2-methylglutaric acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2015-01-05 to 2015-07-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD 414 compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

2013-05-06

Limit test:

no

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

Details on test material:

Denomination: Methylglutaric acid.
CAS number: 617-62-9.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint Germain-Nuelles, France.
- Age at study initiation: 10 to 13 weeks old
- Weight at study initiation: 187 g to 255 g
- Fasting period before study: No
- Housing: One air-conditioned room in a barrier protected unit (building K2). Females were individually housed in plastic cages (dimensions 365 x 225 x 180 mm) in compliance with European Regulations (Directive 2010/63/EU).
- Diet (e.g. ad libitum): Rat pelleted commercial complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian. The rats had free access to the food throughout the study. Bedding: Dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood, analysed at least twice a year for chemical and bacterial contaminants. Enrichment: Shredded paper (SDS/Dietex).
- Water (e.g. ad libitum): Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via bottles). The water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
- Acclimation period: The females were mated at the supplier with a documented day of mating. They were received at the Test Facility on day 0 of gestation (G0). 6 days between animal arrival and the start of treatment.
- Enrichment: Shredded paper (SDS/Dietex).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3 °C (target range).
- Humidity (%): Between 35 and 70 % (target range).
- Air changes (per hr): At least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark (except when required for technical acts).

IN-LIFE DATES: From: 2015-01-12 To: 2015-01-29

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Lavoisier

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was prepared weekly as a solution in the vehicle at concentrations of 40, 80 and 160 mg/mL.
No correction factor was applied.
The formulations were stored refregirated (between +2 and +8 °C) and protected from daylight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Following the study N° 505981 performed at WIL Research Europe B.V., it was showned that The formulations between 20 and 200 mg/mL are stable:
 at room temperature under normal laboratory light conditions for at least 6 hours
 refrigerated (between +2 and +8 °C) and protected from daylight for at least 8 days,
 frozen (-20°C) for 13 days.

Method for analysis:
Quadruplicate 1 mL samples were taken in glass vials from each formulation prepared during main study phase only, including the control, used on the first day of treatment (G 6), as follows:
 groups 2 to 4: samples were collected from the top, middle and bottom of each formulation
 control group: samples were collected from the middle.
The samples were kept frozen (between -15 and -25 °C).

Two of four samples were dispatched on dry ice to the Test Site for analysis. The Principal Investigator was notified by email on the time of sample shipment. At the Test Site the samples were stored in the freezer (≤ -15 °C) until the day of analysis.

The formulation analysis was performed at the Test Site according to the validated method of Project 505911. The Test Site Project number of the formulation analysis phase was 507419.

The accuracy of preparation is considered acceptable if the mean measured concentrations are 90-110 % of the target concentration for solutions, or 85-115 % for suspensions. Homogeneity is demonstrated if the coefficient of variation is ≤ 10 %.

The duplicate formulation samples kept frozen (between -15 and -25 °C) at the Test Facility was only delivered to the Test Site and analysed if requested by the PI and in agreement with the Study Director and the Study Sponsor.

Method:
Reagents:
- Water: Tap water purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA)
- Acetonitrile: Biosolve, Valkenswaard, The Netherlands
- ortho-Phosphoric acid, 85% (H3PO4) Merck, Darmstadt, Germany
All reagents were of analytical grade, unless specified otherwise

Samples:
Samples of 1 ml in glass containers received on 14 January 2015 from WIL Research Europe-Lyon were stored in a freezer (≤ -15°C) until analysis. The duplicate samples (deputy samples) were received on 04 March 2015. Storage stability of samples in a freezer (≤ -15°C) for at least 68 days was demonstrated in this project.

On the day of analysis, the samples were defrosted at room temperature and vortex mixed for 5 seconds. Samples of approximately 500 mg, which were taken using a pipette, were accurately weighed into volumetric flasks of 10, 20, 50 or 100 ml. The volumetric flasks were filled up to the mark with 50/50 (v/v) acetonitrile/water. The solutions were 10-200 times further diluted to obtain an end solution of 0.1% H3PO4 in 5/95 (v/v) acetonitrile/water and concentrations within the calibration range.

