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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
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
Principles of method if other than guideline:
Remark: A mutagenicity analysis was performed within this OECD 422 study as a follow-up on the results obtained in a previous in vivo micronucleus assay performed with dimethyl 2-methyl glutarate (RCC-CCR study no. 1060302, see section 7.6.2 Genetic toxicity in vivo) where a significant increase in micronuclei was observed at the highest treatment dose (1500 mg/kg bw).
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 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.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of copulation.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually.
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)
Details on study schedule:
During the pairing period females were housed with males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females.

Females were removed and housed individually if:
a) a copulation plug was observed, and / or
b) the daily vaginal smear was sperm-positive.
This day was designated day 0 post coitum.

If a female was not mated during the 14-day pairing period, the female was paired with a male of the same group which already mated successfully. If mating was not recorded during this additional pairing period of maximum 14 days the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum.Day 0 of lactation was designated as the day on which a female had delivered all her pups.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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.
Parental animals: Observations and examinations:
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/animal/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).

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.

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.

OTHER:
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).

Oestrous cyclicity (parental animals):
During pre-pairing period, cages of males were interspersed among those holding females to promote the development of regular estrus cycles.
Sperm parameters (parental animals):
Parameters examined in parental males: testis and epididymis weights
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- The sex ratio of the pups was recorded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: litter size, stillbirths, live births and any gross anomalies, weight gain (Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum).

GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
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).
Postmortem examinations (offspring):
SACRIFICE
Pups were sacrificed on day 4 post partum.

GROSS NECROPSY
Pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occured.

HISTOPATHOLOGY / ORGAN WEIGTHS: no observation.
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
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.

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.


Reproduction data:
All mated females were pregnant.
The mating performance was considered to be not influenced by treatment.
The fertility index was 100% in all groups.
The pre-implantation loss, implantation rate, post-implantation loss and gestation length were not influenced by the treatment.


OTHERS:
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.

Micronucleus induction: see section 7.6.2 Genetic toxicity in vivo.
Key result
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.
Key result
Dose descriptor:
NOEL
Remarks:
(Reproduction/developmental)
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No observed effects
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
The number of living pups at first litter check and on day 4 Post Partum (p.p.) was not influenced by treatment with the test item.

No survival and behavioural abnormalities were noted during nesting and nursing until day 4 p.p..

Sex ratios were considered to be not influenced by treatment with the test item.

The mean body weight development of pups up to day 4 p.p. was considered to be not influenced by treatment.

At scheduled necropsy, no treatment-related findings were noted.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No observed effects
Key result
Reproductive effects observed:
no

Reproduction and breeding data: summary of performance:

Group

(mg/kg/day)

1

(0)

2

(100)

3

(300)

4

(1000)

Female numbers

41 - 50

51 - 60

61 - 70

71 - 80

Number of females paired

10

10

10

10

Number of females mated (A)

10

10*

10

10

Number of non-pregnant females

0

0

0

0

Spontaneous death of dams (B)

0

0

0

2

Number of females, which lost their litters

0

0

0

0

Number of females which reared their pups until day 4 post partum

10

10

10

8

 

(A) = In Female No. 60*, first mating was not detected However, pups data were considered and not found specifically different from other litters of the group.

 

(B) = Female No. 74 died on day 2 p.p., dam no. 78 died on day 22 p.c. (prolonged parturition)

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

The NOEL for reproduction/developmental toxicity was considered to be 1000 mg/kg/day, i.e. the highest dose tested without any observed effect.
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.

In pups, the number of living pups at first litter check and on day 4 Post Partum (p.p.) was not influenced by treatment with the test item. No survival and behavioural abnormalities were noted during nesting and nursing until day 4 p.p. The sex ratios was also not affected. The mean body weight development of pups up to day 4 p.p. was considered to be not influenced by treatment. At necropsy, no test item-related findings were noted.

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.

The NOEL for reproduction/developmental toxicity was considered to be 1000 mg/kg/day, i.e. the highest dose tested without any observed effect.

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

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and OECD 422 compliant study (Klimisch 1)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction:

On study of reliability 1 according to Klimisch cotation critera, is available ((Pössnecker A. et al., 2007) and was selected as a key study.

In this study (OECD 422), 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 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).

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

In pups, the number of living pups at first litter check and on day 4 Post Partum (p.p.) was not influenced by treatment with the test item. No survival and behavioural abnormalities were noted during nesting and nursing until day 4 p.p. The sex ratios was also not affected. The mean body weight development of pups up to day 4 p.p. was considered to be not influenced by treatment. At necropsy, no test item-related findings were noted.

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.

The NOEL for reproduction/developmental toxicity was considered to be 1000 mg/kg/day, i.e. the highest dose tested without any observed effect.


Short description of key information:
No effects on mating performance, fertility, gestation duration, litter size and pup viability in rats by oral gavage.

