2-methylglutaric acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2014-05-22 to 2014-12-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD 474 compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2013-05-06

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methylglutaric acid

EC Number:

210-521-7

EC Name:

2-methylglutaric acid

Cas Number:

617-62-9

Molecular formula:

C6H10O4

IUPAC Name:

2-methylglutaric acid

Test material form:

solid: crystalline

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 - 8 weeks old at the start of the treatment
- Weight at study initiation: The mean body weights were for males 34.7 ± 1.5 g and for females 27.7 ± 1.4 g and the range was for males 32 - 37 g and for females 25 - 31 g.
- Assigned to test groups randomly: yes
- Fasting period before study: Feed will be withheld 3 - 4 hours prior to dosing
- Housing: Group housing of maximum 5 animals per sex in labeled Macrolon cages (type MIII height: 18 cm,length 380 mm and width 220 mm ) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and a shelter (disposable paper corner home, MCORN 404, Datesand Ltd, USA) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to tap-water
- Acclimation period: The acclimatisation period was at least 6 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C, (actual range: 21.0 - 22.6°C)
- Humidity (%): 40 - 70% (actual range: 39 - 69%)
- Air changes (per hr): approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: physiol. saline (Eurovet Animal Health, Bladel, the Netherlands)
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No correction was made for the purity/composition of the test substance.

2-Methylglutaric acid was dissolved in physiological saline (Eurovet Animal Health, Bladel, the Netherlands). The specific gravity of physiological saline is 1.0 g/ml. 2-Methylglutaric acid concentrations were dosed within 2.5 hours after preparation.

Duration of treatment / exposure:

Once

Frequency of treatment:

Once

Post exposure period:

24 and 48 depending on the dose

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 500, 1000 and 2000 mg/kg
Basis:
actual ingested
Males

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 38, 875 and 1750 mg/kg
Basis:
actual ingested
Females

No. of animals per sex per dose:

At least five male and five female mice were used per sampling time in each treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 40 mg/kg 10 mL/kg

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a dose range finding study, in total three male animals were dosed with 2000 mg Methylglutaric acid per kilogram body weight. No signs of toxicity were observed within 2 days after dosing (2 male animals). One animal had a hunched posture within 1 hour after dosing. Within 21 hours after dosing the animal had recovered from the treatment.
One female dosed with 2000 mg/kg body weight had a hunched posture, was lethargic and showed ataxia and died within 21 hours after dosing. No signs of toxicity were observed within 2 days after dosing in one female dosed with 1500 mg/kg body weight. In total three female animals were dosed with 1750 mg/kg body weight. One animal had no signs of toxicity. Both other animals had a hunched posture. One animal recovered from the treatment within 21 hours after dosing. The other animal had the following toxic signs within 21 hours after dosing: hunched posture, rough coat, eyes closed and a blue staining of the tail.
Based on the results of the dose range finding study dose levels of 2000, 1000 and 500 mg/kg body weight (male animals) and 1750, 875 and 438 mg/kg body weight (female animals) were selected as appropriate doses for the micronucleus main test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
See table 1: sampling scheme

DETAILS OF SLIDE PREPARATION:
Isolation of bone marrow
Bone marrow of the groups treated with 2-Methylglutaric acid was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing. The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min.

Preparation of bone marrow smears
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. Two slides were prepared per animal.

Staining of the bone marrow smears
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V., Rijswijk, The Netherlands).


METHOD OF ANALYSIS:
Analysis of the bone marrow smears for micronuclei
To prevent bias, all slides were randomly coded before examination. An adhesive label with the study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 2000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme: REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA).

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range.
b) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
c) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

Equivocal results should be clarified by further testing using modification of experimental conditions.

Statistics:

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

See additional information on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range:males 2000 mg/kg (3 animals) and females 2 animals at 2000 mg/kg and 1 animal at 1500 mg/kg then 3 animals at 1750 mg/kg
- Solubility: yes
- Clinical signs of toxicity in test animals:
Males: 1 animal with hunched posture within 1 hour after dosing. Within 21 hours after dosing the animal had recovered from the treatment and 2 animals without clinicals signs
Females: One female dosed with 2000 mg/kg body weight had a hunched posture, was lethargic and showed ataxia and died within 21 hours after dosing. No signs of toxicity were observed within 2 days after dosing in one female dosed with 1500 mg/kg body weight. at 1750 mg/kg, One animal had no signs of toxicity. Both other animals had a hunched posture. One animal recovered from the treatment within 21 hours after dosing. The other animal had the following toxic signs within 21 hours after dosing: hunched posture, rough coat, eyes closed and a blue staining of the tail.

