2-methylglutaric acid

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

other: 90-days study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2014-07-04 to 2015-06-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD 408 compliant. Evaluation of reproductive organs, estrous cycle and spermatogenesis staging during the 90-days toxicity study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 408

Deviations:

no

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

Constituent 2

Reference substance name:

2-Methyglutaric acid

IUPAC Name:

2-Methyglutaric acid

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Fasting period before study: No
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS
- J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage- enrichment or bedding material.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food.
- Water (e.g. ad libitum): Free access to tap water except during motor activity measurements.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18°C to 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From:2014-07-10 to 2014-10-16

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Elix, Millipore S.A.S., Molsheim, France

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. No correction was made for the purity of the test substance. the formulation was stored at ambiant temperature.

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200/ 120 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on three occasions during the treatment phase, according to a validated method (project 505911). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations), in Weeks 1, 6 and 13. Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration, in Week 1). Stability in vehicle over 8 days at room temperature under protection from light was also determined (highest and lowest concentration, in Week 1).

Method:

Samples:
Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 10, 20 or 50 ml. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50% height and stored at room temperature under normal laboratory light conditions for 6 hours and in a refrigerator for 8 days.
The volumetric flasks were filled up to the mark with 50/50 (v/v) acetonitrile/water. The solutions were further diluted to obtain an end solution of 0.1% H3PO4 in 5/95 (v/v) acetonitrile/water andconcentrations within the calibration range.

Analytical conditions:
Quantitative analysis was based on the analytical method validated for the test substance in project505911.
- 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 x 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 and spiking solutions: Stock and spiking solutions of the test substance were prepared in 50/50 (v/v) acetonitrile/water at concentrations of 1000 and 3000 mg/l.

Calibration solutions:
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 blank Elix water (vehicle) 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 sample.

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

Electronic data capture:
System control, data acquisition and data processing were performed using the following programme:
- Empower version 7.00 (Waters, Milford, MA, USA).
Temperature, relative humidity and/or atmospheric pressure during sample storage and/or performance of the studies was monitored continuously using the following programme:
- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA).

Results:

Calibration curves:
Calibration curves were constructed using five concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration2 weighting factor. If necessary, two responses were excluded from the curve the back calculated accuracy was > 15% from the nominal concentration. The coefficient of correlation (r) was > 0.99 for each curve.

Procedural recovery samples:
The mean recoveries of the procedural recovery samples fell within the criterion of 90-110%. It demonstrated that the analytical method was adequate for the determination of the test substance in the test samples.

Accuracy of preparation:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the Group 1 formulations.

Homogeneity:
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability:
Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of ≤ 10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours and in a refrigerator for at least 8 days.

Duration of treatment / exposure:

At least 90 days

Frequency of treatment:

Once a day

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 1000/600 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10 per sex and dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were based on results of a 14-day oral range finding study with 2-Methylglutaric acid by daily gavage in the rat.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately (0-15 minutes) after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly. In order to monitor the health status, some animals were weighed more often.

FOOD CONSUMPTION: Weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before start of treatment and during week 13. Since no treatment-related ophthalmologic findings were noted in Week
13 for grpup 1 a,nd 4, the eyes of the rats of Groups 2 and 3 were not examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13
- Anaesthetic used for blood collection: Yes isoflurane (Abbott B.V., Hoofddorp, The Netherlands)
- Animals fasted: Yes: overnight
- How many animals: All
- Parameters examined.:
White blood cells WBC unit: 10E9/L
Differential leucocyte count unit: % WBC (neutrophils, lymphocytes, monocytes, eosinophils, basophils)
Red blood cells unit: 10e12/L
Reticulocytes unit: %RBC
Red blood cell distribution width RDW unit: %
Haemoglobin unit: mmol/L
Haematocrit Unit:L/L
Mean corpuscular volume MCV unit: fL
Mean corpuscular haemoglobin MCH unit:fmol
Mean corpuscular haemoglobin concentration MCHC unit: mmol/L
Platelets unit: 10e9/L
Prothrombin time PT unit: s
Activated Partial thromboplastin time APTT unit: s