Analytical conditions
Instrument Acquity UPLC system (Waters, Milford, MA, USA)
Detector Acquity UPLC TUV detector or Acquity UPLC PDA detector (Waters)
Column Acquity UPLC HSS T3, 100 mm  2.1 mm i.d., dp = 1.8 µm (Waters)
Column temperature 40°C +/- 1°C
Injection volume 20 µl
Mobile phase 0.1% H3PO4 in 5/95 (v/v) acetonitrile/water
Flow 0.6 ml/min
UV detection 210 nm

Preparation of solutions
- Stock solutions of the test substance were prepared in 50/50 (v/v) acetonitrile/water at concentrations of 1000 - 3000 mg/l.
- Calibration solutions in the concentration range of 0.35 – 20 mg/l were prepared from two stock solutions. The end solution of the calibration solutions was 0.1% H3PO4 in 5/95 (v/v) acetonitrile/water.
- Procedural recovery samples: Approximately 500 mg water was spiked with the test substance at a target concentration of 1 or 200 mg/g. The accuracy samples were treated similarly as the test samples.
- Storage stability samples: Accuracy samples of about 5 ml were prepared with the test substance in water at target concentrations of 1 and 200 mg/g. Samples of 1 ml were taken and transferred into glass containers. The samples were stored in the freezer (≤ -15°C) for 13 days. To cover the storage period of the deputy samples additional samples were stored in the freezer (≤ -15°C) for 68 days.
On the day of analysis, duplicate samples were defrosted at room temperature, vortex mixed for 5 seconds and treated similarly as the test samples.

Sample injections: Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

Formulas
Response (R) Peak area test substance [units]
Calibration curve: R = a CN +b
where:
CN = nominal concentration [mg/l]
a = slope [units x l/mg]
b = intercept [units]

Analysed concentration (CA): CA = ((R-b)/a) x ((V x d)/w) [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [ml]
d = dilution factor

Recovery (CA/CN) x 100 [%]
where:
CN = nominal concentration [mg/g]

Accuracy (CA/CT) x 100 [%]
Where:
CT = target concentration [mg/g]

Specifications: Preparation of formulations was considered acceptable if the mean accuracy was in the range 90-110% of the target concentration and was considered homogeneous if the coefficient of variation was ≤ 10%.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

From G6 to G19 included

Frequency of treatment:

Once daily

Duration of test:

20 days

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
+ Based on the DRF phase in pregnant rats performed in the present study (WIL Research Europe Lyon AB20756) where treatment was not associated with any maternal or embryo-foetal effects at doses of 400, 600 and 800 mg/kg/day.
+ Also based on a 90-day oral rat study (WIL Research Europe B.V. study no. 505981) at the dose levels of 100, 300 and 600/1000 mg/kg/day, where three females were found dead or moribund after one week of treatment at 1000 mg/kg/day.

Despite no maternal toxicity at 800 mg/kg/day in the DRF phase in pregnant rat study, the high dose level in the main study was selected at 800 mg/kg/day, due to the mortality noted at 1000 mg/kg/day in the 90-day oral rat study. The other doses of 200 and 400 mg/kg/day were chosen according to a geometric progression using a factor of 2.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Day 6, 9, 12, 15, 18 and 20 of gestation

FOOD CONSUMPTION : Yes
- Individual food consumption was measured for the periods (days) G 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20 during gestation

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: individual foetal weights, foetal sex

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Statistics:

Statistical analysis was performed, where appropriate, by the data acquisition software, as follows:
The best transformation for the data (none, log or rank) were determined depending upon
- the normality of the data distribution tested by the Shapiro-Wilk's test
- the homogeneity of the variances across groups tested by the Bartlett's test.