Justification for selection of Effect on fertility via oral route:
only one study available (GLP and OECD 422 guideline compliant)

Effects on developmental toxicity

Description of key information
A reduction in fetal body weight was noted in offspring from parent rats and rabbits exposed by oral gavage and associated, in rabbits only, with minor variations at the visceral and skeletal examinations. These effects were considered not to be adverse since they were noted at maternally toxic doses only.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
GLP and OECD 414 compliant study (Klimisch 1)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity:

Two prenatal developmental toxicity studies (OECD414), of reliability 1 according to Klimisch cotation critera, are available and were selected as key studies (Adamska M., 2010, rat and rabbit).

Rat study:

In order to detect effects on the pregnant rat and development of the embryo and fetus, Dimethyl 2-methylglutarate was administered orally by gavage once daily to pregnant females from day 6 through to day 20 post coitum at dose levels of 0 (vehicle alone i.e. water), 100, 300 and 1000 mg/kg bw/day.

 

No test item-related deaths occurred during the study at any dose level. The test item did not show any toxic potential on pregnant females up to and including the dose level of 300 mg/kg/day. At 1000 mg/kg bw/day, adverse effects of Dimethyl 2-Methylglutarate were observed. These were characterized by a reduction in mean food consumption and mean body weights noted from the beginning of the treatment, when compared to the respective control values. These effects were mostly due to a severe reduction of food consumption and reduced corrected body weight gain in three females. In two of these females reduction of food consumption was accompanied by body weight loss. One of these females pushed head through the bedding material and had ruffled fur on day 20 post coitum. During macroscopical examination pale and enlarged kidneys were found in all three females. Dimethyl 2-Methylglutarate had no influence at the relevant reproduction data (postimplantation loss and the mean number of fetuses per dam) up to and including the dose level of 1000 mg/kg bw/day. Treatment of pregnant females with Dimethyl 2-Methylglutarate at the dose level of 1000 mg/kg/day caused statistically significant reduction of body weights of fetuses. This effect was mostly due to reduction of fetal body weights in three liters from females which shown signs of toxicity. Therefore effect on fetal body weights at the high dose was considered to be a secondary effect to the test item-related maternal toxicity. External, visceral or skeletal examinations of fetuses gave no further indication of any test-item related effects on fetal development. Therefore reduction of body weights of fetuses was considered not to be adverse.

Based on these observations, NOEL (No Observed Effect Level) for pregnant rat was considered to be 300 mg/kg body weight/day.

NOEL (No Observed Effect Level) for embryo and fetal development was considered to be 300 mg/kg body weight/day whereas NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/day.

 

Rabbit study:

In order to detect effects on the pregnant rabbit and development of the embryo and fetus, Dimethyl 2-Methylglutarate was administered orally by gavage to pregnant females from day 6 (implantation) through to day 27 post coitum (the day prior to Caesarean section) once daily at dose levels of 0 (vehicle alone i.e. water) 30, 100 and 300 mg/kg/day. 

 

Treatment with the test item at the dose level of 300 mg/kg bw/day resulted in adverse effects (severely reduced food consumption and body weights, lateral recumbency, diarrhea) leading to pretermination of one female on day 26 post coitum. In remaining females at this dose level food consumption, body weights and body weight gain as well as corrected body weights were adversely reduced. During macroscopical examination, an increased incidence of changes in the stomach and digestive tract was observed which further indicated adverse action of the test item at the high dose level. There were no test item-related maternal adverse effects at the dose levels of 100 and 30 mg/kg/day. The relevant reproduction data (post-implantation loss and the mean number of fetuses per dam) were not affected by the treatment with the test item at any dose level. At the dose level of 300 mg/kg bw/day, a moderate but statistically significant reduction of fetal body weights was noted in parallel to the decreased body weight observed in dams and might be secondary to the maternal decrease in food consumption and body weight gain.

During the fresh visceral examination, an increased incidence of white internal opacity of fetal eyes was noted. This finding had no histopathological correlates. During the skeletal examination a higher number of supernumerary ribs and additional ossifications within limbs of fetuses were noted. Number of these findings was statistically significant if calculated on a fetus basis, not significant if calculated on a litter basis.

Overall, effects on fetal body weights, fetal eyes and skeletal development were of minor nature and indicated a non-specific disturbance of fetal development. Therefore these effects were considered to most probably be a secondary effect to the maternal toxicity and would not be expected to adversely affect further development in early postnatal life. No evidence for test item-related developmental effects was observed at the dose levels of 100 and 30 mg/kg bw/day.

 

Based on these data, NOEL (No Observed Effect Level) for pregnant rabbit was considered to be 100 mg/kg body weight/day, whereas NOAEL (No Observed Adverse Effect Level) for reproduction and fetal development was considered to be 300 mg/kg bw/day.


Justification for selection of Effect on developmental toxicity: via oral route:
The rabbit study was selected because this species appears to be more sensitive to the registered substance than the rat (NOAEL rabbit < NOAEL rat).

Justification for classification or non-classification

Based on the absence of any adverse effects on reproduction or development in rats or rabbits up to the dose of 300 mg/kg by oral gavage, taking account of the classification criteria of Annex VI Directive 67/548/EEC or EU Regulation 1272/2008 (CLP), no classification is warranted for reproductive and developmental toxicity.