3-methyl-1,3-butandiol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-08-12 to 2023-03-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1,3-butandiol

EC Number:

459-270-7

EC Name:

3-methyl-1,3-butandiol

Cas Number:

2568-33-4

Molecular formula:

C5 H12 O2

IUPAC Name:

3-methylbutane-1,3-diol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Kuraray Co., Ltd. Otemachi, Chiyoda-ku, Tokyo (Japan)
- Batch (Lot) Number: 23527
- Purity (GC): ≥99.9%
- Expiry date: 14 December 2023

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- No correction was made for the purity/composition of the test material. A solubility test in the vehicle RPMI 1640 culture medium was performed based on visual assessment. The test material formed a clear light orange/pink or a pink solution in RPMI-C medium (Life Technologies, Bleiswijk, The Netherlands). Test material concentrations were used within 0.5 hours after preparation.

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Cultured peripheral human lymphocytes
- Suitability of cells: Peripheral human lymphocytes are recommended in the international OECD guideline 487

For lymphocytes:
- Sex, age and number of blood donors:
Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2021) are presented below:
Dose-range finding study: age 28, gender male, AGT = 12.5 h
First cytogenetic assay: age 29, gender male, AGT = 12.5 h
Second cytogenetic assay: age 27, gender male, AGT = 12.5 h

- Whether whole blood or separated lymphocytes were used: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively) and cultured in a PYREX® 25 mL Round Media Storage Bottle
- Whether blood from different donors were pooled or not: No
- Mitogen used for lymphocytes: Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel Europe Ltd., Dartford, United Kingdom) was added.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100%, containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C. Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Cytokinesis block (if used):

Cytochalasin B (5 µg/mL) was used, cell were incubated for 24 hours (1.5 times normal cell cycle).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH, Giessen, Germany
- method of preparation of S9 mix: prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- concentration or volume of S9 mix and S9 in the final culture medium: 1.8% (v/v)
- quality controls of S9: Alkoxyresorufin-O-dealkylase activity (EROD, PROD, BROD, MROD), test for presence of contaminating microorganisms, promutagen activation (ethidium bromide and cyclophosphamide tested in TA98 and TA1535 strains and benzo(a)pyrene and 2-aminoanthracene in TA100 strain)

Test concentrations with justification for top dose:

In the dose-range finding test a concentration of 1041.5 µg/mL (= 0.01 M) showed no precipitation in the culture medium and was used as the highest concentration of the test material. Minimal cytotoxic effects were observed across all dose levels (cytostasis < 50%), therefore the highest recommended dose level according to OECD 487 (= 0.01 M) was used in both cytogenetic assays.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).)

- Justification for choice of solvent/vehicle: solubility

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

The negative control used in the test system was the vehicle for the test material (culture medium).

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: Two independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 ± 2 hours
- Exposure duration/duration of treatment: first cytogenetic assay: 3 hours (absence and presence of S9-mix; second cytogenetic assay: 24 hours (absence of S9-mix)
- Harvest time after the end of treatment: 24 hours (1st assay) and directly (2nd assay)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- cytokinesis blocked method: After 3 hours exposure in the first cytogenetic assay, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasin B (5 µg/mL) and incubated for another 24 hours.
In the second cytogenetic assay, lymphocytes were cultured for 48 ± 2 hours and thereafter exposed in duplicate to selected doses of the test material with cytochalasin B (5 µg/mL) for 24 hours in the absence of S9-mix.

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
Cell cultures were harvested by centrifugation (5 min, 365 g) and the supernatant removed. The remaining cell pellet was re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), cells were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately thereafter, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v). The fixed cells were dropped onto slides. At least two slides were prepared per culture.
Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).
- Number of cells spread and analysed per concentration: A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): In case the test material was not cytotoxic, the highest concentration analyzed was the recommended 0.01 M.
The following criteria for scoring of binucleated cells were used
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
• Mononucleated, Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1)
• The diameter of micronuclei should be less than one-third of the main nucleus
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI).

Rationale for test conditions:

Minimal cytotoxic effects were observed across all dose levels (cytostasis < 50%), therefore the highest recommended dose level according to OECD 487 (= 0.01 M) was used in both cytogenetic assays.

Evaluation criteria:

A test material is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test material is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range

Statistics:

Graphpad Prism version 8.4 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
The data was analyzed by the Fisher’s exact test (one-sided, p < 0.05). All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: cultured human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Minimal cytotoxic effects were observed across all dose levels (cytostasis < 50%), therefore the highest recommended dose level according to OECD 487 (= 0.01 M) was used in both cytogenetic assays.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A concentration of 1041.5 µg/mL (= 0.01 M) showed no precipitation in the culture medium and was used as the highest concentration of the test material.
The pH and osmolarity of a concentration of 1041.5 µg/mL were 8.4 and 269 mOsm/kg
respectively (compared to 8.4 and 237 mOsm/kg in the solvent control).
In the dose-range finding test blood cultures were treated with 31, 63, 125, 250, 500 and 1041.5 µg test material/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. Minimal cytotoxic effects were observed across all dose levels (cytostasis < 50%), therefore the highest recommended dose level according to OECD 487 (= 0.01 M) was used in both cytogenetic assays (for more information, please refer to section "Any other information on results incl. tables").

HISTORICAL CONTROL DATA
The number of binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database. In addition, the number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database (For more information, please refer to section "Any other information on results incl. tables").

