2-methylglutaric acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

other: OECD 408

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2013-05-06

Limit test:

no

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

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

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

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.

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

Key result

Dose descriptor:

NOAEL

Generation:

F1

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reasonning is made on a Weight of evidence strategy based on OECD 408 and GLP compliant study and a GLP OECD 414 study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No specific study has been performed on fertility. However reproductive parameters (estrous cycle determination and spermatogenesis staging) have been added to a GLP 90 days toxicity study by the oral route in rats performed according to OECD 408 guideline and compliant with good laboratory practices. In addition a GLP prenatal developmental toxicity study by the oral route in the pregnant rat (OECD 414)has been performed.

 

Repeated dose 90-day oral toxicity study with 2-Methylglutaric acid by daily gavage in the rat.

2-Methylglutaric acid, formulated in water (Elix), was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. The dose levels were selected to be 0, 100, 300 and 1000 mg/kg. Dose level of Group 4 was lowered from 1000 mg/kg (Days 1-6) to 600 mg/kg from Day 7 onwards based on the results during Days 1-6.

The following parameters were evaluated: clinical signs daily; functional observation tests in Week 12-13; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues including male and female reproductive organs..

In addition to standard parameters, specific reproductive parameters have been added:

-      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.

-      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).

The results of these study showed no systemic treatment related adverse effects. The main observed toxicity was local effects on the GI tract at 1000 mg/kg related to corrosive properties of the substance.

The results on the reproduction parameters were :

-      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.

The NOAEL was consequently set at 600 mg/kg bw/day (for more details see repeated dose toxicity section).

 

2-Methylglutaric acid - Prenatal developmental toxicity study by the oral route (gavage) in the pregnant rat. 

Daily oral (gavage) administration of Methylglutaric acid to the pregnant Wistar rat at 200, 400 and 800 mg/kg/daywas 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.

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.

 

Based on the absence of any adverse finding concerning reproductive parameters in both studies, 2-Methylglutaric is not expected to be hazardous for fertility.

 

As indicated in REACH (CE 1907/2006) annexe VIII section 8.7.1 column 2 and annex IX section 8.7.3, no Reproduction/Developmental Toxicity Screening Test (OECD 421) is required if a prenatal developmental toxicity study is available and no two-generation reproduction toxicity study (OECD 416) is required if no adverse effects on reproductive organs have been observed in a 90 days toxicity study. Therefore, no further fertility study is required.

Short description of key information:
No effect observed on reproductive organs, estrous cycle and spermatogenesis staging in a GLP 90-days oral study in rat.
No maternal or developmental adverse effect observed in a prenatal developmental toxicity study by the oral route in the pregnant rat

Justification for selection of Effect on fertility via oral route:
Only available study

Effects on developmental toxicity

Description of key information

No maternal or developmental adverse effects observed in a GLP compliant prenatal developmental toxicity study by the oral route in the pregnant rat (OECD 414).

Link to relevant study records

Reference

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

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

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

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.

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.

Details on maternal toxic effects:

Maternal toxic effects:yes

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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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

 

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

800 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and OECD 414 compliant.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A GLP compliant prenatal developmental toxicity study by the oral route in the pregnant rat (OECD 414) has been performed

 

Daily oral (gavage) administration ofMethylglutaric acid tothe pregnant Wistar rat at 200, 400 and 800 mg/kg/daywas 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.

Justification for selection of Effect on developmental toxicity: via oral route:
Only available study

Justification for classification or non-classification

Based on the absence of developmental or reproductive effect observed in the performed studies (Beerens,2015. Key study, Kr: 1 and Reynaud, 2015, Key Study, Kr: 1)) no classification for reprotoxicity is required according to EU criteria.