Registration Dossier

Administrative data

Description of key information

Based on the clinical signs of discomfort, the organ weight changes and the death of one female (whose imputability to the test substance remained doubful) noted at 1000 mg/kg/day in a rat OECD422 study , the No Observed Adverse Effect Level (NOAEL) of Dimethyl 2-Methylglutarate given by the oral route was considered to be 300 mg/kg body weight/day .
Based on the microscopic findings in the respiratory tract, especially in the nasal cavities (degeneration/regeneration of the respiratory or olfactory epithelium), noted at the highest dose tested (5 mg/L air i.e. 5000 mg/m3) in a rat OECD413 study, the No Observed Adverse Effect Concentration (NOAEC) of Dimethyl 2-Methylglutarate given by the inhalation route was considered to be 0.04 mg/L air (i.e. 40 mg/m3) for the local effects. The NOAEC for the systemic effects was considered to be 5 mg/L air (5000 mg/m3).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 07 March 2007 to 20 December 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to international test guidelines and to GLP.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 "Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (guideline from the Office of prevention, pesticides and toxic substances, US EPA, July 2000)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:RCC Ltd Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland.
- Age at study initiation: 9 weeks for males, 11 weeks for females and 13 weeks for positive control (for micronucleus evaluation) males
- Weight at study initiation: 219 to 236g for males and 178 to 202g for females (at first treatment) and 322 to 331g for positive control males (at arrival).
- Fasting period before study: no
- Housing:
Animals were housed in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, 4132 Muttenz/Switzerland).
During pre-pairing period, males and females were housed individually. Cages of males were interspersed among those holding females to promote the development of regular estrus cycles.
During pairing period, rats were housed one male/ one female in Makrolon pairing cages.
After mating or at the end of the pairing period, the males and the females were housed individually again.
During the lactation period (until day 4 of lactation), dams were housed together with their litters.
- Diet (e.g. ad libitum):Pelleted standard Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst/Switzerland) was available ad libitum (Batch No. 89/06).
- Water (e.g. ad libitum):Tap water from Füllinsdorf from an automatic system was available ad libitum.
- Acclimation period: 7 days, under test conditions with an evaluation of the health status.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): air-conditioned with 10-15 air changes per hour.
- Photoperiod (hrs dark / hrs light):12 hours artificial fluorescent light / 12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period.

IN-LIFE DATES: From 28 March to 21 May 2007
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Dose levels were in terms of test item as supplied by the Sponsor. Therefore a correction factor was not used for dose formulations.

PREPARATION OF DOSING SOLUTIONS:
Frequency of dose formulation: daily
Storage of dose formulations: room temperature (20 °C +/- 5°C), under nitrogen.

The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle were added (w/v). Using an appropriate homogenizer a homogenous mixture was prepared. Having obtained a homogenous mixture, vehicle was added until the required final volume was achieved. Separate formulations were prepared for each concentration.
During the daily administration period homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical phase was conducted at RCC Ltd, Itingen, Switzerland under GLP-compliant conditions to verify the identity of the test item dimethyl-2-methyl glutarate administered and to determine the content, homogeneity and stability of application formulations.
Several application formulations were prepared at the test facility and representative analytical samples were collected and dispatched to the test site internally. The test item concentrations were determined by GC coupled to a FID detector and quantified with the area under the peak.
The identity of dimethyl-2-methyl glutarate was confirmed by its retention time which was similar to that measured in the working standards. The test item content in all samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution of dimethyl-2-methyl glutarate in purified water was demonstrated. The application formulations were considered to be stable for at least 4 hours when kept under storage conditions.
In conclusion, the results obtained within the analytical part confirm the correct preparation and storage of application formulations during the conduct of this study.
Duration of treatment / exposure:
Males: 28 days
Females: approximately 40 to 45 days.
Frequency of treatment:
once daily (7 days/week)
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
other: nominal conc.
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose volume: 10 mL/kg body weight (including vehicle control)
- Dose selection rationale: Dose levels were selected in agreement with the Sponsor, based on the results of a preliminary study (RCC Study No. B05668), where 1000 mg/kg/day was used as highest dose level.
Positive control:
In order to carry out an evaluation of the micronucleus induction., an additionnal group of 5 male rats serving as a positive control was given one oral (gavage) administration (20 mg/kg) of Cyclophosphamide monohydrate about 24 hours prior to necropsy.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:All animals were checked at least twice daily for any mortalities and at least twice daily for signs of reaction to treatment and/or symptoms of ill health. Additionally, the females were observed for signs of difficult or prolonged parturition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first test item administration and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily during the entire study.

FOOD CONSUMPTION (g food/kg body weight/day): Yes
Males: Food consumption was recorded weekly during the prepairing and post-pairing period.
Females: Food consumption was recorded for the following periods: days 1-8 and 8-14 of the pre-pairing period; days 0-7, 7-14 and 14-21 post coitum and days 1-4 post partum.
Food consumption was not recorded during the pairing period (mixed values of males and females).

OPHTHALMOSCOPIC EXAMINATION: No

CLINICAL LABORATORY INVESTIGATIONS:
Blood samples were obtained on the day before or on the day of scheduled necropsy from all P generation males after they had been fasted overnight. Blood samples of P generation females were obtained on day 5 post partum after the females had been fasted overnight. Blood samples were collected sublingually with the animal under light isoflurane anaesthesia.
HEMATOLOGY: Yes
The following haematology parameters were determined: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin , Mean corpuscular haemoglobin concentration, Haemoglobin concentration distribution width, Platelet count, Total leukocyte count, Differential leukocyte count.
Coagulation: Thromboplastin time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
The following parameters were determined: Glucose, Urea, Creatinine, total Bilirubin , total Cholesterol, Aspartate aminotransferase, Alanine aminotransferase, Bile acids, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, total Protein, Albumin, Globulin, Albumin/Globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
At one time during the study (males: shortly before scheduled sacrifice; females: on day 3 or 4 post partum) relevant parameters were performed for five P generation males and five P generation females randomly selected from each group. This Functional observation battery (FOB) assessment was conducted following the daily dose administration. Animals were observed for the following:
a) Cage side observations: unusual body movements (e.g. tremors, convulsions), abnormal behaviour (e.g. circling, stereotypy) and posture as well as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex, and reactivity to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer's reflex), urine or faeces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (basing on beam count) was recorded for 6- minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.
Sacrifice and pathology:
SACRIFICE:
Males were sacrificed after at least 28 days of administrapostmortem examination tion of test item. Females were sacrificed on day 5 post partum. Pups were sacrificed on day 4 post partum. Males and females were killed by exsanguination following an intraperitoneal injection of sodium pentobarbital. Pups were killed by an intraperitoneal injection of sodium pentobarbital. If birth did not occur on the expected date (day 21 post coitum), the female was treated until day 24 post coitum, sacrificed on day 25 post coitum.

