Butan-2-one O,O',O''-(vinylsilylidyne)trioxime

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test method according to EPA OTS 798.4700. No data on GLP.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4700 (Reproduction and Fertility Effects)

Version / remarks:

(40 CFR 798.4700)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butanone oxime

EC Number:

202-496-6

EC Name:

Butanone oxime

Cas Number:

96-29-7

Molecular formula:

C4H9NO

IUPAC Name:

butan-2-one oxime

Details on test material:

- Name of test material (as cited in study report): Methylethyl Ketoxyme (2-butanone oxime; MEKO)
- Physical state: Liquid

Test animals

Species:

rat

Strain:

other: CD (Sprague-Dawley) rats (Crl:CD[SD]BR) VAF/Plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Raleigh, NC
- Age at study initiation: 8 weeks (F0); 11 weeks (F1)
- Fasting period before study: No
- Housing: Individually housed or housed in mating pairs or with litters in solid bottom polycarbonate cages with stainless steel wire lids with Ab-Sorb-Dri cage litter
- Diet (e.g. ad libitum): Purina Certified Rodent Chow; ad libitum
- Water (e.g. ad libitum): deionezed/filtered tap water; ad libitum
- Acclimation period: at least 1 week

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(deionized/distilled water CAS no. 7732-18-5)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 0 (control), 5, 50 and 100 mg/ml at a dosing volume of 2 mg/kg.

VEHICLE
- Amount of vehicle (if gavage): Dosing volume of 2.0 mg/kg.

Details on mating procedure:

- Mating period: 3 weeks
- M/F ratio per cage: 1:1 within groups
- Proof of pregnancy: Observations of vaginal sperm and/or copulation plug was considered evidence of successful mating; the date of insemination was designated Gestation day 0.
- After 1 week of unsuccessful pairing replacement of first male by another male.

Due to unexpectedly poor F1 reproductive performance, not treatment- or dose-related, F1 mating pairs not successful in producing F2 litters (designated F2a) were rebred (after a 2-week vaginal cytology evaluation for unsuccessful F1 females) with change in partners within groups to produce F2b litters. This additional mating of initially unsuccessful animals follows the suggested approach in the TSCA testing guidelines (U.S. EPA, 1985a). The decision to rebreed initially unsuccessful F1 parental animals was based on unexpectedly low mating indices: 50.% (15/30), 57.1% (16/28), 63.3% (19/30), and 72.1% (19/26) at 0, 10, 100, and 200 mg/kg/day, respectively. Fertility indices were normal: 93.3% (14 pregnant/15 sperm-positive) at 0 mg/kg/day and 100% for 10, 100, and 200 mg/kg/day and there were 14, 16, 19, and 19 F2a litters on pnd 0 at 0, 10, 100, and 200 mg/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations were homogeneous and stable for at least 30 days at ambient or refrigerated temperatures and were analyzed for the first four formulations and then at least monthly throughout the study. Formulations were 91.2-109.0% of target concentrations throughout the study. No MEKO was detected in the vehicle control solutions with an estimated detection limit of 0.29 mg/ml.

Duration of treatment / exposure:

F0, males: 13 weeks: 10 weeks of prebreeding period and 3 weeks of mating period.
F0, females: 19 weeks: 10 weeks of prebreeding period, 3 weeks of mating period, 3 weeks of gestation and 3 weeks of lactation.
F1, males: 14 weeks: 11 weeks of prebreeding period and 3 weeks of mating period.
F1, females: 20 weeks: 11 weeks of prebreeding period, 3 weeks of mating period, 3 weeks of gestation and 3 weeks of lactation.

Frequency of treatment:

F0: 5 days/week for 10 weeks (prebreeded dosing period) and after 7 days/week dosing (mating, gestation, lactation) until scheduled euthanization.
F1: 5 days/week for at least 11 weeks (initiated on posnatal day 22) and after 7 days/week dosing (mating, gestation, lactation) until the demise of F1 parents.
Observation: the 5 days per week dosing regimen for the prebreed period was utilized with EPA approval and is not uncommon in reproductive toxicity studies when the route of administration is other than dosed feed or dosed water and ad libitum exposure.