RESULTS OF DEFINITIVE STUDY
see result tables

Any other information on results incl. tables

Mean number of micronucleated polychromatic erythrocytes and ratio of polychromatic or normochromatic erythrocytes

Sexe	Group	Treatment	Dose (mg/kg)	Sampling time (hours)	Number of micronucleated polychromatic erythrocytes (mean ± S.D.) (1,2)	Ratio polychromatic/ normochromatic erythrocytes (mean ± S.D.)^(1,3)
Males	A	Vehicle control	0	24	0.6 ± 0.5	0.93 ± 0.22
	B	Methylglutaric acid	2000	24	1.4 ± 0.9	0.82 ± 0.40
	C	Methylglutaric acid	2000	48	1.2 ± 1.8	0.60 ± 0.32
	D	Methylglutaric acid	1000	24	0.4 ± 0.5	1.08 ± 0.15
	E	Methylglutaric acid	500	24	2.8 ± 0.4^(4)	1.10 ± 0.06
	F	Cyclophosphamide	40	48	32.4 ± 12.9	0.60 ± 0.17
Female	A	Vehicle control	0	24	1.2 ± 1.6	1.21 ± 0.22
	B	Methylglutaric acid	1750	24	2.0 ± 1.6	0.88 ± 0.18
	C	Methylglutaric acid	1750	48	1.4 ± 1.5	1.33 ± 0.11
	D	Methylglutaric acid	875	24	1.6 ± 0.9	1.07 ± 0.35
	E	Methylglutaric acid	438	24	2.4 ± 0.9	1.28 ± 0.13
	F	Cyclophosphamide	40	48	24.4 ± 10.8^(4)	0.85 ± 0.9

Vehicle control = physiological saline

CP = Cyclophosphamide.

(1)         Five animals per treatment group.

(2)         At least 2000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.

(3)         The ratio was determined from at least the first 1000 erythrocytes counted.

(4)         Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P = 0.01).

Individual data (Males)

 

Group	Animal number	Number of micronucleated polychromatic erythrocytes	Number of polychromatic erythrocytes scored for micronuclei	Number of poly-chromatic erythrocytes^(1)	Number of normo-chromatic erythrocytes^(1)	Ratio polychromatic/ normochromatic erythrocytes^(1)
A	1	0	2011	490	510	0.96
A	2	0	2011	561	439	1.28
A	3	1	2007	474	526	0.90
A	4	1	2001	413	587	0.70
A	5	1	2005	443	557	0.80
B	11	0	2000	329	671	0.49
B	12	2	2012	266	734	0.36
B	2	2	2018	541	459	1.18
B	1	1	2016	558	442	1.26
B	2	2	2016	447	553	0.81
C	21	0	2003	487	513	0.95
C	22	0	2020	276	724	0.38
C	23	2	2001	377	623	0.61
C	24	4	2009	465	535	0.87
C	25	0	2001	151	849	0.18
D	31	0	2021	564	436	1.29
D	32	1	2023	535	465	1.15
D	33	1	2008	480	520	0.92
D	34	0	2007	494	506	0.98
D	35	0	2007	517	483	1.07
E	41	3	2014	519	481	1.08
E	42	3	2036	543	457	1.19
E	43	3	2010	525	475	1.11
E	44	3	2002	527	473	1.11
E	45	2	2009	502	498	1.01
F	51	30	2003	402	598	0.67
F	52	16	2006	346	654	0.53
F	53	26	2004	337	663	0.51
F	54	49	2015	464	536	0.87
F	55	41	2003	298	702	0.42

(1)     The ratio was determined from the first 1000 erythrocytes counted.

(group A         : oral intubation of the vehicle)

(group B & C  : oral intubation of Methylglutaric acid at 2000 mg/kg body weight)

(group D         : oral intubation of Methylglutaric acid at 1000 mg/kg body weight)

(group E         : oral intubation of Methylglutaric acid at 500 mg/kg body weight)

(group F         : oral intubation of cyclophosphamide at 40 mg/kg body weight)

 

 Individual data (Female)

 

