2-methylglutaric acid

Endpoint summary

Description of key information

A 90 days oral repeated dose toxicity study (OECD408) on rat was performed.
The highest dose was initially 1000 mg/kg bw/day and was lowered to 600 mg/kg bw/day because of strong local effects on the GI tract.
No systemic effects were observed up to 600 mg/kg bw/day and the NOAEL was over 600 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

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 oral 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 project 505911.
- 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 and 1000/600 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10 animals 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

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once a day 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 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 unit: %
Haemoglobin unit: mmol/L
Haematocrit unit:L/L
Mean corpuscular volume unit: fL
Mean corpuscular haemoglobin unit:fmol
Mean corpuscular haemoglobin concentration unit: mmol/L
Platelets unit: 10e9/L
Prothrombin time unit: s
Activated Partial thromboplastin time unit: s

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During week 13
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters cexamined:
Alanine aminotransferase unit: U/L
Aspartate aminotransferase unit: U/L
Alkaline phosphatase 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 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.

OTHER:
Estrous cycle determination 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.

Sacrifice and pathology:

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.

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.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

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. Results on the decedent animals are reported in "other information on result" section.

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.

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.

REPRODUCTIVE ORGANS
histopathological examination of the male and female reproductive organs (including spermatogenesis staging) did not show treatment-related lesions. Therefore these changes were considered not adverse in this study.

Dose descriptor:

NOAEL

Effect level:

> 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Strong local effect on the GI tract observed at 1000 mg/kg

Critical effects observed:

not specified

RESULTS ON THE DECEDENT ANIMALS AT 1000 MG/KG

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 animals found dead or sacrificed for ethical reasons.

The male no.33 and females no.71, 74 and 76 were found dead or sacrificed for ethical reasons.

The results of these animals will be reported in this appendix. The decedent animals numbers will be reported as no. 101, 102, 103 and 104 (= decedent animal no. 33, 71, 74 and 76 respectively).

Mortality

One 1000 mg/kg w/day female was found dead on Day 4 prior to dosing. One 1000 mg/kg bw/day male and two females were sacrificed for ethical reasons on Days 6 or 8.

Clinical signs

Male no. 101 showed laboured respiration, swelling of abdomen and piloerection on Days 6 and/or 7. Female no. 102 showed hunched posture, laboured respiration and piloerection on Day 3.

Female no. 103 showed laboured respiration on Day 7.

Female no. 104 showed hunched posture labored respiration, piloerection and dehydrated appearance between Days 3 and 5.

 

Body weight

The four animals show severe body weight loss during the first week (maximum 23%).

Animal^1	Day 1	Day 7	Day8priortosacrifice	Bodyweightloss(%)
101	164	135	126	23.1
102	132	/^2	/	/
103	140	127	120	14.3
104	128	99 (Day 6)^3	/	22.7

¹The decedent animals numbers will be reported as no. 101, 102, 103 and 104 (= decedent animal no. 33, 71, 74 and 76 respectively).

²Animal no. 102 found dead on Day 3

³Animal no. 104 sacrificed on Day 6

Pathology: macroscopic and microscopic examination

Male no. 101: Main macroscopic findings included emaciation, distension with gas in the GI-tract (without microscopic correlate) and a reduction in size of the thymus (microscopic correlate lymphoid depletion), the spleen (microscopic correlate lymphoid atrophy and decreased red pulp), the prostate and the seminal vesicles (microscopic correlate immature/premature). In the kidneys, moderate basophilia of the tubules with slight degeneration and cellular sloughing was observed. These kidney findings may have contributed to the moribund condition of the animal. Lymphoid atrophy in the thymus, lymphoid depletion in the spleen and possibly the ductal degranulation of the submandibular glands, reflect the moribund condition of the animal.

Female no. 102: Main macroscopic findings included emaciation, many red foci in glandular mucosa of the stomach, distension with gas in the GI-tract (without microscopic correlate) and organs appeared autolytic. The marked lymphocytolyis in the thymus and possibly the ductal degranulation of the submandibular glands as well, reflect the moribund condition of the animal. No indications for the

cause of death was found on the sections examined.

Female no. 103: Main macroscopic findings consisted of emaciation and distension with gas in the GI- tract (without microscopic correlate). The marked lymphocytolyis in the thymus and possibly the ductal degranulation of the submandibular glands, reflect the moribund condition of the animal. No indications for the cause of death was found on the section examined.