Non- or log-transformed data was analysed by parametric methods.

Rank transformed data was analysed using non-parametric methods.

Data was then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.

Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.

The number of resorptions, number of dead foetuses and all litter-based percentages were analysed using non-parametric methods, i.e. Kruskal-Wallis test followed by non-parametric Dunnett’s test if the Kruskal-Wallis was significant.

Selected incidence data was analysed using a chi2 test for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.

Indices:

Individual data were presented for all females. Group mean values and standard deviations are calculated for pregnant females with a terminal caesarean section.
For caesarean data, the group mean values were calculated on a litter basis. Foetal observation data are presented as the percentage of affected foetuses and percentage of affected litters.

Foetal abnormalities are categorised as follows:
 Malformations - structural defects which are rare in the control population and are thought to be life threatening or of major physiological consequence.
 Anomalies - minor abnormalities or defects which are relatively rare in the control population and/or are considered not to be of major physiological consequence.
 Variations - minor abnormalities, defects or alternative forms which are either common in the control population or are of no known physiological consequence.

For each group, the following parameters were calculated:
Pre-implantation loss (in %): ((Number of corpora lutea - Number of implantations)/ Number of corpora lutea) x 100
Post-implantation loss (in %): ((Number of implantations - Number of viable foetuses)/ Number of implantations)x 100

Historical control data:

The data concerning the control pregnant females and the historical data were used to evaluate the effects of the test item.
The data are presented in a addendum of the report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
noisy breathing observed in the 800 mg/kg/day group

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Body weight (g)

day		0 mg/kg	200 mg/kg	400 mg/kg	800 mg/kg
0	Mean	213.93	207.21	214.40	215.35
	SD	13.96	7.77	12.80	11.51
	N	21	22	22	22
	% variation	/	-3.14	0.22	0.66
6	Mean	248.7	239.82	244.96	250.42
	SD	16.28	11.05	13.12	14.33
	N	21	22	22	22
	% variation	/	-3.57	-1.50	0.69
9	Mean	261.65	252.15	256.30	262.01
	SD	18.50	13.00	14.29	15.99
	N	21	22	22	22
	% variation	/	-3.63	-2.04	-0.24
12	Mean	277.61	267.32	272.20	278.42
	SD	18.60	13.00	14.29	15.99
	N	21	22	22	22
	% variation	/	-3.71	-1.95	0.29
15	Mean	295.30	282.45	288.96	294.86
	SD	20.39	14.58	19.12	17.87
	N	21	22	22	22
	% variation	/	-4.35	-2.14	-.014
18	Mean	328.77	314.24	321.47	327.47
	SD	23.48	14.97	23.53	19.70
	N	21	22	22	22
	% variation	/	-4.42	-2.22	-0.40
20	Mean	353.27	335.94	343.33	349.90
	SD	26.85	17.24	26.95	23.61
	N	21	22	22	22
	% variation	/	-4.90	-2.81	0.95

 

Food consumption (g)

day		0 mg/kg	200 mg/kg	400 mg/kg	800 mg/kg
0 -6	Mean	19.81	18.77	18.77	20.47
	SD	2.58	2.17	1.57	2.41
	N	21	22	22	22
	% variation	/	-5.28	-5.28	3.34
6 -9	Mean	22.58	21.25	21.53	22.64
	SD	2.67	2.18	1.97	3.07
	N	21	22	22	22
	% variation	/	-5.87	-4.64	0.29
9 - 12	Mean	24.64	22.17	23.12	24.73
	SD	2.92	2.05	2.24	3.35
	N	21	22	22	22
	% variation	/	-10.01	-6.15	0.38
12 - 15	Mean	25.88	23.83	25.14	26.33
	SD	3.25	2.17	2.51	2.95
	N	21	22	22	22
	% variation	/	-7.94	-2.87	1.71
15 - 18	Mean	27.48	25.15	27.02	27.36
	SD	2.69	2.15	2.95	4.86
	N	21	22	22	22
	% variation	/	-8.13	-3.75	0.48
6 - 18	Mean	25.14	23.10	24.20	25.27
	SD	2.73	1.92	2.21	2.88
	N	21	22	22	22
	% variation	/	-8.13	-3.75	0.48
18.20	Mean	26.82	24.39	26.00	25.68
	SD	2.47	2.99	3.41	6.04
	N	21	22	22	22
	% variation	/	-9.08	-3.06	-4.27

