[No public or meaningful name is available]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 20 May 2009 to 17 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to international test guidelines and to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Harlan Laboratories Ltd.,Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Age at study initiation (day 0 post coitum): 11 weeks
- Weight at study initiation (day 0 post coitum): 156 to 219 g
- Fasting period before study: no
- Housing:Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland).
- Diet:Pelleted standard Kliba Nafag 3433 rodent maintenance diet (batch no. 15/09, Provimi Kliba SA, 4303 Kaiseraugst / Switzerland), ad libitum.
- Water: Community tap-water from Füllinsdorf in water bottles, ad libitum.
- Acclimation period: minimum 7 days

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

IN-LIFE DATES: From 08 June 2009 to 26 June 2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Dimethyl 2-Methylglutarate was weighed into a glass beaker on a tared precision balance and 100% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Dose formulations were stored refrigerated at 2 - 8 °C in glass beakers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical phase was conducted at Harlan Laboratories 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 flame ionization detector (FID) and quantified with the area under the peak.

With the exception for one sample, the application formulations investigated during the study were found to comprise Dimethyl 2-Methylglutarate in the range of 97.9% to 103.6%. Eleven out of 12 samples met the required content limit of ±20% with reference to the nominal concentration. The homogeneous distribution of Dimethyl 2-Methylglutarate in the preparations was approved because single results found did not deviate more than 3.4% (<15%) from the
corresponding mean.

One of the samples taken during the last week of the treatment, the dose formulation of 100 mg/ml, contained the test item at a calculated content of 39.3% of the nominal concentration. Thorough examination of raw data concerning the preparation of this sample as well as of the analytical raw data did not reveal any irregularity in dose preparation and analysis. Therefore the dose formulation for group 4 from the last week of treatment was considered to be prepared and used in proper way although analytical results showed unsatisfactory content.

In addition, the test item was found to be stable in application formulations when kept 7 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item Dimethyl 2-Methylglutarate and milli-Q-water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Details on mating procedure:

After acclimatization, females were housed with sexually mature males (1:1) 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. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day of mating was designated day 0 post coitum.

Male rats of the same source and strain were used only for mating. These male rats are in the possession of Harlan Laboratories and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored.

Duration of treatment / exposure:

from Day 6 to 20 post coitum

Frequency of treatment:

once daily

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:

22 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
The dose levels were selected based on a previous Combined Repeated Dose Toxicity Study with Reproduction / Developmental Toxicity Screening Test in the Wistar Rat (OECD 422), RCC Study Number B05670, using dose levels of 0, 100, 300 and 1000 mg/kg/day, resulting in a NOAEL of
300 mg/kg/day (due to one unexplained death at 1000 mg/kg/day).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for viability/Mortality and once daily (during acclimatization and up to day of necropsy) for clinical signs.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily from day 0 until day 21 post coitum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day
- Time schedule for examinations: food consumption was recorded at 3-day intervals: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [half per litter]

Statistics:

The following statistical methods were used to analyze food consumption, body weights, reproduction and skeletal examination data:
• Means and standard deviations of various data were calculated.
• 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 was applied if the variables could be dichotomized without loss of information.

Indices:

Terminology Used in the Assessment of the Data:
- Empty Implantation Site: Very early resorption or aborted implantation
- Embryonic Resorption: Amorphous mass being resorbed
- Fetal Resorption: Clearly defined fetal body being resorbed
- Dead Fetus: Appearance of live fetus but without induced respiration or movement
- Live Fetus: Breathing and/or moving fetus
- Abnormality: A structural change in a fetus that would probably impair its health or development.
- Variation: A fetal change that is unlikely to adversely affect survival or health. This includes a delay in growth or morphogenesis that has otherwise followed a normal pattern of development.

Historical control data:

Historical control data were provided to allow comparison with concurrent controls.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No test-item related death occured during the study.

Clinical observation:
At the dose level of 1000 mg/kg bw/day, pushing head through the bedding material and ruffled fur were observed in one female on day 20 post coitum. These symptoms were considered to be test item-related and indicate a bad condition of the female. No further clinical signs which were considered to be test item-related were observed at this dose level.

Food Consumption and Body Weights:
At the dose level of 1000 mg/kg bw/day, mean food consumption and mean body weights were slightly lower when compared to the respective control values from the beginning of the treatment. Reduction of food consumption and body weights was statistically significant toward the end of the gestation period. These effects were mostly due to a severe reduction of food consumption in three females. In two of these females reduction of food consumption was accompanied by body weight loss. Effects on food consumption and body weights were considered to be test item-related and adverse effects.

Macroscopical Findings:
At the dose level of 1000 mg/kg bw/day, pale and enlarged kidneys were found in 3 females. This finding was considered to be test item-related adverse effect.

No treatment-related effects on clinical condition, food consumtion or body weight were noted at 100 and 300 mg/kg/day and on reproduction parameters (post-implantation loss and the mean number of fetuses per dam) at any dose levels.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: other:

3

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The only treatment-related effect was a statistically significant reduction of fetal body weights noted at 1000 mg/kg/day. This was considered to be secondary to the maternal toxicity and not an adverse effect.

No test item-related abnormal findings were noted at the external, visceral, skeletal and cartilage examinations and no differences in fetal sex ratios were noted, at any dose level.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Summary of Performance of Mated Females:

Group Dose (mg/kg)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	1 - 22	23 - 44	45 - 66	67 - 88
Number of females mated	22	22	22	22
Number of females not pregnant (A)	1	0	0	0
Number of females death prior schedule (B)	0	0	0	1
Number of females with live fetuses at scheduled termination*	21	22	22	21

* Only dams with at least one live fetus at Caesarean section were used for the calculations of food consumption,

body weight gain and corrected body weight gain data.

(A) Female no. 11 was not pregnant

(B) Female no. 76 was found dead on day 20 post coitum. Because during necropsy fluid was found in its thorax, the death of female no. 76 was considered to be a result of an application error and not a test item-related effect.

 

Applicant's summary and conclusion

Conclusions:

Under the conditions described for this study, Dimethyl 2-Methylglutarate did not reveal teratogenic potential up to and including the dose level of 1000 mg/kg/day in rats.

Executive summary:

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.

This study is classified as acceptable and satisfies the guideline requirement for a prenatal developmental toxicity study OECD 414 in rats.