Female no. 104: Main macroscopic findings consisted of emaciation, some red foci in glandular mucosa of the stomach, distension with gas in the GI-tract (without microscopic correlate) and a reduction in size of the thymus (microscopic correlate lymphoid depletion). In the kidneys, moderate basophilia of the tubules with slight degeneration and cellular sloughing was observed. In the larynx granulocytic infiltration and an erosion/ulceration was observed. There were no further indications for a possible procedural error. Findings in both the kidney and larynx may have contributed to the moribund condition of the animal. The marked lymphoid depletion in the thymus and possibly the ductal degranulation of the submandibular glands, reflect the moribund condition of the animal.

 

 

Conclusions:

Based on the experimental result, the NOAEL for the 2-methylglutaric acid is at least 600 mg/kg

Executive summary:

Title

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

 

Guidelines

The study was based on the following guidelines.

- EC No 440/2008, B.26 Repeated Dose (90 days) Toxicity (oral), 2008.

- OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998.

- OPPTS 870.3100, EPA 712-C-98-199, 90-Day Oral Toxicity in Rodents, 1998.

 

Rationale for dose levels

Based on the results of a 14-day range finding study (Project 505982), the dose levels for this 90-day oral gavage study 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.

 

Study outline

The test substance, 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.

 

Evaluated parameters

Chemical analyses of formulations were conducted three times during the study (Weeks 1, 6 and 13) to assess accuracy, homogeneity and stability over 6 hours.

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.

 

Results

 

Formulation analyses confirmed that formulations of test substance in water (Elix) were prepared accurately and homogenously, and were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours and in a refrigerator for at least 8 days.

 

Initially the highest dose group was started at 1000 mg/kg. Based on the mortality, severe body weight loss and clear clinical signs during Days 1-6 it was decided to lower the dose level to 600 mg/kg from Day 7 onwards and to replace the animals found dead or sacrificed for ethical reasons on Day 8.

Three new females were dosed from Day 8 up to and including Day 98. One male was dosed from Day 8 up to scheduled day of necropsy. The slightly shorter exposure period of 84 days in this single animal was considered not to influence the results of the study.

All animals of the highest dose group, surviving up to the scheduled necropsy, will be reported as 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. Rales were seen in surviving females at 1000 mg/kg.

After lowering the dose level, the animals at 600 mg/kg showed rales during the treatment period. Other clinical signs noted in these animals at lower incidence comprised of hunched posture, laboured respiration, piloerection, chromodacryorrhoea, dehydrated appearance. Based on the low incidence and the expected reversibility no toxicological significance was ascribed to these clinical signs.

 

Body weight and body weight gain were slightly lower for males and females after treatment at 1000 mg/kg for 6 days. After lowering the dose to 600 mg/kg the slightly lower body weights and body weight gains recovered to normal in females. In males at 600 mg/kg the lower body weight was accompanied by lower absolute food consumption.

Since these changes in body weights at 600 mg/kg are slight and within the range expected for rats of this age and strain in this kind of study, these changes are considered not to be adverse. 

 

Three females at 600 mg/kg (3/10) showed irregular cycle length or were acyclic between Day 69 up to and including Day 91. Two animals (control 1/10 and 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 male and female reproductive organs (including spermatogenesis staging) did not show treatment-related lesions. Therefore these changes were considered not adverse in this study.

 

Very slight changes in clinical biochemistry parameters were considered to be related to treatment based on the dose related response. These findings included: higher aspartate transferase and total bilirubin level and lower total protein level in males at 600 mg/kg and lower urea level in females at 600 mg/kg. Since all values were within the range expected for rats of this age and strain in this kind of study and since no corroborative adverse findings were noted at histopathological examination, these changes are considered to be not adverse.

 

At histopathological examination an increased incidence of vacuolation in the zona glomerulosa in the adrenal glands of females treated with 600 mg/kg/day was noted. Since in some studies it may be observed as background finding and the severity is only minor, this finding is not considered to be adverse.

 

No toxicologically significant changes were noted in the remaining parameters observed in this study: functional observations, ophthalmoscopy, heamatological parameters, macroscopic examination and organ weights.

 

 

Conclusion

 

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for 2 -Methylglutaric acid of at least 600 mg/kg was established.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Subchronic (90 days) performed according to the OECD 408 and in compliance with GLP (Kr: 1)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Only one study was available and was considered as the key study. Subchronic toxicity of 2-Methylglutaric acid was evaluated in a 90 days oral repeated dose study performed according to OECD 408 and in compliance with Good Laboratory Practices.