GROSS PATHOLOGY:
structural abnormalities or pathological changes, with special attention paid to the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible haemorrhagic areas of implantation sites2. Dead pups (except if excessively cannibalized) and pups killed at day 4 of lactation were examined macroscopically.

ORGAN WEIGHTS:
The testes and epididymides of all parental males were weighed as pairs.
The ovaries and the uterus of five randomly selected parental females of each group were weighed (as pairs for ovaries).

In addition for five adult males and females, randomly selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:adrenals, kidneys (weighed as pairs), liver, spleen, brain, thymus and heart.

HISTOPATHOLOGY:
Of all parental males the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: prostate, seminal vesicles with coagulation gland, testes and epididymides (in Bouin's fixative).

Of all parental females the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: ovaries.

In addition, of the five males and females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: heart, brain, thymus, spinal cord, thyroid, small and large intestines (incl. Peyer's patches), trachea and lungs (preserved by inflation with fixative), stomach, uterus (with vagina), liver, urinary bladder, kidneys, lymph nodes (mesenterial, mandibular), adrenals, peripheral nerve, spleen and bone marrow.

Full histopathology was carried out on the preserved organs and tissues of the animals in the vehicle control and high dose group (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure).
Other examinations:
ANALYSIS OF THE MICRONUCLEUS INDUCTION:
The preparation of the bone marrow smears and the analysis of the micronucleus frequency were performed by RCC-CCR (see details in section 7.6.2 Genetic toxicity in vivo).
Statistics:
The following statistical methods were used to analyze body weights, food consumption, reproduction and skeletal examination data:
• Means and standard deviations of various data were calculated.
• If the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for inter-group comparisons (i.e. single treatment groups against the control group).
• The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution.
• Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
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
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:

Mortality:
Except 2 dams in group 4 (1000 mg/kg/day), all males and all other females animals survived until scheduled necropsy.
In group 4 (1000 mg/kg/day):
- one female was noted with ruffled fur, quick breathing and sedation about one hour prior to the death on day 22 p.c. after prolonged parturition. As no sign of misadministration could be detected during necropsy, this death was considered test-item related.
- one female died on day 2 p.p.. A body weight loss of 39g and a decreased bw gain of 38% between days 20 and 21 of the gestation period indicated an unperceived injury during intubation on day 20 p.c. This death was considered incidental.

Clinical signs and symptoms:
In group 4, one dam was noted with salivation that started on day 11 of the gestation period. All males and females moved their head through the bedding material as a sign of discomfort starting on day 9 of the pre-pairing period until the last administration.
In group 3, some males and females were noted moving their head through the bedding material and this sign was observed in all animals at the end of the study.
In group 2, only 1 female was noted with moving head through the bedding material, starting on day 5 of gestation.

Body weight, body weight gain, food consumption: These were considered not influenced by treatment with the notified substance.

Functional Observational Battery: None of the parameters investigated was considered to be affected by treatment.

Laboratory findings: Hematology and clinical biochemistry parameters were considered not affected by the treatment.

Effects in organs:
Necropsy: No substance related findings were noted during macroscopic examination at scheduled necropsy.

Organ weights: In group 4 animals (M+F), the mean liver weight was slightly increased, as well as the liver/body weight ratio (but not statistically significant). In females of group 4, the mean kidney weight and the relative kidney weight were statistically significantly increased. All the other mean organ weights were considered to be not influenced by the treatment.

Histopathological examinations:
There were no treatment-related lesions recorded at histopathological examination. All lesions recorded during the microscopic observation were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.
Qualitative staging of testes: There were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations. Individual lesions recorded were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.

Micronucleus induction: see in section 7.6.2 Genetic toxicity in vivo
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the premature death of one dam at 1000 mg/kg/day due to prolonged parturition.
Critical effects observed:
not specified
Conclusions:
Based on the death of one female noted at 1000 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) was considered to be 300 mg/kg body weight/day.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Dimethyl-2-methyl glutarate on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Dimethyl-2-methyl glutarate was administered once daily orally (by gavage) to male rats for 28 days and to female rats throughout the pre-pairing, the pairing, the gestation and the lactation periods until day 4 post partum.

The following dose levels were applied:

Group 1: 0 mg/kg body weight/day (vehicle control)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

In order to carry out an evaluation of the micronucleus induction., an additionnal group of 5 male rats serving as a positive control was given one oral (gavage) administration (20 mg/kg) of Cyclophosphamide monohydrate about 24 hours prior to necropsy.

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (purified water).

The following results were obtained:

In parent animals, with the exception of two female animals in group 4, all male and female animals survived until scheduled necropsy. One female rat died due to an unperceived injury during intubation. The other dam that died was noted with signs of a prolonged parturition.

A sign of discomfort was noted in all animals at 1000 mg/kg/day and in some animals at 300 mg/kg/day in the way animals moved their head throught the bedding material after the daily administration, and this effect was considered to be non-adverse.

Food consumption, body weight, and body weight gain were considered not to be influenced by treatment with the test item.

None of the parameters of the Functional Observational Battery was considered to be affected by exposure to the test item.

None of the parameters under investigation for hematology and clinical biochemistry was considered to be affected by exposure to the test item.

In comparison to the corresponding vehicle controls there was no biologically relevant enhancement in the frequency of the detected micronuclei at any dose level after administration of the test item.

No test item-related macroscopic or microscopic findings were noted at any dose levels.

In group 4 males and females, the mean absolute and relative liver weights were slightly increased. In group 4 females, the mean absolute and relative kidney weights were statistically significantly increased.

Based on the death of one female noted at 1000 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) was considered to be 300 mg/kg/day.

This developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study OECD422 in rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study performed according to the OECD 422 guideline (Klimish score = 1)

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 28 October 2009 to 20 July 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to international test guidelines and to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands.
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: males: 291.7 to 337.7 g ; females: 181.4 to 209.1 g
- Fasting period before study: no
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland).
- Diet: Pelleted standard Kliba Nafag 3433 (batch nos. 35/09 and 87/09) rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum except for the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles except for the periods when the animals were restrained in the exposure tubes.
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 with music during the light period.