Details on study schedule:

- F1 parental animals not mated until 11 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 18 weeks (F0) and 14 weeks (F1)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

30 animals per sex and per dose to yield at least 20 pregnant females/group at or near term.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were based on those employed in a subchronic study in rats to allow direct comparison endpoints, although the top dose in this study was lower to prevent excessive stress on pregnant females.
- Rationale for animal assignment: Sentinel animals examined for fecal parasites, serum viral antibody titers and gross necropsy. Animals were suitable for use. The animals were placed by randomization procedures stratified by body weights, 30 animals per sex and per group.

Examinations

Parental animals: Observations and examinations:

PARENTAL ANIMALS (F0 and F1)

CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
Prebreeding period (10-11 weeks): clinical observations taken at and after dosing daily (mortality checks twice daily).

BODY WEIGHT: Yes
Preebreeding period (10-11 weeks): weekly
Gestational period (3 weeks): dams were weighed on gestation days 0, 7, 14 and 20.
Postanatal period (3 weeks): dams with litters were weighed on postnatal day 0, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Preebreeding period (10-11 weeks: Food consumption for each animal determined and mean weekly diet consumption calculated as mg/kg/day.
Gestational period (3 weeks): dams were weighed on gestation days 0, 7, 14 and 20.
Postanatal period (3 weeks): dams with litters were weighed on postnatal day 0, 4, 7, 14 and 21. Maternal feed consumption was unavoidably confounded after postnatal day 10 by the pups as they began to self feed.

OTHER: HEMATOLOGICAL EVALUATION:
10 animals per sex and per dose were randomly selected for hematological evaluation by retroorbital sinus puncture under C02 anesthesia (nonfasted) before necropsy. Hematologic evaluation consisted of methemoglobin concentration white blood cell (WBC) count (absolute and corrected for nucleated erythrocytes), red blood cell (RBC) count, nucleated RBC count (nRBC), hemoglobin concentration (Hgb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, reticulocyte count, and WBC differential count(segmented neutrophils, band neutrophils, lymphocytes, monocytes, and eosinophils).

Oestrous cyclicity (parental animals):

For the last 2-3 weeks of the prebreed period and 2 weeks of postwean, F0, F1a and F1b females were evaluated for estrous cyclicity by vaginal lavage.
Number (5) cycling, cycle length (days), number not cycling/number with abnormal and number not cycling/cycle parameters were analyzed.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Litters were randomly culled to eight (with as equal sex ratio as possible) on postnatal day 4. At weaning on postnatal 21, 30 F1 wealings per sex and per dose were randomly selected to be parents of the F2 generation.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring:
Number of live litters, gestational length (days), number of implantation sites per litter, number of pups per litter, prenatal mortality, stilbirths, livebirth and survival were analyzed on days 4, 7, 14, 21 and lactation.
Litter size, sex ratio, and body weights were measured and recorded during lactation on postnatal days 0, 1, 4, 7, 14 and 21.
10 weanling per sex and per day were randomly selected for hematologic evaluation and necropsy and remaining weanlings were examined externally, euthanized, and discarded (inguinal mammary glands from nonselected F1 female weanlings were examined histopathologically).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, after mating.
- Maternal animals: All surviving animals, after weaning of their F1 litters and after 2-week postwean vaginal cytological evaluation for estrous cyclicity.

GROSS NECROPSY / ORGAN WEIGHTS
- F0 parental necropsy included organ weights as follows (absolute and relative to terminal body weight): testes plus epididymides (males), ovaries (females), liver (both sexes), spleen (both sexes), and pituitary (fixed, both sexes).