 

Mean Gravid Uterus Weight and Maternal Body Weight Change

		0 mg/kg	200 mg/kg	400 mg/kg	800 mg/kg
Gravid uterus (g)	Mean	60.68	60.82	56.82	62.56
	SD	14.85	9.01	16.03	8.26
	N	21	22	22	22
	% variation	/	0.23	-6.36	3.11
Necropsy BW (g)	Mean	345.51	329.28	338.41	344.25
	SD	27.26	18.21	27.01	24.18
	N	21	22	22	22
	% variation	/	-4.70	-2.06	-0.37
Adjusted bw (g)	Mean	284.83	268.46	281.59	281.68
	SD	21.10	18.05	18.26	22.64
	N	21	22	22	22
	% variation	/	-5.75	-1.14	-1.11
Net BWC from G6 (g)	Mean	96.81	89.46	93.45	93.83
	SD	15.52	11.96	18.29	18.89
	N	21	22	22	22
	% variation	/	-7.59	-3.48	-3.09
Net BWC – Uterine Wt (g)	Mean	36.14	28.65	36.63	31.26
	SD	8.87	10.44	9.84	17.32
	N	21	22	22	22
	% variation	/	-20.73	1.35	-13.49
Mean foetal Wt ( M & F) (g)	Mean	3.54	3.41	3.46	3.34
	SD	0.19	0.16	0.24	0.41
	N	21	22	22	22
	% variation	/	-3.58	-2.14	-5.61
Nb of live foetuses	Mean	10.7	11.2	10.1	11.8
	SD	2.7	1.7	3.0	1.7
	N	21	22	22	22
	% variation	/	4.8	-5.0	10.4

 

 

Cesarean observations

		0 mg/kg	200 mg/kg	400 mg/kg	800 mg/kg
Female mated	N	21	22	22	22
Dams with viable foetuses	N	21	22	22	22
Nb of corpora lutea	Mean	12.5	12.7	12.2	13.4
	SD	1.9	1.5	1.7	1.9
	Sum	263	280	269	295
Nb of implantations	Mean	11.5	11.9	11.2	12.9
	SD	2.8	1.4	3.1	1.8
	Sum	242	261	246	284
Pre Implantation Loss	Mean	1	0.86	1.05	0.50
	SD	1.48	1.08	2.36	1.06
	Sum	21	19	23	11
Pre Implantation Loss (%)	Mean	9.19	6.47	8.91	3.38
	SD	16.78	7.73	20.39	8.11
Nb of early resorptions	Mean	0.8	0.6	1.0	1.1
	SD	1.3	0.8	1.1	1.5
	Sum	16	14	22	25
Early resorptions (%)	Mean	5.86	5.36	8.87	8.23
	SD	9.94	7.19	11.04	10.06
Nb of late resorptions	Mean	0.1	0.0	0.0	0.0
	SD	0.3	0.2	0.2	0.0
	Sum	2	1	1	0
Late resorptions (%)	Mean	0.80	0.45	0.32	0.00
	SD	2.53	2.13	1.52	0.00
Nb of dead foetuses	N	0	0	0	0
Post implantation loss	Mean	0.86	0.68	1.05	1.14
	SD	1.42	0.89	1.09	1.49
	Sum	18.0	15.0	23.0	25.0
Post implantation loss (%)	Mean	6.66	5.81	9.20	8.23
	SD	10.91	7.79	10.87	10.06
Nb of live foetuses	Mean	10.7	11.2	10.1	11.8
	SD	2.7	1.7	3.0	1.7
Nb of male foetuses	Mean	5.1	6.0	5.4	5.4
	SD	2.4	1.1	2.3	1.9
	Sum	107	132	119	118
	%	45.24	54.41	53.27	45.02
Nb of female foetuses	Mean	5.6	5.2	4.7	6.4
	SD	1.9	1.6	2.1	1.5
	Sum	117	114	104	141
Total litter weight (g)	Mean	37.73	38.081	35.345	39.042
	SD	9.866	5.517	10.956	6.010
	N	21	22	22	22
	% diff	/	0.929	-6.321	3.476
Mean foetal weight (M & F) (g)	Mean	3.54	3.41	3.46	3.34
	SD	0.19	0.16	0.24	0.41
	N	21	22	22	22
	% diff	/	-3.58	-2.14	-5.61