IN-LIFE DATES: From 11 November 2009 to 11 February 2010
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Mass Median Aerodynamic Diameters and Geometric Standard Deviations were calculated on the basis of the results from the impactor, using Microsoft Excel Software.

The mean MMAD was below 3 μm (target range was 1 to 3 µm) and therefore the aerosol was considered to be respirable for rats (see summary table below) . In addition, the test item atmosphere of groups 2 and 3 was respirable as the test item was vaporous.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Inhalation exposure was performed using a system similar to that originally described by Sachsse et al.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes which were positioned radially around the flow-past, nose-only exposure chamber as described by Cannon et al.. The design of this chamber is based upon the fluid dynamic modeling of the test aerosol flow.
- Source and rate of air: The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 1.0 L/min, which is sufficient to minimize re-breathing of the test atmosphere as it is more than twice the respiratory minute volume of a rat.
- System of generating particulates/aerosols:The test aerosol was generated using a Hudson nebulizer connected to a syringe pump. The polyethylene injector inside model was replaced by a stainless steel injector. Aerosol concentrations for groups 2 and 3 were achieved by serial dilution of the aerosol generated for exposure of group 4 with compressed, filtered, dry air using an air vacuum device.
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity of the test atmosphere was measured continuously and recorded hourly during each exposure using a calibrated device (Rotronic Probe Serie I-200, Rotronic AG).
- Air flow rate: The airflow rate was adjusted before the start of the exposure using a calibrated flow-meter (TSI 4000 series). The actual airflow rate through the exposure chamber was checked hourly during each exposure.
- Method of particle size determination:The cumulative particle size distribution of the test aerosol was determined using a 7 stage cascade Mercer Impactor (Model 02-130, In-Tox. Products Inc., Albuquerque, New Mexico, U.S.A.). The test aerosol was impacted at each stage onto stainless steel slips and the particle size distribution was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor. In addition the particle size distribution was determined by chemically analyzing the test item deposited on each stage of the cascade impactor. Immediately after
weighing the steel slip of each stage was put into an appropriately labeled vial and covered with an appropriate amount of Acetonitril. The samples were analyzed using a GC method.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric determinations of aerosol concentration were performed once per exposure for groups 2 and 3 and three times per exposure for group 4. The test atmosphere samples were collected on a Millipore®durapore filter, Type HVLP, loaded in a 47 mm in-line stainless steel filter sampling device. The filters were weighed before and immediately after sampling using a calibrated balance. The test aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical phase was conducted at Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland under GLP-compliant conditions.

Chemical determinations of aerosol concentration were performed twice per week for groups 2 to 4.
The samples were collected on a Millipore®durapore filter, Type HVLP filter loaded in a stainless steel filter device and in a solvent trap mounted after the filter (groups 2 and 3 only). The solvent trap contained an appropriate amount of Acetonitril and was cooled in an ice/water and covered with 5 mL of Acetonitril.
The samples were analyzed using a GC method. The results were corrected for purity.

The chemical aerosol concentrations were as targeted and stable during the whole treatment period (see summary table below).
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0, 0.01, 0.04, 5 mg/L air
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Dose selection rationale:
Target aerosol concentrations were selected by the Sponsor based on the results from the previous 2- week inhalation study performed at Harlan Laboratories (study C51304, non-GLP) and information on related substances.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during treatment and once daily during acclimatization for mortality/viability ; once daily during treatment and once weekly during acclimatization for clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly (each individual animal) during acclimatization and twice weekly during treatment.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule for examinations: once weekly (per cage) during acclimatization and at least once weekly (per cage) during treatment.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: At acclimatization (all animals) and at week 13 (control and high dose animals), using a direct ophthalmoscope.

CLINICAL LABORATORY INVESTIGATIONS:
Blood and urine sampling was performed after 13 weeks of treatment for all animals.
Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

HEMATOLOGY: Yes
The following haematology parameters were determined: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin , Mean corpuscular haemoglobin concentration, Haemoglobin concentration distribution width, Reticulocyte count, Reticulocyte maturity index (low, medium, high fluorescence), Platelet count, Total leukocyte count, Differential leukocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells).
Coagulation: Thromboplastin time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
The following parameters were determined: Glucose, Urea, Creatinine, total Bilirubin , total Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, total Protein, Albumin, Globulin, Albumin/Globulin ratio.

URINALYSIS: Yes
The following parameters were determined: Urine volume (18 hours), Specific gravity (relative density), Color, Appearance, pH value, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes.
Sacrifice and pathology:
SACRIFICE:
All animals were sacrificed after 13 weeks of treatment. They were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded.

ORGAN WEIGHTS:
The following organs were weighed before fixation and recorded on the scheduled dates of necropsy: Adrenal glands, Brain (including section of medulla/pons, cerebral and cerebellar cortex), Epididymides, Heart including auricles, Kidneys, Liver, Lungs, Ovaries, Spleen, Testes, Thymus, Thyroid incl. parathyroid gland and Uterus with vagina. Relative organ weights were calculated on the basis of the body weight and brain weight.

GROSS PATHOLOGY: Yes
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution except for eyes with optic nerve which were fixed in Davidson's solution or epididymides and testes which were fixed in Bouin’s solution: Adrenal glands,Aorta,Bone (sternum, femur including articular surface), Bone marrow (femur), Brain (including section of medulla/pons, cerebral and cerebellar cortex), Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Heart including auricles, Kidneys, Large intestine (cecum, colon, rectum), Larynx, Lacrimal gland (exorbital), Liver, Lungs (filled via trachea with formalin at approximately 30 cm H2O pressure), Lymph nodes (mesenteric, mandibular and mediastinal), Mammary gland area (females only), Nasal cavity, Nasopharyngeal duct and pharynx, Olfactorial bulb, Ovaries, Pancreas,
Peyers Patches, Pituitary gland, Prostate gland including coagulating glands, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin and subcutaneous tissue, Small intestine (duodenum, jejunum, ileum), Spinal cord (cervical, midthoracic, lumbar segments), Spleen, Stomach, Teeth, Testes (fixed in Bouin's solution), Thymus, Thyroid incl. parathyroid gland, Tongue, Trachea (adjacent to larynx and carina and bifurcation), Urinary bladder (filled with formalin at necropsy), Uterus with vagina, All gross lesions)

HISTOPATHOLOGY: Yes
All organs and tissues (except for the Peyers Patches and tongue) which were collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin. The slides were then examined by the study pathologist. As test item-related changes were recorded in organs of the high dose group, these organs from the mid and low dose group were examined as well.
Statistics:
The following statistical methods were used to analyze food consumption, body weight, opthalmoscopic findings, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY:
All animals survived the scheduled treatment period.
Salivation was recorded in all males and females during most of the treatment period at 5 mg/L air. Breathing noise was recorded in one male of this group during the last five days of treatment.

