3-methyl-1,3-butandiol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three reliable in vitro genotoxicity key studies and one supporting in vitro study are available for 3-methyl-1,3-butanediol. The test substance 3-Methyl-1,3-butanediol was negative in all in vitro genotoxicity assays.

3-methyl-1,3-butandiol was found to be negative in a reverse gene mutation assay in bacteria (AMES test, GLP) conducted in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2uvrA (pKM101).

3-Methyl-1,3-butandiol (MBD) was negative in an in vitro mammalian cell gene mutation study (OECD 476, GLP) conducted in L5178Y mouse lymphoma (3.7.2c) cells up to the maximum recommended concentration of 10 mM. Additionally MBD did not demonstrate mutagenic potential in a supporting in vitro mammalian cell gene mutation study (OECD 476, GLP) up to a maximum recommended concentration of 10 mM.

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

Link to relevant study records

Reference

Reference 1

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

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

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.

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

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.

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").

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2005 - 9 August 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Appearance: Clear and colourless liquid
Storage conditions: Room temperature (ca. 20ºC), in the dark
Batch number: 52834
Expiry date: December 2006
Purity: >98%

Target gene:

thymidine

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: heterozygous at the thymidine kinase locus, TK +/-

MEDIA USED
- Type and identity of media:
R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 µg/mL gentamicin.
R10p: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p: R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Concentration preliminary toxicity test: 2, 4.1, 8.1, 16.3, 32.5, 65.1, 130.1, 260.3, 520.5, 1041 µg/mL
Concentration range in the main test: 32.53, 65.06, 130.13, 260.25, 520.5, 1041 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the test item was found to be soluble

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

In the absence of S9 mix; solvent: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

In the presence of S9 mix; solvent: DMSO

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 24 hours

Expression time:
2 days

Selection time:
10-14 days

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

Data are presented for concentrations tested up to the maximum exposure of 1041 µg/mL (10mM) for freely soluble compounds, in accordance with current guidelines.

Evaluation criteria:

The test agent was regarded as negative if: The Induced mutation frequency (test concentration MF minus mean control MF) for any test concentration was less than the Global Evaluation Factor (126 x 10-6).
If the IMF of any test concentration exceeded the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test (Relative suspension growth):
Concentrations from 2 to 1041 µg/mL:
3 hours exposure :
- without S9 mix:127% to 44%
- with S9 mix: 98% to 89%
24 hours exposure :
- without S9 mix: 93% to 78%

Remarks on result:

other: after 3 hour treatment

Conclusions:

MBD did not demonstrate mutagenic potential up to limit concentrations (1041 µg/mL; 10mM) in this in vitro mammalian cell gene mutation assay (OECD 476, GLP), under the experimental conditions described.

Executive summary:

In a mammalian cell gene mutation assay (the thymidine kinase (tk) locus (TK^+/-), OECD 476, GLP) L5178Y mouse lymphoma (3.7.2c) cells cultured in vitro were exposed to 3-Methyl-1,3-butandiol (MBD, purity >98%) dissolved in sterile water at concentrations of 32.53, 65.06, 130.13, 260.25, 520.5, 1041 µg/mL in the presence and absence of mammalian metabolic activation (S9 mix). The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

3-Methyl-1,3-butandiol was tested up to the maximum recommended concentration specified in OECD TG 476 (10 mM). There were no increases in induced mutation frequency that exceeded the Global Evaluation Factor (GEF) or the mutant frequency of the concurrent solvent control at any of the concentrations tested within acceptable levels of cytotoxicity. The positive controls induced an acceptable increase in mutation frequency. The results for the solvent controls were within acceptable ranges of historical control data.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

7 June 2006 - 19 June 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In a previous study (Huntingdon Life Sciences report number RIH 498/052850), the test substance did not demonstrate any mutagenic potential, when dosed at a final concentration of 10 mM following a 3 and 24 hour treatment in the absence of S9 mix and a 3 hour treatment in the presence of S9 mix. Following submission of the previous study to the Health and Safety Executive, United Kingdom, it was deemed necessary that a confirmatory test at 3 hours, in the presence of S9 mix would be required. The mutagenic potential of the test item was assessed in this in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Appearance: Clear and colourless liquid
Storage conditions: Room temperature (ca. 20 C), in the dark
Batch number: 52834
Expiry date: December 2006
Purity: >98%

Target gene:

thymidine

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: heterozygous at the thymidine kinase locus, TK +/-

MEDIA USED
- Type and identity of media:
R10p: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine, 50 µg/mL gentamicin, 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 µg/mL gentamicin, 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with

Metabolic activation system:

S9

Test concentrations with justification for top dose:

73.20, 146.39, 292.78, 585.56, 780.75, 1041 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 2.4 x 10^6 cells/mL

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 24 and 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells):7 days for viability plates and approximately 10-14 days for mutant plates

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS:

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

Guideline study
See 'Administrative data -Justification for type of information'

Evaluation criteria:

The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (ie. relative total growth reduced to approximately 10 to 20% of the concurrent solvent control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The positive control, 3-methylcholanthrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
There were no increases in induced mutation frequency that exceeded the Global Evaluation Factor plus the induced mutant frequency of the concurrent solvent control following exposure to the test item up to 1041 µg/mL.

Conclusions:

It was concluded that the test item did not demonstrate mutagenic potential following a 3 hour treatment in the presence of S9 mix in this in vitro mammalian cell gene mutation assay, under the experimental conditions described.

