2-[(2-methyl-1-oxoallyl)oxy]ethyl acetoacetate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 April - 16 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted in accordance with international guidelines

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

other: Colourless to yellowish clear liquid

Details on test material:

- Name of test material (as cited in study report): LZ649
- Physical state: Colourless to yellowish clear liquid
- Expiration date of the lot/batch: 23 November 2014
- Storage condition of test material: Room temperature in the dark, desiccated

Specific details on test material used for the study:

Retest Date: 23 November 2014

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD) strain was used

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Reputable supplier
- Age at study initiation: (P) Males 71 days; Females 65
- Weight at study initiation: (P) Males: 342-407 g; Females: 219-259
- Fasting period before study: No
- Housing: Polycarbonate cages, the gridded cages used during pairing were suspended over trays covered with absorbent paper which was
changed daily. For cages with solid floors, wood based material was used as bedding and was sterilised by autoclaving and changed at least twice
each week
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): SDS VRF1 certified diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40 to 70
- Air changes (per hr): Not given
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 18 April 2012 To: 8 June 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The LZ649 was prepared for administration as a series of graded concentrations in the vehicle. Approximately 50% of the final volume of vehicle was added to the test substance and mixed by magnetic stirring until dissolution was achieved. The volume was made up with the remaining vehicle and magnetically stirred until homogenous.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is a commonly used vehicle, test substance dissolves in this vehicle
- Concentration in vehicle: 0, 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Lot/batch no. (if required): NA
- Purity: NA

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A method of analysis of the test substance and formulation preparation, homogeneity and stability, was validated in this study.
The first and last preparations for dosing were analysed using the validated method to confirm the dose concentrations.

Duration of treatment / exposure:

F0 genearation only were dosed, both males and females from the start of the study until day 7 of lactation. F0 females were not dosed if parturition was in progress at the scheduled time of administration.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

In a previous 13 week study dose levels of 50, 150 and 500 mg/kg/day were investigated using a five day/week dosing regime and 500 mg/kg/day was found to be the No Observed Adverse Effect Level (NOAEL). Therefore dose levels were selected for this reproduction screening study taking into consideration the requirement to dose reproducing animals on a seven day/week regime, doses of no higher than those administered in the 13 week study were selected.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, also for F0 females on Days 0, 3, 7, 10, 14, 17 and 20 after mating and days 1, 4 and 7 of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/rat/day: Yes

Oestrous cyclicity (parental animals):

For 15 days before pairing, vaginal smears taken, eaxamined to establish duration and regularity of the oestrous cycle

Sperm parameters (parental animals):

Parameters examined in P male parental generation:

The following organs, taken from each male, were dissected free of adjacent fat and other contiguous tissue and the weights recorded:
Epididymides (L&R)
Testes (L&R)

L&R Bilateral organs weighed individually
Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities, possible cause of death was determined in some casesfor pups born or found dead
Missing offspring and those grossly autolysed or grossly cannibalised could not be examined.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Surviving F0 males were killed after the first Day 7 of lactation of the females (after confirmation that a second mating was not required).
- Maternal animals: F0 females were killed on Day 7 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination:
Epididymides - caput, corpus and cauda
Ovaries - qualitative evaluation of one section from each ovary

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 7 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

A careful external examination was performed for gross abnormalities and externally normal offspring were discarded without further internal examination.
Externally abnormal offspring were internally examined and any abnormal tissues were retained in an appropriate fixative.

Statistics:

All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no post dose signs. Clinical signs observed were of a type commonly observed in this strain of rat, at this age, in this laboratory and did not indicate any adverse reaction to the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One Group 3 male died following dosing on Day 30 of study. Necropsy did not give any indication of the cause of death and no evidence of dosing trauma was observed. Abnormalities at necropsy were limited to clear fluid present in the stomach. The cause of death was therefore undetermined following pathology examination. In the absence of any signs or deaths in the high dose group it was not attributed to the test material.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Following the first week of study lower bodyweight gain in females receiving LZ649 at 500 mg/kg/day was apparent, individual responses were variable and some females at each dose level lost weight. During the second week of study the females receiving 500 mg/kg/day had similar weight gain to Control animals. There was no effect of LZ649 on bodyweight in males at any dose level.
Bodyweight on Day 0 of gestation was slightly lower in animals receiving 500 mg/kg/day but following superior bodyweight gains by Day 20 of gestation no differences were apparent.
Bodyweight gain was decreased during lactation in females receiving 150 mg/kg/day compared with Controls, there was no similar effect in the high dose group. As the 150 mg/kg/day animals started with the bodyweight values greater than controls, and ended with values similar to Controls no test article relationship was attached.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect of LZ649 on food consumption prior to pairing, during gestation or lactation.
It was apparent that food consumption was slightly low in females receiving 500 mg/kg/day after the first week of study, an effect of this degree was not considered to be adverse. The food consumption during week two of dosing was similar to Controls in both males and females at all dose levels. Food consumption during the period Days 0-2 of gestation and throughout lactation was also slightly lower than control for females receiving 500 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological evaluation was performed on tissues with gross abnormalities, ovaries, testes and epididymides.
There were no microscopic findings attributable to administration with LZ649.
One control male showed slight degeneration/atrophy of the epithelium in a few seminiferous tubules, but this is not an uncommon finding in rats of this age and strain at these laboratories.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of LZ649 on oestrous cycle length. All animals mated at the first oestrus opportunity following pairing.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Percentage mating, conception rate and fertility index was 100% in each dose group up to 500 mg/kg/day. All animals gave birth to a live litter.
Gestation length was within the expected timeframe observed at this laboratory.