BODY WEIGHT AND WEIGHT GAIN:
Body weight loss was recorded in males and females during the first four days of exposure at 5 mg/L air. Slight body weight loss or stagnation in body weight was also recorded in the other groups treated with the test item, but also in females of the control group.
Statistically significantly decreased body weight gain and absolute body weight was observed in males and females exposed at 5 mg/L air during the whole treatment period. Lower body weight compared to control animals was also recorded in animals at 0.01 mg/L air during several weeks.

FOOD CONSUMPTION:
Statistically significantly decreased food consumption was recorded in males (-15.4% below the control group) at 5 mg/L air during most of the treatment period and in females (-9% compared to controls) at this aerosol concentration during the first week.

OPHTHALMOSCOPIC EXAMINATION:
There were no ophthalmic changes that were considered to be related to treatment with the test item.

HAEMATOLOGY:
There were no changes in hematology parameters that were considered to be related to treatment with the test item.

CLINICAL CHEMISTRY:
Statistically significant increase in urea, sodium and chloride values was observed in males exposed at 5 mg/L air. These values were also increased in females at this aerosol concentration with statistical significance of sodium only.
Statistically significant increase in sodium and chloride was also observed in males treated at 0.04 mg/L air. There was no dose-dependence pattern observed in females for urea, sodium and chloride increase.

URINALYSIS:
In males treated with the test item the following group mean values were modified: decreased urine volume (with statistical significance in groups 3 and 4 only) as well as increased relative density (statistically significant in all groups) and protein values (with statistical significance in groups 2 and 4). These values were also modified in females (statistical significance for protein values only) at 5 mg/L air. Statistically significantly decreased pH was recorded in males at 5 mg/L air.

ORGAN WEIGHTS:
Statistically significant increase in relative weights of adrenals and kidneys were recorded in males and females after exposure at 5 mg/L air. Absolute adrenal weight was statistically significantly increased in females at 5 mg/L air. Statistically significant increase in lung weight ratios and decrease in thymus/brain weight ratio was observed in males of this group.

GROSS PATHOLOGY:
There were no macroscopic findings that were considered to be related to treatment with the test item.

HISTOPATHOLOGY (NON-NEOPLASTIC):
Minimal epithelial atrophy in the trachea was recorded in some animals exposed at 5 mg/L air.

Squamous metaplasia of the respiratory epithelium, inflammatory exudates, necrosis of the epithelium, inflammation of the submucosa, respiratory metaplasia of the olfactory epithelium and ulceration was recorded in the nasal cavity of some animals at 5 mg/L air with minimal to moderate intensity.

Slightly increased severity of thymus atrophy / involution was recorded in a few animals treated at 5 mg/L air.

Degeneration / regeneration of the respiratory or olfactory epithelium was observed at section levels 1 to 4 of the nasal cavity with a minimal to moderate intensity in most of the animals at 5 mg/L air, and marked effects in one male at section Level 2.
Four females at 0.04 mg/L air had minimal (2 females) to slight (2 females) effects, at level 3 of the nasal cavity (olfactory epithelium).
In the group treated at 0.01 mg/L air, degeneration / regeneration of the olfactory epithelium of minimal intensity was recorded in 3 males (sections levels 3 or 4) , and with slight intensity in 1 female (at Level 3).
Incidence and mean severity of degeneration / regeneration of the respiratory or olfactory epithelium are summarized in the table below.

Dose descriptor:
NOAEC
Effect level:
0.04 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the microscopic findings in the respiratory tract, especially in the nasal cavities (degeneration/regeneration of the respiratory or olfactory epithelium), observed at the high dose of 5.0 mg/L air.
Critical effects observed:
not specified

Incidence and mean severity of degeneration / regeneration of the respiratory or olfactory epithelium:

 

Finding

Group 1

Group 2

Group 3

Group 4

Incidence /

Mean Severity

10 M

10 F

10 M

10 F

10 M

10 F

10 M

10 F

Nasal Cavity, Level 1

Degener. / Regener.

-

-

-

-

-

-

8/1.9

6/1.3

Nasal Cavity, Level 2

Degener. / Regener.

-

-

-

-

-

-

10/2.7

9/2.3

Nasal Cavity, Level 3

Degener. / Regener.

-

-

2/1.0

1/2.0

-

4/1.5

9/2.1

9/1.9

Nasal Cavity, Level 4

Degener. / Regener.

-

-

1/1.0

-

-

-

8/2.0

7/1.7

 

The severity is expressed as the mean of the individual grading (1: minimal, 2: slight, 3: moderate, 4: marked).

Conclusions:
Based on the microscopic findings in the respiratory tract, especially in the nasal cavities (degeneration/regeneration of the respiratory or olfactory epithelium), observed at the high dose of 5.0 mg/L air, the overall no-observed-adverse-effect-concentration (NOAEC) of Dimethyl 2-Methylglutarate for this study was established at 0.04 mg/L air.

For systemic effects the NOAEC was considered to be 5 mg/L air.
Executive summary:

In this study, Dimethyl 2-Methylglutarate was administered to rats by nose-only inhalation for 6 hours a day for a period of 13 weeks.

The study comprised four groups containing ten male and ten female rats each. The animals of the control group (group 1) were exposed to air only. The following aerosol concentrations were administered:

Group 1: 0 mg/L air

Group 2: 0.01 mg/L air

Group 3: 0.04 mg/L air

Group 4: 5.0 mg/L air

Throughout the study all animals were observed for viability and clinical signs. In addition body weights and food consumption were recorded. Ophthalmoscopic examinations were performed and blood and urine samples were taken for clinical laboratory investigations. Following completion of the treatment period, a detailed necropsy was performed on all animals and selected organs were weighed. Various tissues and organs were placed in fixative, processed and examined microscopically.

The achieved chemical aerosol concentrations were between 100 and 112 % of the target. Temperature, relative humidity and oxygen concentration in the inhalation chamber were considered to be satisfactory for this study type.