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of the study, oral (gavage) administration of Methylglutaric acid at 200, 400 and 800 mg/kg/day in the pregnant Wistar rat was well tolerated in all groups (no effect on body weight gain and food consumption) but was associated with post-dose hypersalivation, principally at 400 and 800 mg/kg/day, and a low incidence of noisy breathing in the high dose group only.

The No Observed Adverse Effect Level (NOAEL) for maternal toxicity is conservatively set at 400 mg/kg/day based on the low incidence of noisy breathing observed in the 800 mg/kg/day group.

In the absence of any adverse effect of treatment, the No Observed Adverse Effect Level (NOAEL) for embryo-foetal toxicity is set at 800 mg/kg/day.

There was no obvious evidence of a teratogenic potential of Methylglutaric acid in any group.

Executive summary:

The effect of Methyl glutaric acid was assess during an OECD 414 study.

Daily oral (gavage) administration of Methylglutaric acid to the pregnant Wistar rat at 200, 400 and 800 mg/kg/day was well tolerated in all groups (no effect on body weight gain and food consumption) but was associated with post-dose hypersalivation, principally at 400 and 800 mg/kg/day, and a low incidence of noisy breathing in the high dose group only.

Evidence of treatment-related embryo-foetal effects was restricted to slightly lower mean foetal weight in the 800 mg/kg/day group and a slightly higher incidence of fetuses with an unossified metacarpal of the 5^thdigit in the 400 and 800 mg/kg/day groups compared with the control. These minor changes were considered to be of no toxicological significance.

There were 2, 1 and 1 malformed foetuses from a single litter in each of the 200, 400 and 800 mg/kg/day groups compared with none in the control. One foetus in each of the 200 and 400 mg/kg/day groups had cardiovascular changes and one foetus in each of the 200 and 800 mg/kg/day groups had an omphalocele. In the absence of any dose-related increase in their incidence, and since these findings are part of the background of recent changes noted for the strain of rat, these isolated cases in each group were considered to be incidental. In addition, there were no other changes supportive of a possible association with treatment amongst the caesarean data (embryo-foetal survival and foetal data) or the less severe fetal visceral and skeletal anomalies or variations in the treated groups compared with the control.

Conclusion

Under the experimental conditions of the study, oral (gavage) administration of the Methylglutaric acid at 200, 400 and 800 mg/kg/day in the pregnant Wistar rat was well tolerated in all group (no effect on body weight gain and food consumption) but was associated with post-dose hypersalivation, principally at 400 and 800 mg/kg/day, and low incidence of noisy breathing in the high dose only.

The NOAEL for maternal toxicity is conservatively set at 400 mg/kg/day based on the low incidence based on the low incidence of noisy breathing observed in the 800 mg/kg/day group.

In the absence on any adverse effect of treatment, the NOAEL for embryo-foetal toxicity is set at 800 mg/kg/day.

There was no obvious evidence of a teratogenic potential of methylglutaric acid in any group.