Details on results (P0)

SIGNS and MORTALITY:
One Group 3 male (number 29) died following dosing on Day 30 of study. Necropsy did not give any indication of the cause of death and no evidence of dosing trauma was observed. Abnormalities at necropsy were limited to clear fluid present in the stomach. The cause of death was therefore undetermined following pathology examination. In the absence of any signs or deaths in the high dose group it was not attributed to the test material.
There were no post dose signs. Clinical signs observed were of a type commonly observed in this strain of rat, at this age, in this laboratory and did not indicate any adverse reaction to LZ649.
BODYWEIGHT:
Bodyweight on Day 0 of gestation was slightly lower in animals receiving 500 mg/kg/day but following superior bodyweight gains by Day 20 of gestation no differences were apparent.
FOOD CONSUMPTION:
There was no adverse effect of LZ649 on food consumption prior to pairing, during gestation or lactation.
OESTROUS CYCLE LENGTH:
There was no effect of LZ649 on oestrous cycle length.
PRE-COITAL INTERVAL:
All animals mated at the first oestrus opportunity following pairing.
MATING PERFORMANCE and FERTILITY:
Percentage mating, conception rate and fertility index was 100% in each dose group up to 500 mg/kg/day.
GESTATION LENGTH and GESTATION INDEX:
All animals gave birth to a live litter. Gestation length was within the expected timeframe observed at this laboratory.
MACROPATHOLOGY:
There were no changes detected at macroscopic examination in any of the males or females receiving LZ649 that were related to exposure.
ORGAN WEIGHTS:
There were no effects of LZ649 on epididymides or testes weights.
HISTOPATHOLOGY:
There were no microscopic findings attributable to administration with LZ649.
One control male showed slight degeneration/atrophy of the epithelium in a few seminiferous tubules, but this is not an uncommon finding in rats of this age and strain at these laboratories.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There was no effect of test article on the number of implantations and total litter size on Day 1, and live litter sizes on Days 1, 4 and 7 were similar to Control values.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The post implantation survival and offspring survival up to Day 7 was not affected by LZ649.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Offspring bodyweight on Day 1 of age and subsequent bodyweight gain up to Day 7 of age was unaffected by parental administration with LZ649 at levels up to 150 mg/kg/day. Bodyweight gain for the period Days 1-7 of lactation of male and female offspring derived from females receiving 500 mg/kg/day was lower than Control though this was not considered adverse at the degree observed.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring dying before scheduled termination or killed at scheduled termination on Day 7 of age did not reveal any findings that were attributed to parental test material exposure.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

LITTER SIZE:
There was no effect of test article on the number of implantations and total litter size on Day 1, and live litter sizes on Days 1, 4 and 7 were similar to Control values.
OFFSPRING SURVIVAL INDICES:
The post implantation survival and offspring survival up to Day 7 was not affected by LZ649.
SEX RATIO:
Sex ratio was not affected by parental test material exposure.
BODYWEIGHT:
Offspring bodyweight on Day 1 of age and subsequent bodyweight gain up to Day 7 of age was unaffected by parental administration with LZ649 at levels up to 150 mg/kg/day. Bodyweight gain for the period Days 1-7 of lactation of male and female offspring derived from females receiving 500 mg/kg/day was lower than Control though this was not considered adverse at the degree observed.
OFFSPRING MACROPATHOLOGY:
Macroscopic examination of offspring dying before scheduled termination or killed at scheduled termination on Day 7 of age did not reveal any findings that were attributed to parental test material exposure.

Effect levels (F1)

1

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The results observed in this screening study conclude that LZ649 has no effect on reproductive performance and offspring survival or development at doses up to 500 mg/kg/day.

Executive summary:

The toxic potential of LZ649 on basic reproductive processes was assessed in this OECD 421 screening study in which Crl:CD(SD) rats were dosed for two weeks prior to pairing, throughout gestation and lactation at dose levels of 50, 150 or 500 mg/kg/day. The administration of LZ649 was well tolerated up to and including the maximum level of 500 mg/kg/day assessed within this study. There was no adverse effect of administration with LZ649 on clinical condition, bodyweight, and food consumption, macroscopic and microscopic appearance. Reproductive performance assessments of oestrous cycles, gestation length and parturition for F0 females also showed no test article related change. Organ weights for F0 males were similar throughout the groups. The clinical condition, litter size and survival and sex ratio of offspring, exposed in utero or via the milk, were not affected by doses up to and including 500 mg/kg/day. The results observed in this screening study conclude that LZ649 has no effect on reproductive performance and offspring survival or development at doses up to and including 500 mg/kg/day.