The particle size distribution was determined for group 4 only as part of the atmosphere of group 2 and 3 was vaporous. The chemical mean mass median aerodynamic diameter (MMAD) was 2.50 μm and the mean geometric standard deviation (GSD) was 2.90. Therefore the aerosol of all groups was considered to be respirable for rats.

All animals survived the scheduled treatment period. Salivation was recorded in all animals at 5 mg/L air. Breathing noise was recorded in one male of this group during the last five days of treatment.

Reduced food consumption associated with a decreased body weight gain was recorded in animals exposed at 5.0 mg/L air.

Increase in urea, sodium and chloride values was observed in animals exposed at 5 mg/L air.

In males treated with the test item the following group mean values were statistically significantly modified: decreased urine volume (groups 3 and 4 only) as well as increased relative density and protein values (groups 2 and 4 only). These values were also modified in females at 5 mg/L air. Statistically significantly decreased pH was recorded in males at 5 mg/L air.

There were no ophthalmic changes and no changes in hematology that were considered to be related to treatment with the test item.

Increase in relative weights of the kidneys were recorded in males and females after exposure at 5 mg/L air. Increase in lung/body weight ratio was observed in males of this group.

There were no macroscopic findings that were considered to be related to treatment with the test item.

The following microscopic changes were recorded:

- Epithelial atrophy in the trachea of animals at 5 mg/L air.

- Squamous metaplasia of the respiratory epithelium, inflammatory exudates, necrosis of the epithelium, inflammation of the submucosa, respiratory metaplasia of the olfactory epithelium and ulceration in the nasal cavity of animals at 5 mg/L air.

- Degeneration / regeneration of the respiratory or olfactory epithelium in the nasal cavity (nasal section levels 1 to 4) of minimal to marked intensity in animals treated at 5 mg/L. Minimal effects on the olfactory epithelium (lower severity and lower incidence) were observed in the lower dose groups. However, the effects observed at 0.01 and 0.04 mg/L air are considered to be unspecific since similar variations were observed in some control animals of the preliminary Dose range finding study (2 -week repeated dose study) as well as in Harlan Laboratories Ltd historical control data.

For systemic effects the NOAEC was considered to be 5 mg/L air.

This 13 -week inhalation toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a subchronic (90 days) inhalation study OECD413 in rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
5 000 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Study performed according to the OECD 413 guideline (Klimish score = 1)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 28 October 2009 to 20 July 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to international test guidelines and to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands.
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: males: 291.7 to 337.7 g ; females: 181.4 to 209.1 g
- Fasting period before study: no
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland).
- Diet: Pelleted standard Kliba Nafag 3433 (batch nos. 35/09 and 87/09) rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum except for the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles except for the periods when the animals were restrained in the exposure tubes.
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 with music during the light period.

IN-LIFE DATES: From 11 November 2009 to 11 February 2010
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Mass Median Aerodynamic Diameters and Geometric Standard Deviations were calculated on the basis of the results from the impactor, using Microsoft Excel Software.

The mean MMAD was below 3 μm (target range was 1 to 3 µm) and therefore the aerosol was considered to be respirable for rats (see summary table below) . In addition, the test item atmosphere of groups 2 and 3 was respirable as the test item was vaporous.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Inhalation exposure was performed using a system similar to that originally described by Sachsse et al.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes which were positioned radially around the flow-past, nose-only exposure chamber as described by Cannon et al.. The design of this chamber is based upon the fluid dynamic modeling of the test aerosol flow.
- Source and rate of air: The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 1.0 L/min, which is sufficient to minimize re-breathing of the test atmosphere as it is more than twice the respiratory minute volume of a rat.
- System of generating particulates/aerosols:The test aerosol was generated using a Hudson nebulizer connected to a syringe pump. The polyethylene injector inside model was replaced by a stainless steel injector. Aerosol concentrations for groups 2 and 3 were achieved by serial dilution of the aerosol generated for exposure of group 4 with compressed, filtered, dry air using an air vacuum device.
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity of the test atmosphere was measured continuously and recorded hourly during each exposure using a calibrated device (Rotronic Probe Serie I-200, Rotronic AG).
- Air flow rate: The airflow rate was adjusted before the start of the exposure using a calibrated flow-meter (TSI 4000 series). The actual airflow rate through the exposure chamber was checked hourly during each exposure.
- Method of particle size determination:The cumulative particle size distribution of the test aerosol was determined using a 7 stage cascade Mercer Impactor (Model 02-130, In-Tox. Products Inc., Albuquerque, New Mexico, U.S.A.). The test aerosol was impacted at each stage onto stainless steel slips and the particle size distribution was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor. In addition the particle size distribution was determined by chemically analyzing the test item deposited on each stage of the cascade impactor. Immediately after
weighing the steel slip of each stage was put into an appropriately labeled vial and covered with an appropriate amount of Acetonitril. The samples were analyzed using a GC method.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric determinations of aerosol concentration were performed once per exposure for groups 2 and 3 and three times per exposure for group 4. The test atmosphere samples were collected on a Millipore®durapore filter, Type HVLP, loaded in a 47 mm in-line stainless steel filter sampling device. The filters were weighed before and immediately after sampling using a calibrated balance. The test aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical phase was conducted at Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland under GLP-compliant conditions.

Chemical determinations of aerosol concentration were performed twice per week for groups 2 to 4.
The samples were collected on a Millipore®durapore filter, Type HVLP filter loaded in a stainless steel filter device and in a solvent trap mounted after the filter (groups 2 and 3 only). The solvent trap contained an appropriate amount of Acetonitril and was cooled in an ice/water and covered with 5 mL of Acetonitril.
The samples were analyzed using a GC method. The results were corrected for purity.

The chemical aerosol concentrations were as targeted and stable during the whole treatment period (see summary table below).
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0, 0.01, 0.04, 5 mg/L air
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Dose selection rationale:
Target aerosol concentrations were selected by the Sponsor based on the results from the previous 2- week inhalation study performed at Harlan Laboratories (study C51304, non-GLP) and information on related substances.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during treatment and once daily during acclimatization for mortality/viability ; once daily during treatment and once weekly during acclimatization for clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly (each individual animal) during acclimatization and twice weekly during treatment.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule for examinations: once weekly (per cage) during acclimatization and at least once weekly (per cage) during treatment.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: At acclimatization (all animals) and at week 13 (control and high dose animals), using a direct ophthalmoscope.