HISTOPATHOLOGY
Histopathologic examination was performed on the following tissues from high dose and control parental animals: pituitary, ovaries (2), testes (2), vagina, uterus, mammary glands (females), epididymides, seminal vesicles, and prostate; testes, liver, and spleen were examined from parental animals in all groups. Specified histological procedures included ovaries serially sectioned at 5 µm, mounting, and staining every 10th section, with 10 sections of each ovary examined for oocyte and follicular morphology; testes fixed in 10% BNF (buffered neutral formalin), embedded in plastic (GMA; glycol methacrylate), one section through the center cut at 2–3 µ, stained with PAS (periodic acid Schiff)/hematoxylin, and examined for spermatogenic cycle; and mammary glands fixed at the ventral region, right and left inguinal mammary glands evaluated histologically (5-m sections, hematoxylin and eosin stain) for F0 and F1 parental females.

Postmortem examinations (offspring):

SACRIFICE
F1 males were terminated after the second mating; all F1 females were terminated after a 2-week postwean vaginal cytology evaluation. F2a weanlings, 10/sex/dose, were randomly selected for hematology and necropsy, and mammary glands from remaining F2a females and all F2b females were examined histologically after external examination and termination of the weanlings.

GROSS NECROPSY / ORGAN WEIGTHS
- F1 parental necropsy included organ weights as follows (abso lute and relative to terminal body weight): testes plus epididymides (males), ovaries (females), liver (both sexes), spleen (both sexes), and pituitary (fixed, both sexes).

HISTOPATHOLOGY
Histopathologic examination was performed on the following tissues from high dose and control parental animals: pituitary, ovaries (2), testes (2), vagina, uterus, mammary glands (females), epididymides, seminal vesicles, and prostate; testes, liver, and spleen were examined from parental animals in all groups. Specified histological procedures included ovaries serially sectioned at 5 µm, mounting, and staining every 10th section, with 10 sections of each ovary examined for oocyte and follicular morphology; testes fixed in 10% BNF (buffered neutral formalin), embedded in plastic (GMA; glycol methacrylate), one section through the center cut at 2–3 µ, stained with PAS (periodic acid Schiff)/hematoxylin, and examined for spermatogenic cycle; and mammary glands fixed at the ventral region, right and left inguinal mammary glands evaluated histologically (5-m sections, hematoxylin and eosin stain) for F1 and F2 (a and b) nonselected female weanlings.

Statistics:

Both parametric and nonparametric statistical procedures were applied to selected measures from this reproductive toxicity study. Parametric evaluations were as follows: Appropriate General Linear Models (GLM) procedures for the analyses of variance (ANOVA) were employed. Prior to GLM analysis, an arcsine square root transformation was performed on all litter-derived percentage data and Bartlett’s test for homogeneity of variance (alpha level = 0.001) was performed on all data to be analyzed by ANOVA. GLM analyses were used to determine the significance of the dose–response relationship (test for linear trend) and to determine whether significant dose effects had occurred for selected measures (ANOVA). When a significant (p<0.05) main effect for dose occurred, Dunnett’s multiple comparison test was used to compare each chemical exposed group to the vehicle control group for that measure. A one-tailed test (i.e., Dunnett’s test) was used for all pairwise comparisons except for adult body and organ weight parameters, maternal feed consumption, pup body weight, and percentage males per litter, for which a two-tailed test was used. Nominal scale measures were analyzed by the X2 test for independence for differences among treatment groups, and by a test for linear trend on proportions. When X2 revealed significant (p<0.05) differences among groups, then a one-tailed Fisher’s exact probability test was used for pairwise comparisons between each treated group and the vehicle control group. Nonparametric data were statistically evaluated using the Kruskal–Wallis one-way analysis of variance by ranks to test for differences among dose groups. Whenever the result of a Kruskal–Wallis test was significant (p<0.05), the Mann–Whitney U test was used to make individual comparisons between vehicle and chemical dose groups for that measure. Jonckheere’s test for k independent samples was used to identify significant dose–response trends for these parameters.

Reproductive indices:

Number of mating pairs, female mating index, female fertility index, male mating index, male fertility index, gestational index, number of live litters (postnatal day 0), number of live litters (postnatal day 21), gestational length (days), number of implantation sites per litter, number of total pups/litter, number of live pups/litter (postnatal day 0), prenatal mortality index, stilbirth index, livebirth index and survival indices (day 4, 7, 14, 21 and lactation).

