1: Plate incorporation

Conc. µL/plate	Number of revertant colonies/plate (mean of 3 plates(±SD))
	-S9 Mix					+S9 Mix
	TA98	TA100	TA1535	TA1537	TA102	TA98	TA100	TA1535	TA1537	TA102
0	21	85	9	13	238	30	87	9	12	297
	(±9.0)	(±15.7)	(±2.0)	(±2.1)	(±6.0)	(±4.0)	(±6.7)	(±1.2)	(±1.0)	(±3.0)
0.0316	25	87	9	10	244	24	79	9	11	304
	(±4.0)	(±5.5)	(±1.5)	(±0.0)	(±5.5)	(±1.0)	(±11.3)	(±2.0)	(±1.2)	(±11.0)
0.100	23	93	10	10	242	28	99	10	11	303
	(±10.0)	(±3.5)	(±1.5)	(±1.5)	(±4.0)	(±10.1)	(±20.6)	(±2.1)	(±3.0)	(±12.1)
0.316	20	74	10	10	244	24	79	9	12	305
	(±8.5)	(±14.6)	(±1.0)	(±0.6)	(±12.0)	(±5.0)	(±14.6)	(±1.7)	(±3.0)	(±5.1)
1.0	19	85	11	9	247	27	83	9	11	309
	(±3.1)	(±13.3)	(±1.0)	(±0.6)	(±8.6)	(±1.5)	(±3.5)	(±1.5)	(±2.6)	(±10.3)
2.5	22	82	10	13	249	24	86	9	10	305
	(±2.0)	(±14.3)	(±1.5)	(±2.0)	(±13.7)	(±11.0)	(±18.1)	(±2.1)	(±2.5)	(±11.4)
5.0	21	77	10	13	249	19	81	10	13	311
	(±3.1)	(±2.3)	(±2.1)	(±3.8)	(±14.0)	(±0.6)	(±5.0)	(±1.0)	(±0.6)	(±11.2)
Positive control.	NOPD	NaN3	NaN3	NOPD	MMS	2AA	2AA	2AA	2AA	2AA
Conc. µg/plate	10	10	10	40	1 µL/plate	2.5	2.5	2.5	2.5	10
Colonies /plate	696	549	203	209	1101	2532	1430	165	201	1058
	(±70.1)	(±94.0)	(±35.8)	(±17.6)	(±61.7)	(±348.9)	(±41.9)	(±65.3)	(±72.2)	(±140.7)

NOPD:4-nitro-o-phenylenediamine; NaN_s: Sodium azide; MMS:methyl methanesulfonate;
2AA:2-aminoanthracene
Control: Purifiedwater

 

 

Experiment 2: Pre-incubation

Conc. µL/plate	Number of revertant colonies/plate (mean of 3 plates(±SD))
	-S9 Mix					+S9 Mix
	TA98	TA100	TA1535	TA1537	TA102	TA98	TA100	TA1535	TA1537	TA102
0	25	77	11	14	240	23	83	10	11	302
	(±2.1)	(±5.1)	(±1.5)	(±1.5)	(±13.7)	(±2.6)	(±3.6)	(±3.2)	(±1.5)	(±8.1)
0.0316	23	65	9	11	236	21	75	7	11	290
	(±4.2)	(±6.9)	(±1.5)	(±3.1)	(±6.1)	(±3.2)	(±9.2)	(±2.5)	(±2.1)	(±11.0)
0.100	24	70	10	14	235	23	7.6	8	12	304
	(2.6)	(±6.5)	(±1.0)	(±1.0)	(±8.1)	(±4.0)	(±7.2)	(±3.2)	(±5.9)	(±3.6)
0.316	23	71	9	13	239	23	74	9	15	304
	(±7.6)	(±7.8)	(±2.5)	(±5.2)	(16.3)	(±3.8)	(±10.0)	(±2.5)	(±4.0)	(±13.1)
1.0	21	69	9	13	243	24	79	9	13	302
	(±2.9)	(±3.5)	(±2.1)	(±2.1)	(5.9)	(±4.0)	(±6.0)	(±2.3)	(±1.0)	(±7.2)
2.5	24	80	9	13	249	23	84	9	14	307
	(±1.7)	(±1.5)	(±1.5)	(±1.5)	(±10.7)	(±9.1)	(±6.0)	(±2.3)	(±3.8)	(±9.0)
5.0	27	79	10	15	249	25	80	9	15	308
	(±9.8)	(±3.5)	(±0.6)	(±1.5)	(±5.9)	(±4.0)	(±4.2)	(±1.5)	(±1.7)	(±8.0)
Positive control.	NOPD	NaN3	NaN3	NOPD	MMS	2AA	2AA	2AA	2AA	2AA
Conc. µg/plate	10	10	10	40	1 µL/plate	2.5	2.5	2.5	2.5	10
Colonies /plate	261	448	235	196	1093	292	493	166	159	1030
	(±8.5)	(±22.1)	(±33.3)	(±23.6)	(±30.0)	(±17.3)	(±61.9)	(±11.6)	(±19.6)	(±141.5)

NOPD:4-nitro-o-phenylenediamine; NaN3: Sodium azide; MMS:methyl methanesulfonate;
2AA:2-aminoanthracene
Control: Purified water

Applicant's summary and conclusion

Conclusions:

It was concluded that IPD-AC did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this test guideline (OECD 471) GLP compliant study. These conditions included treatments at concentrations up to 5.0 µL/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S9), utilising plate incorporation and pre-incubation methodologies.

Executive summary:

In order to investigate the potential of 3-Methyl-1,3-butanediol-1-acetate (IPD-AC) was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and presence of a metabolic activation by a phenobarbitone/ß-naphthoflavone induced rat liver post-mitochondrial fraction (S9), in two separate experiments. This study utilised both plate incorporation and pre-incubation methodologies.

 

All IPD-AC treatments in this study were performed using formulations prepared in purified water.

 

In the plate incorporation experiment I treatments, all the tester strains were performed in the absence and in the presence of S9, using final concentrations of IPD-AC at 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate, plus negative (vehicle) and positive controls. Following these treatments, no evidence of toxicity in any of the strains were observed in either the absence or presence of S9.

 

In the pre-incubation experiment II treatments, all the tester strains were performed in the absence and in the presence of S9 using the same final concentrations of IPD-AC previously. Following these treatments, no evidence of toxicity in any of the strains were observed in either the absence or presence of S9.

 

No precipitation was observed on the test plates up to the maximum concentration tested, in either experiment.

 

Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for negative control treatments. The positive control chemicals induced large increases in revertant numbers of =2.0-fold (in strains TA98, TA100 or TA102) or =3.0-fold (in strains TA1535 and TA1537) confirming discrimination between different strains and an active S9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

 

No evidence of mutagenic activity was seen at any concentration of IPD-AC in either mutation test.

 

It was concluded that IPD-AC did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5.0 µL/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S9), utilising plate incorporation and pre-incubation methodologies.