CLINICAL LABORATORY INVESTIGATIONS:
Blood and urine sampling was performed after 13 weeks of treatment for all animals.
Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

HEMATOLOGY: Yes
The following haematology parameters were determined: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin , Mean corpuscular haemoglobin concentration, Haemoglobin concentration distribution width, Reticulocyte count, Reticulocyte maturity index (low, medium, high fluorescence), Platelet count, Total leukocyte count, Differential leukocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells).
Coagulation: Thromboplastin time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
The following parameters were determined: Glucose, Urea, Creatinine, total Bilirubin , total Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, total Protein, Albumin, Globulin, Albumin/Globulin ratio.

URINALYSIS: Yes
The following parameters were determined: Urine volume (18 hours), Specific gravity (relative density), Color, Appearance, pH value, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes.
Sacrifice and pathology:
SACRIFICE:
All animals were sacrificed after 13 weeks of treatment. They were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded.

ORGAN WEIGHTS:
The following organs were weighed before fixation and recorded on the scheduled dates of necropsy: Adrenal glands, Brain (including section of medulla/pons, cerebral and cerebellar cortex), Epididymides, Heart including auricles, Kidneys, Liver, Lungs, Ovaries, Spleen, Testes, Thymus, Thyroid incl. parathyroid gland and Uterus with vagina. Relative organ weights were calculated on the basis of the body weight and brain weight.

GROSS PATHOLOGY: Yes
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution except for eyes with optic nerve which were fixed in Davidson's solution or epididymides and testes which were fixed in Bouin’s solution: Adrenal glands,Aorta,Bone (sternum, femur including articular surface), Bone marrow (femur), Brain (including section of medulla/pons, cerebral and cerebellar cortex), Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Heart including auricles, Kidneys, Large intestine (cecum, colon, rectum), Larynx, Lacrimal gland (exorbital), Liver, Lungs (filled via trachea with formalin at approximately 30 cm H2O pressure), Lymph nodes (mesenteric, mandibular and mediastinal), Mammary gland area (females only), Nasal cavity, Nasopharyngeal duct and pharynx, Olfactorial bulb, Ovaries, Pancreas,
Peyers Patches, Pituitary gland, Prostate gland including coagulating glands, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin and subcutaneous tissue, Small intestine (duodenum, jejunum, ileum), Spinal cord (cervical, midthoracic, lumbar segments), Spleen, Stomach, Teeth, Testes (fixed in Bouin's solution), Thymus, Thyroid incl. parathyroid gland, Tongue, Trachea (adjacent to larynx and carina and bifurcation), Urinary bladder (filled with formalin at necropsy), Uterus with vagina, All gross lesions)

HISTOPATHOLOGY: Yes
All organs and tissues (except for the Peyers Patches and tongue) which were collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin. The slides were then examined by the study pathologist. As test item-related changes were recorded in organs of the high dose group, these organs from the mid and low dose group were examined as well.
Statistics:
The following statistical methods were used to analyze food consumption, body weight, opthalmoscopic findings, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY:
All animals survived the scheduled treatment period.
Salivation was recorded in all males and females during most of the treatment period at 5 mg/L air. Breathing noise was recorded in one male of this group during the last five days of treatment.

BODY WEIGHT AND WEIGHT GAIN:
Body weight loss was recorded in males and females during the first four days of exposure at 5 mg/L air. Slight body weight loss or stagnation in body weight was also recorded in the other groups treated with the test item, but also in females of the control group.
Statistically significantly decreased body weight gain and absolute body weight was observed in males and females exposed at 5 mg/L air during the whole treatment period. Lower body weight compared to control animals was also recorded in animals at 0.01 mg/L air during several weeks.

FOOD CONSUMPTION:
Statistically significantly decreased food consumption was recorded in males (-15.4% below the control group) at 5 mg/L air during most of the treatment period and in females (-9% compared to controls) at this aerosol concentration during the first week.

OPHTHALMOSCOPIC EXAMINATION:
There were no ophthalmic changes that were considered to be related to treatment with the test item.

HAEMATOLOGY:
There were no changes in hematology parameters that were considered to be related to treatment with the test item.

CLINICAL CHEMISTRY:
Statistically significant increase in urea, sodium and chloride values was observed in males exposed at 5 mg/L air. These values were also increased in females at this aerosol concentration with statistical significance of sodium only.
Statistically significant increase in sodium and chloride was also observed in males treated at 0.04 mg/L air. There was no dose-dependence pattern observed in females for urea, sodium and chloride increase.

URINALYSIS:
In males treated with the test item the following group mean values were modified: decreased urine volume (with statistical significance in groups 3 and 4 only) as well as increased relative density (statistically significant in all groups) and protein values (with statistical significance in groups 2 and 4). These values were also modified in females (statistical significance for protein values only) at 5 mg/L air. Statistically significantly decreased pH was recorded in males at 5 mg/L air.

ORGAN WEIGHTS:
Statistically significant increase in relative weights of adrenals and kidneys were recorded in males and females after exposure at 5 mg/L air. Absolute adrenal weight was statistically significantly increased in females at 5 mg/L air. Statistically significant increase in lung weight ratios and decrease in thymus/brain weight ratio was observed in males of this group.

GROSS PATHOLOGY:
There were no macroscopic findings that were considered to be related to treatment with the test item.

HISTOPATHOLOGY (NON-NEOPLASTIC):
Minimal epithelial atrophy in the trachea was recorded in some animals exposed at 5 mg/L air.

Squamous metaplasia of the respiratory epithelium, inflammatory exudates, necrosis of the epithelium, inflammation of the submucosa, respiratory metaplasia of the olfactory epithelium and ulceration was recorded in the nasal cavity of some animals at 5 mg/L air with minimal to moderate intensity.

Slightly increased severity of thymus atrophy / involution was recorded in a few animals treated at 5 mg/L air.

Degeneration / regeneration of the respiratory or olfactory epithelium was observed at section levels 1 to 4 of the nasal cavity with a minimal to moderate intensity in most of the animals at 5 mg/L air, and marked effects in one male at section Level 2.
Four females at 0.04 mg/L air had minimal (2 females) to slight (2 females) effects, at level 3 of the nasal cavity (olfactory epithelium).
In the group treated at 0.01 mg/L air, degeneration / regeneration of the olfactory epithelium of minimal intensity was recorded in 3 males (sections levels 3 or 4) , and with slight intensity in 1 female (at Level 3).
Incidence and mean severity of degeneration / regeneration of the respiratory or olfactory epithelium are summarized in the table below.