Offspring viability indices:

Offspring litter size (number of pups/litter), sex ratio (% males/litter) and body weights (body weight/litter) during lactation.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day: Treatment-related clinical signs of toxicity included tremors, salivation, slow respiration, mouth breathing, lethargy, staggers, and rooting in bedding (postdosing, presumably from the ‘‘taste’’ of the dosing solution) in F0 males; tremors, ataxia, and convulsions (only in animals prior to demise), stupor, abnormal respiration (audible, irregular, raspy, labored), dyspnea, dehydration, excessive urination, bright yellow urine, and rooting in bedding in F0 females.
100 mg/kg bw/day: Clinical signs of toxicity included lethargy, staggers, and rooting in bedding for F0 males; weaving, tremors, and rooting in bedding in F0 females.
10 mg/kg bw/day: the only apparent clinical sign of toxicity was rooting in bedding (F0 males)

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

200 mg/kg bw/day: treatment-related mortality was observed in males and females: 4/30 (13.3%) in males, in including 3 which died during the prebreed period (excluding an additional F0 male which died during this period due to an intubation accident) and 1 which died during the mating period; 11/30 (36.7%) in F0 females, all of which died during the prebreed period (no intubation accidents).
100 and 10 mg/kg bw/day: no treatment-related mortality occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day: Both sexes exhibited significant reductions in body weights and weight gains during their prebreed exposure periods. There were no further effects on body weights for F0 females during gestation or lactation. There were also occasional significant changes in feed consumption (decreased in F0 males and females), expressed as g/day or as g/kg/day during the prebreed periods. Maternal feed consumption was not significantly reduced for F0 females during gestation and lactation as g/day.
100 mg/kg bw/day: occasional changes were observed in feed consumption in F0 males (reductions) and females (decreases). No effects during gestation and lactation were noted.
10 mg/kg bw/day: no effects of treatment on body weights, weight gains or feed consumption (F0 males).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day: There were occasional significant changes in feed consumption (decreased in F0 males and females), expressed as g/day or as g/kg/day during the prebreed periods. Maternal feed consumption was not significantly reduced for F0 females during gestation and lactation as g/day.
100 mg/kg bw/day: occasional changes were observed in feed consumption in F0 males (reductions) and females (decreases). No effects during gestation and lactation were noted.
10 mg/kg bw/day: no effects of treatment on body weights, weight gains or feed consumption (F0 males).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day: Hematologic evaluation indicated a consistent picture of anemia, with reduced RBC count, reduced hematocrit, reduced hemoglobin concentration, increased RBC size (MCV), increased nucleated RBC count, increased reticulocyte count, increased MCH (consistent with fewer, larger RBCs), and increased WBC count (with no change in relative differential WBC counts) in F0 males and females, and increased methemoglobin concentration in F0 males.
100 mg/kg bw/day: Hematologically, again there was a consistent picture of anemia in both sexes, an increased number of WBC in F0 males (with no change in differential counts), and an increased methemoglobin concentration (F0 males only).
10 mg/kg bw/day: F0 males only also exhibited reduced RBC count and reduced hemoglobin concentration.

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):

200 mg/kg bw/day: F0 males and females exhibited splenic congestion and extramedullary hematopoiesis and hemosiderosis and hepatic hemosiderosis and hematopoiesis. Possible evidence of mild testicular effects at 10, 100, and 200 mg/kg/day in the F0 males was clearly not treatment related since these lesions, minimal to mild testicular degeneration and atrophy, were observed with no dose–response pattern in F0 males. These findings were also unaccompanied by any changes in relative testis weights. These mild testicular findings are currently frequently observed in CD rat males beginning at 4–5 months of age (Christian et al., 1991). There were no effects on any other organs, including mammary glands of F0 females.
100 mg/kg bw/day: Livers and spleens of F0 males and females exhibited extramedullary hematopoiesis and hemosiderosis.
10 mg/kg bw/day: both spleens and livers exhibited extramedullary hematopoiesis and hemosiderosis.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no significant effects of treatment on number of F0 females, pre- or postbreed, which were cycling, or on cycle length, number of females not cycling, or number of females with abnormal cycles.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects at any dose for F0 generations for any reproductive indices. Prenatal mortality and stillbirth indices exhibited no significant trends or pairwise comparisons appeared to be slight dose-related increases for both parameters in F0 mattings. All values for the two parameters in this study were well within the historical control data for the performing laboratory.

