Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
July 11, 2007 - April 21, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2008
Reference Type:
publication
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: (P) 6 wks, (F1) 3 wks.
- Weight at study initiation: (P) Males: 165 to 245 g; Females: 125 to 197 g
- Fasting period before study: Yes
- Housing: Animals were housed in groups of 4 per sex in solid polycarbonate cages with Lignocel 3/4 wood flakes for breeding. During mating 1, male and 1 female from the same treatment group were housed together. During gestation and lactation, females were housed individually or with their litters.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): Artificial lighting 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required volume of vehicle was measured and added to the test substance and mixed using a whirlmixer for a minimum of one minute and then stirred magnetically whilst sampling (five minutes). Moisture was avoided at all times during formulation. The test substance was used as supplied. All formulations were prepared freshly each week and stored refrigerated until the day of use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): In a previous study corn oil formulations were shown to be homogenous in the vehicle and stable at ambient temperature for two days and for 15 days following refrigerated storage.
- Concentration in vehicle: 0 (control), 5.0, 15.0, 50.0 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: two weeks
- Proof of pregnancy: Ejected copulation plugs. Vaginal smears were prepared and examined for the presence of spermatozoa and the stage of the oestruous cycle. Evidence of mating was referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): Individually during gestation and with litters during post partum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration for Weeks 1, 5, 11 and 14 of treatment of the F0 animals and Week 1 of treatment of the selected F1 animals were analysed for achieved concentration of the test substance. The analytical procedure was validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision. The homogeneity and stability was confirmed for MIBKO in corn oil formulations at a nominal concentration of 50 mg/mL. The mean concentrations of MIBKO in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
Parental rats were dosed 10 weeks prior to mating, for up to 2 weeks during mating, or until signs of mating were noted, and then during both gestation and lactation. Selected F1 animals were treated from weaning (Day 21 of age) to approximately 7 weeks of age.
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: 16 weeks (6 weeks at study initiation + 10 weeks dosing period).
- Offspring were weaned on Day 21 of age and separated from the dam. The allocation of offspring to form the F1 generation was made before weaning on Day 21 of age.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0 gneration: 28 animals per sex and per dose.
F1 generation: 24 animals per sex and per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study with reference to preliminary work with this compound performed by Huntingdon (NBJ0037/070047). MIBKO at dose levels of 10, 30 and 100 mg/kg/day had no adverse effects on reproductive performance or F1 pre- and postnatal development, following treatment of F0 males and F0 females for 28 days before pairing, and during gestation and post partum.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least twice daily
- Observations: evidence of ill-health or reaction to treatment

CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Weeks 1 to 2, twice weekly during Weeks 3 to 5 (middle and end of each week) and once a week from Week 6 onwards for F0 females on Days 0, 6, 13 and 20 after mating and Days 1, 7, 14 and 21 post partum. A more detailed physical examination was performed on each adult F0 animal weekly, F0 females on Days 0, 6, 13 and 20 after mating and Days 1, 7, 14 and 21 post partum.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly until termination. The weight of each F0 female was recorded weekly until mating was detected. After mating
bodyweights were recorded on Days 0, 3, 6, 10, 14, 17 and 20 and on Days 1, 4, 7, 11, 14, 18 and 21 post partum.

FOOD CONSUMPTION:
- Time schedule: For each F0 male the weight of food supplied, that remaining and an estimate of any spilled was recorded weekly until paired for mating. For each F0 female, the weight of food supplied, that remaining and an estimate of any spilled was recorded weekly until paired for mating, Days 0-2, 3-5, 6-9, 10-13, 14-16 and 17-19 after mating and 1-3, 4-6, 7-10, 11-13, 14-17 and 18-20 post partum.
Oestrous cyclicity (parental animals):
Three weeks prior to mating, vaginal smears were prepared to observe the estrous cycle of the F0 and F1 females. Daily vaginal smears taken from females before necropsy (Days 25 to 28 post partum) were used to determine the stage of the oestrous cycle at termination. For females whose litters had previously died, the smears were taken on the theoretical Days 25-28.
Sperm parameters (parental animals):
Sperm analysis was performed in all male parental generations. After termination, the left vas deferens, epididymis and testis was removed and the epididymis and testis were weighed. The following tests were performed: Sperm motilitly, sperm morphology, sperm count and homogenisation-resistan spermatids count.
Litter observations:
STANDARDISATION OF LITTERS
- On postnatal day 4, litters were culled to 8 or 10 pups, with equal numbers of males and females were possible, by random selection. Pups that were selected continued in the study until postnatal day 55.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
- Following delivery the number of pups were determined and the ratio of male to female pups was calculated.
- Litters were examined daily for dead pups.
- Pups were weight on postanal days 1, 4, 7, 11, 14, 18, 21, 25, and 28.
- Total litter size, number of male and female pups, number of stillborn and live births and grossly malformed pups, and pups showing abnormalities were recorded on postanatal day 1.
- The number of live and dead pups, as well as pups showing malformations or abnormalities, was recorded on postnatal days 4, 7, 14 and 21.
- Sexual maturation: Males were examined daily from day 38 until blanopreputial separation occurred and selected females were examined daily from day 28 until the vaginal opening occurred. Body weights were recorded at these events.