Dose descriptor:
NOAEC
Effect level:
0.04 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the microscopic findings in the respiratory tract, especially in the nasal cavities (degeneration/regeneration of the respiratory or olfactory epithelium), observed at the high dose of 5.0 mg/L air.
Critical effects observed:
not specified

Incidence and mean severity of degeneration / regeneration of the respiratory or olfactory epithelium:

 

Finding

Group 1

Group 2

Group 3

Group 4

Incidence /

Mean Severity

10 M

10 F

10 M

10 F

10 M

10 F

10 M

10 F

Nasal Cavity, Level 1

Degener. / Regener.

-

-

-

-

-

-

8/1.9

6/1.3

Nasal Cavity, Level 2

Degener. / Regener.

-

-

-

-

-

-

10/2.7

9/2.3

Nasal Cavity, Level 3

Degener. / Regener.

-

-

2/1.0

1/2.0

-

4/1.5

9/2.1

9/1.9

Nasal Cavity, Level 4

Degener. / Regener.

-

-

1/1.0

-

-

-

8/2.0

7/1.7

 

The severity is expressed as the mean of the individual grading (1: minimal, 2: slight, 3: moderate, 4: marked).

Conclusions:
Based on the microscopic findings in the respiratory tract, especially in the nasal cavities (degeneration/regeneration of the respiratory or olfactory epithelium), observed at the high dose of 5.0 mg/L air, the overall no-observed-adverse-effect-concentration (NOAEC) of Dimethyl 2-Methylglutarate for this study was established at 0.04 mg/L air.

For systemic effects the NOAEC was considered to be 5 mg/L air.
Executive summary:

In this study, Dimethyl 2-Methylglutarate was administered to rats by nose-only inhalation for 6 hours a day for a period of 13 weeks.

The study comprised four groups containing ten male and ten female rats each. The animals of the control group (group 1) were exposed to air only. The following aerosol concentrations were administered:

Group 1: 0 mg/L air

Group 2: 0.01 mg/L air

Group 3: 0.04 mg/L air

Group 4: 5.0 mg/L air

Throughout the study all animals were observed for viability and clinical signs. In addition body weights and food consumption were recorded. Ophthalmoscopic examinations were performed and blood and urine samples were taken for clinical laboratory investigations. Following completion of the treatment period, a detailed necropsy was performed on all animals and selected organs were weighed. Various tissues and organs were placed in fixative, processed and examined microscopically.

The achieved chemical aerosol concentrations were between 100 and 112 % of the target. Temperature, relative humidity and oxygen concentration in the inhalation chamber were considered to be satisfactory for this study type.

The particle size distribution was determined for group 4 only as part of the atmosphere of group 2 and 3 was vaporous. The chemical mean mass median aerodynamic diameter (MMAD) was 2.50 μm and the mean geometric standard deviation (GSD) was 2.90. Therefore the aerosol of all groups was considered to be respirable for rats.

All animals survived the scheduled treatment period. Salivation was recorded in all animals at 5 mg/L air. Breathing noise was recorded in one male of this group during the last five days of treatment.

Reduced food consumption associated with a decreased body weight gain was recorded in animals exposed at 5.0 mg/L air.

Increase in urea, sodium and chloride values was observed in animals exposed at 5 mg/L air.

In males treated with the test item the following group mean values were statistically significantly modified: decreased urine volume (groups 3 and 4 only) as well as increased relative density and protein values (groups 2 and 4 only). These values were also modified in females at 5 mg/L air. Statistically significantly decreased pH was recorded in males at 5 mg/L air.

There were no ophthalmic changes and no changes in hematology that were considered to be related to treatment with the test item.

Increase in relative weights of the kidneys were recorded in males and females after exposure at 5 mg/L air. Increase in lung/body weight ratio was observed in males of this group.

There were no macroscopic findings that were considered to be related to treatment with the test item.

The following microscopic changes were recorded:

- Epithelial atrophy in the trachea of animals at 5 mg/L air.

- Squamous metaplasia of the respiratory epithelium, inflammatory exudates, necrosis of the epithelium, inflammation of the submucosa, respiratory metaplasia of the olfactory epithelium and ulceration in the nasal cavity of animals at 5 mg/L air.

- Degeneration / regeneration of the respiratory or olfactory epithelium in the nasal cavity (nasal section levels 1 to 4) of minimal to marked intensity in animals treated at 5 mg/L. Minimal effects on the olfactory epithelium (lower severity and lower incidence) were observed in the lower dose groups. However, the effects observed at 0.01 and 0.04 mg/L air are considered to be unspecific since similar variations were observed in some control animals of the preliminary Dose range finding study (2 -week repeated dose study) as well as in Harlan Laboratories Ltd historical control data.

For systemic effects the NOAEC was considered to be 5 mg/L air.

This 13 -week inhalation toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a subchronic (90 days) inhalation study OECD413 in rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
40 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Study performed according to the OECD 413 guideline (Klimish score = 1)

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

Repeated dose toxicity:

Two repeated dose toxicity studies of reliability 1 according to Klimisch cotation critera are available and were selected as key studies (Pössnecker A. et al., 2007 (OECD 422, oral route) and Pothmann D., 2010 (OECD 413, inhalation route)).

 

OECD 422 study (oral route):

 In this study, Dimethyl-2-methyl glutarate was administered once daily orally (by gavage) to male rats for 28 days and to female rats throughout the pre-pairing, the pairing, the gestation and the lactation periods until day 4 post partum at the dose levels of 100, 300 and 1000 mg/kg body weight/day. An additional group of control animals received the vehicle alone (purified water). In order to carry out an evaluation of the micronucleus induction, an additional group of 5 male rats serving as a positive control was given one oral (gavage) administration (20 mg/kg) of Cyclophosphamide monohydrate about 24 hours prior to necropsy.

In parent animals, with the exception of two female animals in the high dose group, all male and female animals survived until scheduled necropsy. One female rat died due to an unperceived injury during intubation. The other dam that died was noted with signs of a prolonged parturition. A sign of discomfort was noted in all animals at 1000 mg/kg/day and in some animals at 300 mg/kg/day in the way animals moved their head throught the bedding material after the daily administration, and this effect was considered to be non-adverse. Food consumption, body weight, and body weight gain were considered not to be influenced by treatment with the test item. None of the parameters of the Functional Observational Battery was considered to be affected by exposure to the test item. None of the parameters under investigation for hematology and clinical biochemistry was considered to be affected by exposure to the test item. In comparison to the corresponding vehicle controls there was no biologically relevant enhancement in the frequency of the detected micronuclei at any dose level after administration of the test item. No test item-related macroscopic or microscopic findings were noted at any dose levels. In the high dose males and females, the mean absolute and relative liver weights were slightly increased. In group 4 females, the mean absolute and relative kidney weights were statistically significantly increased.

