[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

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

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

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.

2

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.