Executive summary:

In a mammalian cell gene mutation assay (the thymidine kinase (tk) locus (TK^+/-), OECD 476, GLP) L5178Y mouse lymphoma (3.7.2c) cells cultured in vitro were exposed to 3-Methyl-1,3-butanediol (MBD, purity >98%) dissolved in sterile water at concentrations from 73.20 to 1041 µg/mL. There were no increases in induced mutation frequency that exceeded the Global Evaluation Factor or the induced mutant frequency of the concurrent solvent control following exposure to MBD up to 1041 µg/mL (10 mM). The positive control, 3-methylcholanthrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants. The results for the solvent controls were within acceptable ranges of historical control data.

It was concluded that MBD did not demonstrate mutagenic potential following a 3 hour treatment in the presence of S9 mix in this in vitro cell mutation assay, under the experimental conditions described.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 May 1987 - 17 May 1987

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: Method of Ames et al.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The test substance Lot No .734242, a clear Liquid, was stored in the dark at room temperature.

Target gene:

his G 46 in TA 1535 and TA 100
his C 3076 in TA 1537
his D 3052 in TA 98

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Liver from Aroclor-treated rats (S9 mix).

Test concentrations with justification for top dose:

Concentration range in the main test: 33, 100, 333, 1000, 3333 and 10000 µg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

methylmethanesulfonate

other: 2-Aminoanthracene

Remarks:

With and without metabolic activation

Details on test system and experimental conditions:

DURATION
- Preincubation period: 3 days
- exposure time: 2 days

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

A significant mutagenic response was recorded if there was:
-for S.typhimurium strains TA 1535, TA 1537 and TA 98 and for E.coli, at least a doubling of the mean concurrent vehicle control value at some concentration of the test substances and, for S.typhimurium strain TA 100 a 1.5-fol d increase over the control val ue. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes.
-a dose rel ated response, al though at high dose levels this rel ati onship coul d be i nverted because of, for exampl e, (1) toxicity to the bacteri a generally, (2}) specific toxicity tothe mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
-a reproducible effect in independent tests.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Observations:
Both tests were conducted using the pre-incubation method on agar plates.

Conclusions:

3-Methyl-1,3-butanediol (Lot No. 734242) was not mutagenic in S. typhimurium (strains TA 1535, TA 1537, TA 98, TA 100) and E. coli WP2uvrA (pKM101) when tested at concentrations ranging between 33 µg and 10000 µg per plate neither in the presence nor in the absence of metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (AMES test, GLP), S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2uvrA (pKM101) were exposed to 3-Methyl-1,3-butanediol (Lot No. 734242) dissolved in Dimethyl sulfoxide (DMSO) at concentrations of 0, 33, 100, 333 ,1000, 3333 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (rat liver S9 mix) using the pre-incubation method.

3-Methyl-1,3-butanediol was tested up to concentrations exceeding the maximum recommended test concentration of 5000 µg/plate (10000 µg/plate). There was no precipitation of test item and no toxicity to bacteria up to a concentration of 10000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reverse gene mutation assay in bacteria (AMES test, GLP), S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2uvrA (pKM101) were exposed to 3-Methyl-1,3-butanediol (Lot No. 734242) dissolved in Dimethyl sulfoxide (DMSO) at concentrations of 0, 33, 100, 333 ,1000, 3333 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (rat liver S9 mix) using the pre-incubation method. 3-Methyl-1,3-butanediol was tested up to concentrations exceeding the maximum recommended test concentration of 5000 µg/plate (10000 µg/plate). There was no precipitation of test item and no toxicity to bacteria up to a concentration of 10000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In a mammalian cell gene mutation assay (the thymidine kinase (tk) locus (TK^+/-), OECD 476, GLP) L5178Y mouse lymphoma (3.7.2c) cells cultured in vitro were exposed to 3-Methyl-1,3-butandiol (MBD, purity >98%) dissolved in sterile water at concentrations of 32.53, 65.06, 130.13, 260.25, 520.5, 1041 µg/mL in the presence and absence of mammalian metabolic activation (S9 mix). The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. 3-Methyl-1,3-butandiol was tested up to the maximum recommended concentration specified in OECD TG 476 (10 mM). There were no increases in induced mutation frequency that exceeded the Global Evaluation Factor (GEF) or the mutant frequency of the concurrent solvent control at any of the concentrations tested within acceptable levels of cytotoxicity. The positive controls induced an acceptable increase in mutation frequency. The results for the solvent controls were within acceptable ranges of historical control data.

In a supporting mammalian cell gene mutation assay (the thymidine kinase (tk) locus (TK^+/-), OECD 476, GLP) L5178Y mouse lymphoma (3.7.2c) cells cultured in vitro were exposed to 3-Methyl-1,3-butanediol (MBD, purity >98%) dissolved in sterile water at concentrations from 73.20 to 1041 µg/mL. There were no increases in induced mutation frequency that exceeded the Global Evaluation Factor or the induced mutant frequency of the concurrent solvent control following exposure to MBD up to 1041 µg/mL (10 mM). The positive control, 3-methylcholanthrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants. The results for the solvent controls were within acceptable ranges of historical control data. It was concluded that MBD did not demonstrate mutagenic potential following a 3 hour treatment in the presence of S9 mix in this in vitro cell mutation assay, under the experimental conditions described.

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

Justification for classification or non-classification

3-Methyl-1,3-butanediol did not show genotoxic effects in reliable in vitro genotoxicity assays (AMES test, OECD 476 and OECD 487). Therefore, classification according to Regulation (EC) No 1272/2008 is not warranted.