Based on the death of one female noted at 1000 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) was considered to be 300 mg/kg/day.

 

OECD 413 study (inhalation route):

In this study, Dimethyl 2-Methylglutarate was administered to rats by nose-only inhalation for 6 hours a day for a period of 13 weeks at the dose levels of0.01,0.04 or 5.0 mg/L air. An additional group of control animals was exposed to air.The study comprised four groups containing ten male and ten female rats each. only. Throughout the study all animals were observed for viability and clinical signs. In addition body weights and food consumption were recorded. Ophthalmoscopic examinations were performed and blood and urine samples were taken for clinical laboratory investigations. Following completion of the treatment period, a detailed necropsy was performed on all animals and selected organs were weighed. Various tissues and organs were placed in fixative, processed and examined microscopically.

The achieved chemical aerosol concentrations were between 100 and 112 % of the target. Temperature, relative humidity and oxygen concentration in the inhalation chamber were considered to be satisfactory for this study type. The particle size distribution was determined for high dose group only as part of the atmosphere for group low and mid dose groups was vaporous. The chemical mean mass median aerodynamic diameter (MMAD) was2.50μm and the mean geometric standard deviation (GSD) was 2.90. Therefore the aerosol of all groups was considered to be respirable for rats. All animals survived the scheduled treatment period. Salivation was recorded in all animalsat 5mg/L air. Breathing noise was recorded in one male of this group during the last five days of treatment.

Reduced food consumption associated with a decreased body weight gain was recorded in animals exposed at 5.0 mg/L air. Increase in urea, sodium and chloride values was observed in animals exposedat 5mg/L air. In males treated with the test item the following group mean values were statistically significantly modified: decreased urine volume (mid and high dose groups only) as well as increased relative density and protein values (groups 2 and 4 only). These values were also modified in femalesat 5mg/L air. Statistically significantly decreased pH was recorded in malesat 5mg/L air. There were no ophthalmic changes and no changes in hematology that were considered to be related to treatment with the test item. Increase in relative weights of the kidneys were recorded in males and females after exposureat 5mg/L air. Increase in lung/body weight ratio was observed in males of this group. There were no macroscopic findings that were considered to be related to treatment with the test item. The following microscopic changes were recorded:

- Epithelial atrophy in the trachea of animalsat 5mg/L air.

- Squamous metaplasia of the respiratory epithelium, inflammatory exudates, necrosis of the epithelium, inflammation of the submucosa, respiratory metaplasia of the olfactory epithelium and ulceration in the nasal cavity of animalsat 5mg/L air.

- Degeneration / regeneration of the respiratory or olfactory epithelium in the nasal cavity (nasal section levels 1 to 4) of minimal to marked intensity in animals treatedat 5mg/L.

At lower concentrations, a few animals had also regeneration/degeneration of the olfactory epithelium, with an incidence and severity that were much lower compared to the highest dose group.Although such a change was not observed in controls of the present study, theeffects observed at 0.01and0.04mg/L air are considered to be a consequence of the experimental procedure and unrelated to the administration of the test substance since similar variations were observed in some control animals of the preliminary dose range finding study (2-week treatment period) performed in the same facility (Harlan Laboratories Ltd, study number C51304) and in Harlan Laboratories Ltd historical control data. Similar changes were also reported in some control rats exposed for 7 or 13 weeks to air only in a study with Dibasic esters (Keenan et al., 1990).

Based on these observations, the No Observed Adverse Effect Concentration (NOAEC) for local effects was considered to be0.04mg/L air.

For systemic effects the NOAEC was considered to be 5 mg/L air.

 

Of note, the olfactory epithelium is frequently the target tissue of a variety of compounds (Ref: Harkema et al. - The nose revisited: A brief review of the comparative structure, function, and toxicologic pathology of the nasal epithelium. Toxicol. Pathol. 34:252-269, 2006), including volatile solvants (Hardisty et al. - Histopathology of nasal olfactory mucosa from selected inhalation toxicity studies conducted with volatile chemicals. Toxicol. Pathol. 27:618-627, 1999), and there are multiple accounts of similar changes in the litterature. Other dibasic esters (DBE) are reported to induce degenerative/regenerative changes in the olfactory epithelium that were similar in nature (Lee et al.- Nasal Lesion Development and Reversibility in Rats Exposed to Aerosols of Dibasic Esters. Toxicol. Pathol. 20(3 part 1):376-393, 1992). The mechanism of olfactory toxicity by DBEs has been extensively studied and is believed to be related to an effect on supporting (sustentacular) cells, with subsequent necrosis of sensory cells (Harkema et al., 2006). These sustantacular cells are rich in carboxylesterase which mediates thein situformation of acid metabolites of DBEs, thought to be responsible for the lesion. DBEs are metabolized at a rate that is 100 to 1000 times lower in human nasal tissue compared with rat nasal tissue (Bogdanffy and Frame - Olfactory mucosal toxicity. Integration of morphological and biochemical data in mechanistic studies: Dibasic esters as an example. Inhal. Toxicol. 6 (suppl):205-219, 1994), suggesting a lower concern in the human situation. These effects are thought to be overpredictive in the rat model as compared to humans, because of the morphological and physiological differences.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
only one study available (GLP and OECD 422 guideline compliant)

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
only one study available (GLP and OECD 413 guideline compliant)

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
only one study available (GLP and OECD 413 guideline compliant)

Justification for classification or non-classification

Based on the classification criteria of Annex VI Directive 67/548/EECand on the classification criteria of EU Regulation 1272/2008 (CLP), no R48 or 'STOT – repeated exposure' classification, respectively, is warranted because 1) no clinical symptoms or histological changes indicative of serious respiratory tract damage/dysfunction were observed following repeated exposure to the test item in rats and 2) there was no systemic toxicity and the local effects in the nasal cavities were adverse only at a concentration well above the upper guidance value leading to classification (5 mg/L air vs. 0.2 mg/L air (CLP Regulation), respectively). Furthermore, the toxicological relevance of rat findings to humans is limited based on well-known interspecies differences in the anatomy and physiology of the nasal and olfactory epithelia between rats and humans which support a lower susceptibility of humans to olfactory changes.