Any other information on results incl. tables

Table 1. Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with 3-Methyl-1,3-butanediol in the Dose-range Finding Test

 

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Number of cells with ….nuclei
	1	2	3 or more	CBPI	% cytostasis
0	290	137	95	1.63	0
31.3	305	123	92	1.59	6
62.5	301	110	90	1.58	8
125	307	103	101	1.6	5
250	303	114	84	1.56	10
500	346	110	50	1.42	34
1041.5	343	99	70	1.47	25
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Number of cells with ….nuclei
	1	2	3 or more	CBPI	% cytostasis
0	321	113	78	1.53	0
31.3	343	81	90	1.51	3
62.5	269	127	111	1.69	-31
125	311	130	68	1.52	1
250	328	113	79	1.52	1
500	303	118	99	1.61	-16
1041.5	303	105	104	1.61	-16
Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/mL	Number of cells with ….nuclei
	1	2	3 or more	CBPI	% cytostasis
0	325	93	83	1.52	0
31.3	343	73	87	1.49	5
62.5	358	85	63	1.42	19
125	372	62	67	1.39	24
250	387	65	57	1.35	32
500	389	69	57	1.36	31
1041.5	387	66	54	1.34	34

 

Table 2. Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with 3-Methyl-1,3-butanediol (3 hours exposure time, 27 hours harvest time, without metabolic activation (-S9-mix))

 

Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronucle (1000 binucleated cells were scored for the presence of micronuclei)
		1000	1000	2000
		A	B	A+B
0	0	1	1	2
250	3	1	0	1
500	7	1	1	2
1041.5	17	0	0	0
0.20 MMC-C	30	19	8	27^****
0.25 MMC-C	40	22	12	34^****
0.05 Colch	31	11	8	19^****
MMC-C: mitomycin C; Colch: colchicine; Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001; Duplicate cultures are indicated by A and B

 

Table 3. Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with 3-Methyl-1,3-butanediol (3 hours exposure time, 27 hours harvest time, with metabolic activation (+S9-mix))

 

Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei (1000 binucleated cells were scored for the presence of micronuclei)
		1000	1000	2000
		A	B	A+B
0	0	2	2	4
250	13	0	1	1
500	13	1	1	2
1041.5	18	1	1	2
7.5 CP	53	8	9	17**
CP: cyclophosphamide; Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001Duplicate cultures are indicated by A and B

 

Table 4. Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with 3-Methyl-1,3-butanediol (24 hours exposure time, 24 hours harvest time, without metabolic activation (-S9-mix))

 

Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei (1000 binucleated cells were scored for the presence of micronuclei)
		1000	1000	2000
		A	B	A+B
0	0	2	4	6
250	-16	0	3	3
500	-39	2	1	3
1041.5	-12	3	2	5
0.125 MMC-C	51	13	13	26^***
0.01 Colch	10	2	4	6
0.05 Colch	100	44	42	86^****
MMC-C: mitomycin C; Colch: colchicine; Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001; Duplicate cultures are indicated by A and B.

 

Table 5. Historical Control Data for in vitro Micronucleus Studies of the Solvent Control

 

	Binucleated
	-S9 Mix		+S9 mix
	3 hour exposure	24 hour exposure	3 hour exposure
Mean number of micronucleated cells (per 2000 cells)	3.7	4.5	4.0
SD	2.4	3.0	2.3
n	111	112	115
Lower Control Limit (95% Control Limits)	-1	-1	-1
Upper Control Limit (95% Control Limits)	8	10	9
SD = Standard deviation; n = Number of observations; Distribution historical negative control data from experiments performed between November 2019 and November 2022.

 

Table 6. Historical Control Data for in vitro Micronucleus Studies of the Positive Control Substances

 

	Binucleated			Binucleated
	-S9 Mix (MMC-C)		+S9 mix (CP)	-S9 Mix (Colch)
	3 hour exposure	24 hour exposure	3 hour exposure	3 hour exposure	24 hour exposure
Mean number of micronucleated cells (per 2000 cells)	46.9	40.8	41.3	53.6	67.6
SD	24.5	20.3	33.7	133.4	114.1
n	118	118	123	121	124
Lower Control Limit (95% Control Limits)	-1	1	-25	-208	-156
Upper Control Limit (95% Control Limits)	95	81	107	315	291
SD = Standard deviation; n = Number of observations; Distribution historical negative control data from experiments performed between November 2019 and November 2022.

Applicant's summary and conclusion

Conclusions:

3-Methyl-1,3-butanediol is not clastogenic or aneugenic in human lymphocytes under the experimental conditions of this in vitro micronucleus assay (GLP, OECD TG 487).

Executive summary:

In mammalian cell cytogenetic assay (micronucleus assay, OECD TG 487), cultured human lymphocytes were exposed to 3-Methyl-1,3-butanediol (purity (GC) ≥99.9%) in culture medium at concentrations of 0, 250, 500, and 1041.5 µg/mL (first cytogenetic assay, 3 hours exposure time, 27 hours harvest time) with and/ without metabolic activation (S9-mix) and at 0, 250, 500, and 1041.5 µg/mL (second cytogenetic assay, 24 hours exposure time, 24 hours harvest time) without metabolic activation (S9-mix).

3-Methyl-1,3-butanediol was tested up to the maximum recommended concentration of 0.01 M. The number of binucleated cells with micronuclei in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive controls (mitomycin C, colchicine and cyclophosphamide) induced the appropriate response. After 3 hours exposure time, 3-Methyl-1,3-butanediol did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei both in the absence and presence of metabolic activation. 3-Methyl-1,3-butanediol did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei after 24 hours exposure in the absence metabolic activation.

This study is classified as acceptable This study satisfies the requirement for Test Guideline OECD 487 for in vitro cytogenetic genotoxicity data.

3-Methyl-1,3-butanediol is not clastogenic or aneugenic in human lymphocytes under the experimental conditions of this study.