Diethyl malonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20/06/2003 - 04/03/2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Justification for study design:

Three dose levels of 100, 300 and 1000 mg/kg Bwt/day were selected based on the results of a repeated dose (14-day) oral toxicity study by gavage (Study No.: 3665/03) in consultation with the sponsor. In brief, the results of repeated dose (14-day) oral toxicity study by gavage were as follows:
At 100, 300 and 1000 mg/kg Bwt/day doses, there were no treatment related changes in clinical signs, pre-terminal deaths, body weights, food consumption, terminal fasting body weights, organ weights and organ weight ratios and there were no treatment related gross pathological changes.

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl malonate

EC Number:

203-597-8

EC Name:

Dimethyl malonate

Cas Number:

108-59-8

Molecular formula:

C5H8O4

IUPAC Name:

dimethyl malonate

Details on test material:

purity 99.8 wt%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Animals: HSDCpb-WU rats
- Age at study initiation: 11-12 weeks
- Weight at study initiation:
males: 377-379 g, females: 210-219 g

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on mating procedure:

During mating, two animals were housed in a polypropylene rat cage with stainless steel mesh bottom i.e., 1 :1 (one male: one female).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentration of the test item in the solutions was analysed and documented in the study report.
The test item was found to be stable for 8 days, when fortified and stored at room temperature at both the level of fortification.

Duration of treatment / exposure:

Males:
Test groups: 39 days, male recovery group: 39 exposure days, 45 test days.
Females:
Treatment groups: 51 +/- 7 days, recovery group: 39 exposure days, 45 test days.

Frequency of treatment:

once daily, 7 days a week

Details on study schedule:

Animals were housed under standard laboratory conditions; the room was air conditioned with 12-15 filtered fresh air changes per hour. The maximum and minimum temperature in the experimental room was recorded once daily in the morning hours and the temperature ranged from 20 - 23°C. The humidity in the experimental room was calculated from dry and wet bulb temperature recordings and the humidity ranged between 30-70%. The experimental room had 12 hour fluorescent light (6 a.m. to 6 p.m.) and 12 hour dark (6 p.m. to 6 a.m.) cycle.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Main group: 10 males, 10 females
Recovery groups: 5 males, 5 females

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure recovery period in satellite groups: 14 days
Treatment:
Male rats: The test item was administered once daily, 7 days per week by gavage for 2 weeks prior to mating, during the mating period and approximately 2 weeks post mating.
Female rats: The test item was administered once daily, 7 days per week by gavage for 2 weeks prior to mating, during the mating period, pregnancy and up to lactation day 4. For the high dose recovery group animals (males and females) the test item was administered once daily, 7 days per week by gavage throughout the treatment period.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Twice daily: morbidity and mortality. Daily observations for appearance, behaviour, clinical signs and preterminal deaths. Females were observed for signs of difficult and pronlonged parturition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before exposure and at least once per week thereafter. Signs included were changes in skin, fur, eyes, mucous membranes, occurrence of secretions or excretions and autonomic activity, changes in gait, posture, response to handling, behavioural changes, difficult or prolonged parturition.

BODY WEIGHT: Yes
- Time schedule for examinations:
Body weights were recorded at the beginning of the study, at least weekly thereafter and at termination. All dams were weighed on gestation days 0, 7, 14 and 20 and lactation days 0 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period and the recovery period
- Anaesthetic used for blood collection:No data
- Animals fasted: No data
- How many animals: 5 randomly selected males and females or each group.
- Standard haematological parameters checked

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period and the recovery period
- Anaesthetic used for blood collection:No data
- Animals fasted: No data
- How many animals: 5 randomly selected males and females or each group.
- Standard clinical chemistry parameters checked

URINALYSIS: No data


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the dosing period for males and during the lactation period tor females
- Dose groups that were examined: each group
- Battery of functions tested:
handling observations, open field tests, sensory observations, neuromuscular observations and physiological observations (body temperature)

Litter observations:

All pups from each litter were examined for any external deformities, litter size and sex distribution was determined. Pup weights were recorded on day 0 and 4. All pups were examined for malformations and subject to gross pathological examination. Pup survival index up to lactation day 4 was determined.