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During wek 13
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters cexamined:
Alanine aminotransferase ALAT unit: U/L
Aspartate aminotransferase ASAT unit: U/L
Alkaline phosphatase ALP unit: U/L
Total Protein unit:g/L
Albumin Unit: g/L
Total Bilirubin Unit: µmol/L
Urea Unit:mmol/L
Creatinine Unit: µmol/L
Glucose Unit: mmol/L
Cholesterol Unit:mmol/L
Sodium Unitmmol/L
Potassium Unit: mmol/L
Chloride Unit: mmol/L
Calcium Unit: mmol/L
Inorganic Phosphate Inorg. Phos Unit: mmol/L
Bile acids Unit: µmol/L

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during Weeks 12-13
- Dose groups that were examined: All
- Battery of functions tested:
-hearing ability (HEARING), pupillary reflex (PUPIL L/R) and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent): all Group 1 and 4 animals.
-fore- and hind-limb grip strength was recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands): all Group 1 and 4 animals
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA): all animals (males and females were tested on separate days).
- Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine finer movements like grooming, weaving or movements of the head.

Oestrous cyclicity (parental animals):

All females (except the replaced females) had a daily lavage from 16 September (Day 69) up to and including 8 October (Day 91) to determine the stage of estrous
All replaced females had a daily lavage from 23 September (treatment for 69 days) up to and including 14 October (treatment for 90 days) to determine the stage of estrous.

Sperm parameters (parental animals):

From all males of Group 1 and 4, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes was processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

Litter observations:

not applicable

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution):
Identification marks: not processed
Adrenal glands
Aorta
Brain [cerebellum, mid-brain, cortex]
Caecum
Cervix
(Clitoral gland)
Colon
Duodenum
Epididymides *
Eyes with optic nerve [if detectable] and
Harderian gland *
Female mammary gland area
(Femur including joint)
Heart
Ileum
Jejunum
Kidneys
Larynx
(Lacrimal gland, exorbital)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Oesophagus
Ovaries
Pancreas
Peyer's patches [jejunum, ileum] if detectable
Pituitary gland
(Preputial gland)
Prostate gland
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles including coagulating gland
(Skeletal muscle)
Skin
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes *
Thymus
Thyroid including parathyroid [if detectable]
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions
* Fixed in modified Davidson's solution, prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid - glacial (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA). Tissues were transferred to formalin after fixation for at least 24 hours.
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Organ weights
The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:

Adrenal glands, Spleen Brain, Testes Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid.

Histotechnology
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded in paraffin wax (Klinipath, Duiven, The Netherlands), cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
From all males of Group 1 and 4, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes was processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

Histopathology
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Group 1 and 4 animals,
- all tissues from animal nos. 101, 102, 103 and 104 (Group 4) which died spontaneously or were terminated in extremis,
- the additional slides of the testes of Group 1 and 4 males to examine staging of spermatogenesis,
- adrenals of all females of Groups 2 and 3, based on possible treatment-related changes in this organ in Group 4,
- all gross lesions.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Histopathology was subjected to a peer review.

Postmortem examinations (offspring):

Not applicable

Statistics:

The following statistical methods were used to analyze the data:
−If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many- to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
−The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
−The Fisher Exact-test (Ref. 3) was applied to frequency data.
−The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values

Reproductive indices:

Not applicable

Offspring viability indices:

Not applicable

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

Details on results (P0)

Based on the mortality, severe body weight loss and clinical signs during Days 1-6 at the highest dose level of 1000 mg/kg, it was decided to lower the dose level to 600 mg/kg and to replace the 4 animals found dead or sacrificed for ethical reasons. The decedent animals cause of death is maily related to strong local effects on the GI tract (see repeated dose toxicity section for more details).

REPRODUCTIVE ORGANS
histopathological examination of the male and female reproductive organs from all doses tested in the study did not show treatment-related lesions for male or female.

SPERMATOGENESIS STAGING
histopathological examination of the male spermatogenesis staging did not show treatment-related lesions.