Effect levels (P0)

1

Key result

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on hematopoiesis and hemosiderosis in spleens and livers of F0 males and females.

2

Key result

Dose descriptor:

NOAEL

Effect level:

> 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day: Treatment-related clinical signs of toxicity included tremors, audible breathing, and rooting in bedding in F1 males; and lethargy, abnormal respiration (labored, gasping, and raspy), cyanosis, and rooting in bedding in F1 females.
100 mg/kg bw/day: Clinical signs of toxicity included slight dehydration, audible breathing, and rooting in bedding in F1 males; and labored breathing in F1 females.
10 mg/kg bw/day: the only apparent clinical sign of toxicity was rooting in bedding (F1 males)

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

200 mg/kg bw/day: treatment-related mortality was observed in males and females: 15/30 (50.0%) in F1 males, including 8 during the prebreed period, 1 during the first breed, 3 during the interbreed period prior to the second breed, and 3 during the second breed (no intubation accidents); and 8/30 (26.7%) in F1 females, including 4 during the prebreed period, 3 during the vaginal cytology associated with the first breed, and 1 during the lactation of her F2b litter (no intubation accidents).
100 and 10 mg/kg bw/day: no treatment-related mortality occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day: Both sexes exhibited significant reductions in body weights and weight gains during their prebreed exposure periods. There were no further effects on body weights for F1 females during gestation or lactation. There were also occasional significant changes in feed consumption (decreased in F1 females, increased in F1 males), expressed as g/day or as g/kg/day during the prebreed periods. Maternal feed consumption was significantly reduced for F1 females during gestation (but not lactation) as g/day.
100 mg/kg bw/day: Occasional reductions in body weights and weight gains were observed during the prebreed period in F1 females; occasional changes were observed in feed consumption in F1 males (increases) and females (decreases). Feed consumption during gestation was decreased for F1 females (Table 1), and no effects during gestation and lactation were noted.
10 mg/kg bw/day: no effects of treatment on body weights, weight gains or feed consumption (F1 males).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day: There were occasional significant changes in feed consumption (increased in F1 males and decreased in F1females), expressed as g/day or as g/kg/day during the prebreed periods. Maternal feed consumption was significantly reduced for F1 females during gestation but not lactation as g/day.
100 mg/kg bw/day: Feed consumption during gestation was decreased for F1 females.
10 mg/kg bw/day: no effects of treatment on body weights, weight gains or feed consumption (F1 males).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day: Hematologic evaluation indicated a consistent picture of anemia, with reduced RBC count, reduced hematocrit, reduced hemoglobin concentration, increased RBC size (MCV), increased nucleated RBC count, increased reticulocyte count, increased MCH (consistent with fewer, larger RBCs), and increased WBC count (with no change in relative differential WBC counts) in F1 males and females, and increased methemoglobin concentration in F1 males.
100 mg/kg bw/day: Hematologically, again there was a consistent picture of anemia in both sexes in both generations, an increased number of WBC in F1 males (with no change in differential counts).
10 mg/kg bw/day: No effects observed in F1.

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

Description (incidence and severity):