The test substance, 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 for this 90-day oral gavage study were selected to be 0, 100, 300 and 1000 mg/kg however 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 

 

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

Macroscopic and microscopic observation on the 1000 mg/kg decedent animals revealed that the main cause of death was strong local effects on the gastro-intestinal tract. Some treatment-related slight effects on the kidney were observed on two of the four animals.

 

After lowering the dose level, the animals at 600 mg/kg showed rales during the treatment period. Other clinical signs noted in these animals at lower incidence comprised of hunched posture, laboured respiration, piloerection, chromodacryorrhoea, dehydrated appearance. Based on the low incidence and the expected reversibility no toxicological significance was ascribed to these clinical signs.

 

Body weight and body weight gain were slightly lower for males and females after treatment at 1000 mg/kg for 6 days. After lowering the dose to 600 mg/kg the slightly lower body weights and body weight gains recovered to normal in females. In males at 600 mg/kg the lower body weight was accompanied by lower absolute food consumption.

Since these changes in body weights at 600 mg/kg are slight and within the range expected for rats of this age and strain in this kind of study, these changes are considered not to be adverse. 

 

Three females at 600 mg/kg (3/10) showed irregular cycle length or were acyclic between Day 69 up to and including Day 91. Two animals (control 1/10 and 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 male and female reproductive organs (including spermatogenesis staging) did not show treatment-related lesions. Therefore these changes were considered not adverse in this study.

 

Very slight changes in clinical biochemistry parameters were considered to be related to treatment based on the dose related response. These findings included: higher aspartate transferase and total bilirubin level and lower total protein level in males at 600 mg/kg and lower urea level in females at 600 mg/kg. Since all values were within the range expected for rats of this age and strain in this kind of study and since no corroborative adverse findings were noted at histopathological examination, these changes are considered to be not adverse.

 

At histopathological examination an increased incidence of vacuolation in the zona glomerulosa in the adrenal glands of females treated with 600 mg/kg/day was noted. Since in some studies it may be observed as background finding and the severity is only minor, this finding is not considered to be adverse.

 

No toxicologically significant changes were noted in the remaining parameters observed in this study: functional observations, ophthalmoscopy, haematological parameters, macroscopic examination and organ weights.

 

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for 2-Methylglutaric acid of at least 600 mg/kg was established.

 

Macroscopic and microscopic observation on the 1000 mg/kg decedent animals revealed that the main cause of death was strong local effects on the gastro-intestinal tract. Some treatment-related slight effects on the kidney were observed on two of the four animals. However, as these effects are present only at doses were local effects were sufficient to induce the death of the animals they are not considered as relevant for the evaluation of systemic toxicity of 2-Methylglutaric acid.

 

Therefore, it is concluded that long term exposition to 2-Methylglutaric is not expected to cause systemic toxicity by the oral route.

No study by the dermal route is available. However as 2-Methylglutaric acid is corrosive, long term exposition to the substance is not possible.

No study by the inhalation route is available. However, as 2-Methylglutaric acid is a non-volatile, non-pulverulent solid and as no aerosol generation is foreseen in the expected use, exposition by the inhalation route exceeding the studied oral exposition is highly unlikely. Therefore, 2-Methylglutaric acid is not expected to cause long term systemic toxicity by inhalation.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only available long term exposition study

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Since 2-Methylglutaric acid is classified as corrosive, is a non-volatile solid with a very low volatility and that no generation of aerosol is foreseen in the expected uses, no repeated dose toxicity study by inhalation is necessary.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Since 2-Methylglutaric acid is classified as corrosive, is a non-volatile solid with a very low volatility and that no generation of aerosol is foreseen in the expected uses, no repeated dose toxicity study by inhalation is necessary.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Since 2-Methylglutaric acid is classified as corrosive no dermal repeated dose toxicity study is necessary.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Since 2-Methylglutaric acid is classified as corrosive no dermal repeated dose toxicity study is necessary.

Justification for classification or non-classification

Based on the results described above (Beerens,2015. Key study, Kr: 1) and the observed NOAEL of at least 600 mg/kg bw/day, no classification for Single Target Organ Toxicity - Repeated Exposure is required according to EU criteria.