Group	Animal number	Number of micronucleated polychromatic erythrocytes	Number of polychromatic erythrocytes scored for micronuclei	Number of poly-chromatic erythrocytes^(1)	Number of normo-chromatic erythrocytes^(1)	Ratio polychromatic/ normochromatic erythrocytes^(1)
A	6	1	2000	522	478	1.09
A	7	1	2006	580	420	1.38
A	8	0	2003	478	522	0.92
A	9	4	2000	591	409	1.44
A	10	0	2006	552	448	1.23
B	16	1	2009	524	476	1.10
B	17	0	2019	503	497	1.01
B	18	2	2002	449	551	0.81
B	19	4	2002	449	551	0.81
B	20	3	2011	455	545	0.83
C	26	0	2010	290	410	1.44
C	27	3	2011	557	443	1.26
C	28	0	2003	569	431	1.32
C	29	3	2010	590	410	1.44
C	30	1	2006	546	454	1.20
D	36	1	2000	389	611	0.64
D	37	3	2024	527	473	1.11
D	38	2	2007	521	479	1.09
D	39	1	2009	614	386	1.59
D	40	1	2002	481	519	0.93
E	46	2	2005	563	437	1.29
E	47	4	2001	595	405	1.47
E	48	2	2016	565	435	1.30
E	49	2	2016	528	472	1.12
E	50	2	2007	550	450	1.22
F	56	13	2009	318	682	0.47
F	57	22	2012	391	609	0.64
F	58	30	2005	513	487	1.05
F	59	40	2006	590	410	1.44
F	60	17	2020	395	605	0.65

(1)     The ratio was determined from the first 1000 erythrocytes counted.

(group A         : oral intubation of the vehicle)

(group B & C  : oral intubation of Methylglutaric acid at 2000 mg/kg body weight)

(group D         : oral intubation of Methylglutaric acid at 1000 mg/kg body weight)

(group E         : oral intubation of Methylglutaric acid at 500 mg/kg body weight)

(group F         : oral intubation of cyclophosphamide at 40 mg/kg body weight)

 

HISTORICAL CONTROL DATA FOR MICRONUCLEUS STUDIES

	Number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes
	Males	Females
Range	0-5	0-5
Mean	1.1	1.5
SD	1.2	1.3
N	168	35

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between May 2011 and May 2014

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that 2-Methylglutaric acid is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male and female mice up to a dose of 2000 and 1750 mg/kg, respectively (maximum recommended/tolerated dose in accordance with current regulatory guidelines in males and females, respectively).

Executive summary:

2-Methylglutaric acid was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow. This study was performed according to OECD guideline N° 474 and in compliance with GLP.

 

The test substance was dissolved in physiological saline.

 

In the dose range finding test, in total three male animals were dosed with 2000 mg 2-Methylglutaric acid per kilogram body weight. No signs of toxicity were observed within 2 days after dosing (2 male animals). One animal had a hunched posture within 1 hour after dosing. Within 21 hours after dosing the animal had recovered from the treatment.

One female dosed with 2000 mg/kg body weight had a hunched posture, was lethargic and showed ataxia and died within 21 hours after dosing. No signs of toxicity were observed within 2 days after dosing in one female dosed with 1500 mg/kg body weight. In total three female animals were dosed with 1750 mg/kg body weight. One animal had no signs of toxicity. Both other animals had a hunched posture. One animal recovered from the treatment within 21 hours after dosing. The other animal had the following toxic signs within 21 hours after dosing: hunched posture, rough coat, eyes closed and a blue staining of the tail.

 

In the main study animals were dosed via oral gavage with vehicle or with 2000, 1000 and 500 mg 2-Methylglutaric acid per kg body weight (male animals) or with 1750, 875 and 438 mg Methylglutaric acid per kg body weight (female animals). A positive control group was dosed via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 12 treatment groups were used, each consisting of at least 5 animals. Three additional female animals, treated with 1750 mg 2-Methylglutaric acid /kg body weight, were used to correct for possible deaths. The male animals, dosed with 2000 mg 2-Methylglutaric acid per kg body weight, showed the following toxic signs after dosing: lethargy(2 males), a hunched posture (3 males) and rough coat (2 males). No treatment related clinical signs or mortality were noted all other animals dosed with 2 -Methylglutaric acid or in control animals receiving vehicle or cyclophosphamide.

Bone marrow of the groups treated with 2-Methylglutaric acid was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with 2-Methylglutaric acid compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical vehicle control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met.

Male animals treated for 48 hours and dosed with 2000 mg 2-Methylglutaric acid /kg body weight showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating toxic effects of this test substance on erythropoiesis. The other groups that were treated with 2-Methylglutaric acid showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

 

It is concluded that 2-Methylglutaric acid is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male and female mice up to a dose of 2000 and 1750 mg/kg, respectively (maximum recommended/tolerated dose in accordance with current regulatory guidelines in males and females, respectively) under the experimental conditions described.