GROSS EXAMINATION OF DEAD PUPS:
- Yes, for external and internal abnormalities.



STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Parturition observation and gestation lengh: umbers of live and dead offspring, duration of gestation.
- Clinical observations: All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter. Selected F1 animals were assessed weekly from Day 21 of age for detailed physical examinations.
- Bodyweight: Individual offspring bodyweights were recorded on Days 1, 4 (before culling), 7, 11, 14, 18 and 21 of age. The weight of each selected F1 animal was recorded on Days 21, 25 and 28 of age.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
- Sexual maturation: For all selected males, sexual maturation was assessed by daily examination from Day 38 of age until balano-preputial separation occurred. For all selected females, sexual maturation was assessed by daily examination from Day 28 of age until vaginal opening occurred.

[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:]

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities (see below); possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were killed when majority of litters had been weaned (approx. 16 weeks).
- Maternal animals: Females surviving until the end of the scheduled study period were killed on Day 28 post partum, which failed to produce viable litter on Day 25 and whose litter died before Day 21 post partium on the day the last offspring died.

GROSS NECROPSY
- Gross necropsy consisted of:full macroscopic examination of the tissues, all external features and orífices; the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera, abnormal position, morphology or interaction; external and cut surfaces of the
organs and tissues; abnormalities in the appearance or size of any organ and tissue. For females, the numbers of implantation sites in each uterine horn was counted.

ORGAN WEIGHTS:
- Adrenals, brain, epididymides, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroids, uterus with cervix and oviducts.

HISTOPATHOLOGY
- All parental animals from control and 100 mg/kg bw/day.
- Pathology: adrenals, brain, epididymis, kidneys, liver, mammary area-caudal, ovaries, piruitary, prostate, seminal vesicles, spleen, testis, thyroids, uterus with cervix and oviductus, vagina, any abnormal tissue plus the lymph nodes draining the regions adjacent to masses. Reproductive organs were also analysed in animals at 10 and 30 mg/kg bw/day that showed reduced fertility. Spleen (both sexes) and kidneys (males only) were examined for all F0 adults since were considered to exhibit a reaction to treatment at the high dose.
- Histology: Epididymis, kidneys, liver, ovaries, seminal vesicles, uterus, vagina.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as F1 generation were sacrificed approximately at 21 days of age.
- Selected F1 animals were killed at approximately 7 weeks of age.
- Selected F1 offspring + non selected ones with external abnormalities + premature deaths, were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of: full macroscopic examination of the tissues, all external features and orífices; the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera, abnormal position, morphology or interaction; external and cut surfaces of the
organs and tissues; abnormalities in the appearance or size of any organ and tissue. For females, the numbers of implantation sites in each uterine horn was counted.

ORGAN WEIGHTS:
- Unselected F1 offsprings: Brain, spleen and thymus
- Selected F1 offsprings: Spleen