ESTROUS CYCLE DETERMINATION
Three females at 600 mg/kg (3/10) showed irregular cycle length (no. 77 and 80) or were acyclic (no. 73) during Days 69 - 91. Two animals (no. 46: control (1/10) and no. 57: 100 mg/kg (1/10)) showed an irregular estrous cycle length. All other females showed a normal (regular) estrous cycle of 4 days. The incidence of irregular or acyclic estrous cycle length was slightly higher at 600 mg/kg, however histopathological examination of the female reproductive organs did not show treatment-related lesions. Therefore these changes were considered not adverse in this study.


CLINICAL SIGNS AND MORTALITY:
No mortality occurred in the animals treated at 100, 300 and 600 mg/kg.

The surviving males at 1000 mg/kg showed hunched posture, rales, piloerection, chromodacreorrhoea and/or dehydrated appearance during the first 6 days. The surviving females at 1000 mg/kg showed rales only.

The animals dosed at 600 mg/kg showed rales during the treatment period, more explicit in males than females. Other clinical signs noted at lower incidence comprised of hunched posture, laboured respiration, piloerection, chromodacryorrhoea and/or dehydrated appearance.

No clinical signs of toxicity were noted in control animals and animals treated at 100 and 300 mg/kg during the observation period. And no abnormalities in weekly arena observations were noted in all treated animals during the observation period.

Salivation seen after dosing among animals of the highest dose during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.

Incidental findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed and/or in absence of a dose related response, these were considered signs of no toxicological.

BODY WEIGHT AND WEIGHT GAIN:
Mean body weights and mean body weight gain were slightly lower for males and females after treatment at 1000 mg/kg for one week. After lowering the dose to 600 mg/kg slightly lower body
weights and body weight gains remained in males at 600 mg/kg during the study. The body weight and body weight gain of females were comparable to controls from Day 8 onwards.

The changes observed in body weight and body weight gain of females at 100 mg/kg occurred in absence of a dose response and were slight in nature. Therefore, these changes in females are considered to be not treatment related.

No toxicologically significant changes in body weights and body weight gain were noted in the remaining animals.

OPHTHALMOSCOPIC EXAMINATION
No ophthalmology findings were noted that were considered to be related to treatment.

The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test substance.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.

Minor statistically significant differences arising between controls and animals receiving 300 mg/kg were considered not to represent a change of biological significance.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters were considered to be related to treatment:
-Slightly higher aspartate transferase (ASAT) level in males at 600 mg/kg
-Slightly lower total protein level in males at 600 mg/kg
-Slightly higher total bilirubin level in males at 600 mg/kg
-Lower urea level in females at 600 mg/kg

Values in treated females at 300 mg/kg, achieving a level of statistical significance when compared to controls, were within the range expected for rats of this age and strain which are housed and treated under the conditions in this study and occurred in the absence of a treatment-related distribution. Therefore these changes are considered to be of no biological significance.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.

The variation in motor activity did not indicate a relation with treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

ORGAN WEIGHTS
No toxicologically significant changes were noted in organ weights and organ to body weight ratios of animals treated at 100, 300 and 600 mg/kg.

Any statistically significant changes in organ weights were considered to be of no toxicological significance as these remained within the range considered normal for rats of this age and strain

GROSS PATHOLOGY
Macroscopic observations at necropsy of animals treated at 100, 300 and 600 mg/kg did not reveal any alterations that were considered to have arisen as a result of treatment.

The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and/or had no treatment-related histopathological correlates. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
A test item-related non-adverse microscopic finding was present in the adrenals of half of the females treated at 600 mg/kg/day and consisted of minimal vacuolation in the zona glomerulosa.

All other microscopic findings of animals treated at 100, 300 and 600 mg/kg were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Results: F1 generation

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the experimental result, the NOAEL for the 2-Methylglutaric acid is at least 600 mg/kg.
No significant treatment-related effect were observed on studied reproductive parameters.

Executive summary:

During the 90 days GLP study performed following the OECD 408 guideline, the Estrous Cycle were measured and histopathological examination of male and female reproductive organs were performed (including staging of spermatogenesis for male control and high dose groups).

No significant treatment-related effect were noted on these reproductive parameters.