Postmortem examinations (parental animals):

Organ weights of liver, adrenals, kidneys, thymus, spleen, brain and heart were determined of 5 males and females of each group. Testes and epididymis weights of all adult males of each group were also determined.
Pathology: All adult animals and pups were examined for any structural abnormalities and pathological changes.
Histopathology: The following tissues of 5 males and females of the control and high dose group as well as all animals of the recovery and recovery control groups were examined microscopically: all gross lesions, brain, spinal cord, gastrointestinal tract, liver, kidney, adrenals, spleen, heart, thymus, thyroid, trachea, lungs, testes (fixed in Bouins fluid), epididymes (fixed in Bouins fluid), ovaries, uterus, seminal vesioles, coagulating glands, prostate, urinary bladder, axillary lymph nodes, mesenteric lymph nodes, sciatic nerve, femur with marrow, bone marrow smear. Stages of spermatogenesis and interstitial testicular structure in male gonads were determined additionally. Livers of 5 males and females in the mid and low dose groups and testes of 5 males of the mid and low dose groups were also examined.

Postmortem examinations (offspring):

Pathology: All adult animals and pups were examined for any structural abnormalities and pathological changes.

Reproductive indices:

Fertility index
Gestation index
Number of implantations
Number of corpora lutea
Percentage pre- and post implantation loss

Offspring viability indices:

Number of pubs born
Number of live litters
Mean litter size index
Mean viable litter size
No. of pubs alive on day 0
Live birth index
Sex ratio at birth (no of males/total number born x 100)
No of pups dead or cannibalised up to day 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/ Bwt/day, treatment resulted in lower food intake du ring gestation period but had no effects on general health, neurological findings, body weights, haematological and biochemical parameters, reproductive performance of dams and sires and gross pathology.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: Livers of males and females showed a significantly increased incidence of hepatocellular hypertrophy. The change was considered reversible as the incidence was not significantly increased in the high dose recovery animals.
300 and 100 mg/kg bw: No treatment related changes of the liver were observed.
All other histopathological findings were not considered treatment related.

Histopathological findings: neoplastic:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no treatment rt3lated changes in the reproductive organs.

Effect levels (P0)

1

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reversible hepatocellular hypertrophy.

2

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects have been observed.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Observations and necropsy findings on pups: No treatment related effects were observed.
Fertility indices for males and females were not statistically different from controls in all dose groups. In the low dose group post implantation loss and consequently the percentage of live pubs born was significantly reduced compared to controls (P <= 0.05). These changes were considered incidental and not treatment related as the effects were not observed at the higher dose groups. No statistical significant differences from controls were observed for the number of pregnancies, number littered, number of live litters, mean litter size, mean viable litter size, sex ratio at birth, number of pups dead at first observation or day 2 to 4, number of live pups on day 0, 3 and 4 and the associated survival indices, external abnormalities of life and dead pups at all dose levels.
A significantly higher percentage of male rats in the low dose group on day 4 was considered incidental and not treatment related as a similar change was not found in the higher dose groups.
The mean number and the mean weight of male and female (and both sexes combined) pups during different intervals of the lactation period were not statistically significantly different from controls except from a significantly lower (P <= 0.05) mean number of female pups on lactation day 4 in the low dose group which was considered incidental and not related to treatment. (For details see table 1 "Any other information on results incl. tables.)

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects have been observed.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Reproductive results and indices of various dose groups:

	Dose (mg/kg bw)
	0	100	300	1000
Fertility index (%)	100	100	100	100
Duration of gestation
Days ± SD	22±0.3	23±0.5	23±0.5	22±0.4
Gestation index (%)	100	100	100	100
Parturition (%)	100	100	100	100
Effects on sperm	No treatment related effects	No treatment related effects	No treatment related effects	No treatment related effects
Number of implantations
Number	12.3	11.8	12.0	11.6
%	88.1	84.7	88.9	88.6
Number of corpora lutea
Number	14.0	13.9	13.5	13.1
Percentage preimplantation loss	11.9	15.3	11.1	11.4
Percentage postimplantation loss	8.1	19.1	10.4	15.1
Number of pubs born	103	76	86	84
Number of live litters	9	8	8	8
Mean litter size index	11.4	9.5	10.8	10.5
Mean viable litter size	11.3	9.5	10.8	9.9
Number of pubs alive on day 0	102	76	86	79
Live birth index	99	100	100	94
Sex ratio at birth (Number of males/total number born x 100)	46.6	60.5	54.7	46.4
24 hour survival (%)	100	100	100	100
Number of pubs alive on day 4 of lactation	101	75	83	78
Day 4 survival index	99.0	98.7	96.5	98.7
Sex ratio day 4	44.7	60.5	53.5	41.7
Number of pups dead or cannibalised up to day 4	2	1	3	6

 

Applicant's summary and conclusion

Conclusions:

Based on the study results a LOAEL value of 1000 mg/kg bw for both male and female can be stated for dimethyl malonate.
Based on the study results a NOAEL value of 300 mg/kg bw for both male and female can be stated for dimethyl malonate.