HISTOPATHOLOGY / ORGAN WEIGTHS
- Unselected F1 offsprings: Brain, spleen and thymus.
- Setected F1 offsprings: Spleen.
Statistics:
Statistical analyses were performed by using one or multiple methods, as appropriate. For categorical data, Fisher’s exact probability test was used. For continuous data, one-way analysis of variance (ANOVA), followed by either Dunnett’s multiple comparison or Barlett’s test, was used. Kruskal-Wallis nonparametric ANOVA, followed by the Mann-Whitney U-test, was used for nonparametric data. In addition, postimplantation loss and sex ratio were analyzed by the mixed linear model Wald chi-square test.
Reproductive indices:
Percentage mating, conception rate, fertility index
Offspring viability indices:
Litter size, survival indices (post-implantation survival index, live birth index, viability index, lactation index), sex ratio (percentage males).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
(salivation and chin rubbing, at 100 mg/kg bw/day, both sexes)
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(at 100 mg/kg bw/day, in both sexes)
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male at 30 mg/kg bw/day was found dead on day 32, and another male at 100 mg/kg bw/day was found dead on day 112. One control female was found dead on day 35. The cause of dead was not determined. Salivation and chin rubbing were the only clinical signs attributed to administration of the test article. These effects were observed in 100 mg/kg bw/day group males and females and diminished following 4 weeks of treatment. No animals were found in moribund conditions. One female at 30 mg/kg/day had a total litter loss with no pups born alive. No remarkable clinical signs were observed. One female was sacrificed for welfare reasons on day 1 of lactation. Clinical signs included piloerection, hunched posture, skin pallor, and hair loss. Neither of these deaths was considered to be treatment related. One female in the 100-mg/kg group was killed on day 1 of lactation due to poor clinical condition.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight, body-weight change, and food consumption were similar to controls at all time points.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No effects on estrus cycle were observed.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects on sperm counts or morphology were observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects on precoital time, the number of pregnant females, fecundity, or duration of gestation were seen.
One female each from the control, 10 mg/kg/day, and 100 mg/kg/day groups were not pregnant.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Both the absolute and bodyweight-relative kidney, liver and spleen weights were statistically significantly high amongst males and females at 100 mg/kg/day.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Dark spleens were seen for the majority of males and females which had received at 100 mg/kg/day. Enlarged spleens were seen for the majority of males and three females at 100 mg/kg/day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In the spleens, minimal to moderate hemosiderosis was observed in higher incidence and severity in the 100 mg/kg/day males and females. Minimal congestion was seen in the majority of animals of both sexes treated at 100 mg/kg/day. In the kidneys of males, minimal to moderate cortical tubules with hyaline droplets were seen at a significantly higher incidence in animals treated at 30 mg/kg/day or 100 mg/kg/day. All other findings were considered to be incidental and unrelated to the test substance.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
(parental toxicity)
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
(reproductive toxicity)
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: (Based on no effects at the highest dose tested)

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No remarkable clinical signs were observed in Litter. A higher incidence of chin rubbing, salivation and post salivation staining was seen in both the males and females at 100 mg/kg/day in F1 generation.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight at birth and subsequent weight gain to Day 21 were considered unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
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:
no effects observed
Description (incidence and severity):
There was no effect of treatment with MIBKO on the time of completion of vaginal opening or preputial separation at any dose.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Absolute and bodyweight relative spleen weights were statistically significantly high amongst the offspring of females at 100 mg/kg/day. There were no other obvious effects of treatment for animals receiving 10, 30 or 100 mg/kg/day. Nevertheless, there was no effect of treatment apparent upon the weights of the spleens of F1 generation animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings were considered related to treatment of the F0 generation.
Histopathological findings:
no effects observed
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
(F1 toxicity)
Generation:
F1
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: (Based on no effects at the highest dose tested)
Key result
Dose descriptor:
NOAEL
Remarks:
(developmental and sexual maturation)
Generation:
F1
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: (Based on no effects at the highest dose tested)

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a one-generation reproduction study performed with methyl isobutyl ketoxime (MIBKO) in Sprague-Dawley rats, the NOAEL for parental toxicity was considered to be 30 mg/kg bw/day (based on histopathological effects on the spleen) and the NOAEL for the F1 generataion and reproductive toxicity was considered to be >100 mg/kg bw/day (based on no effects observed on reproduction or developmental parameters).
Executive summary:

A one-generation reproduction study was performed according to OECD guideline 415 with methyl isobutyl ketoxime (MIBKO) in Sprague-Dawley rats. 28 parental rats per sex and per dose, were given methyl isobutyl ketoxime in water by gavage at doses of 0 (control), 10, 30 and 100 mg/kg bw/day during 10 weeks prior to mating, for up to 2 weeks during mating, or until signs of mating were noted. Males were killed following mating, and females were continuously exposed through gestation and lactation. Parental and litter examinations as well as reproduction observations were performed. All surviving male and female parent rats, were sacrificed for postmortem examinations. All stillborn pups, pups found dead, or pups terminated in a moribund condition and those for which the sexual maturation was observed, were subjected to a throrough necropsy. No adverse effects were observed in any of the reproductive or developmental parameters or in the F1 pups. The toxicity profile includes symptoms of minimal to moderate hemosiderosis and minimal congestion in the spleens at 100 mg/kg bw/day males and females. The NOAEL for parental toxicity in the F0 generation was considered to be 30 mg/kg bw/day based on histological effects on the spleen. The minor changes observed in male kidneys at 30 mg/kg bw/day was not considered to be significant. The NOAEL for the F1 generation and reproductive toxicity was considered to be >100 mg/kg bw/day based on no effects observed at the highest dose.