200 mg/kg bw/day: At necropsy, F1 males and females exhibited significantly increased absolute and relative spleen weights and significantly increased relative liver weights in F1 males and F1 females.
100 mg/kg bw/day: At necropsy, F1 males and females exhibited significantly increased absolute and relative spleen weights.
10 mg/kg bw/day: At necropsy, no treatment-related observation.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day: F1 males and females exhibited splenic congestion and extramedullary hematopoiesis and hemosiderosis and hepatic hemosiderosis and hematopoiesis. Minimal to mild testicular degeneration and atrophy were observed with no dose–response pattern in all groups (including the vehicle control group) in F1 males. These findings were also unaccompanied by any changes in relative testis weights. These mild testicular findings are currently frequently observed in CD rat males beginning at 4–5 months of age (Christian et al., 1991). There were no effects on any other organs, including mammary glands of F1 females.
100 mg/kg bw/day: Livers and spleens of F1 males and females exhibited extramedullary hematopoiesis and hemosiderosis.
10 mg/kg bw/day: both spleens and livers exhibited extramedullary hematopoiesis and hemosiderosis.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no significant effects of treatment on number of F1 females, pre- or postbreed, which were cycling, or on cycle length, number of females not cycling, or number of females with abnormal cycles.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects at any dose for F1 (a + b) generations for any reproductive indices. Prenatal mortality and stillbirth indices exhibited no significant trends or pairwise comparisons appeared to be slight dose-related increases for stillbirth index only in F1 (a / b) mattings. All values for the two parameters in this study were well within the historical control data for the performing laboratory.

Details on results (P1)

Results reported under this section (P1) are refered to the effects in second generation as parental while results on next section (F1) are referred to the developmental effects for the same individuals as offspring.

Effect levels (P1)

Key result

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on hematopoiesis and hemosiderosis in spleens and livers of F1 males and females

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical observations for F1 pups during lactation.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no effects of treatment at any dose on total or live litter size, sex ratio, or pup body weights per litter with sexes pooled or separate (pnd 0–21) for F1 litters.

Body weight and weight changes:

not specified

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:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in hematologic parameters or organ weights in F1 weanlings, 10/sex/dose.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related necropsy findings of pups during lactation or of F1 pups, 10/sex/dose, which were necropsied at weaning.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

There were also no histologic effects on F1 weanling female mammary glands.

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on no effects at highest dose tested.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical observations for F2 (a / b) pups during lactation.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no effects of treatment at any dose on total or live litter size, sex ratio, or pup body weights per litter with sexes pooled or separate (pnd 0–21) for F2 (a + b) litters.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects of treatment at any dose on total or live litter size, sex ratio or pup body weights per litter with sexes pooled or separate (pnd 0–21) for F2 (a + b) litters.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in hematologic parameters or organ weights in F2a weanlings, 10/sex/dose.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related necropsy findings of pups during lactation or of F2a pups, 10/sex/dose, which were necropsied at weaning.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

There were also no histologic effects on F2a or F2b weanling female mammary glands.

Other effects:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

> 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on no effects at highest dose tested

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL for reproductive and postnatal toxicity of methylethyl ketoxime in a two-generation study with CD (Sprague-Dawley) rats was at least 200 mg/kg/day under study conditions.

Executive summary:

Methylethyl ketoxime was tested for reproductive toxicity in two-generation study with CD (Sprague-Dawley) rats according to EPA OTS 798.4700. Thirty rats per sex and per dose (F0) were administered MEKO in water by gavage at 0 (control), 10, 100 and 200 mg/kg bw/day, 5 days/week for 10 weeks with vaginal cytology evaluation (VCE) of F0 females during the last 3 weeks of the prebreed period. Animals were mated within groups for 3 weeks with dosing during mating, gestation, and lactation for 7 days/week. F0 parents and F1 weanlings, 10/sex/dose, were necropsied (after a 2-week postwean VCE in F0 females) with hematologic evaluation (including methemoglobin) and histology of adult livers, spleens, and reproductive organs. F1 weanlings, 30/sex/dose, were dosed for 11 weeks and mated as described above. Because of poor reproductive performance, not treatment related, F1 animals with no F2a litters were rebred to produce F2b litters. F1 parents and F2a weanlings, 10/sex/dose, were necropsied and evaluated as described above. Inguinal mammary glands were examined histologically from all nonselected F1 and F2 (a and b) female weanlings. Adult toxicity was observed in both generations and both sexes at all doses. Treatment-related parental deaths occurred at 200 mg/kg/day. At 100 and 200 mg/kg/day, parents exhibited dose-related reduced body weights and weight gains, reduced feed consumption, clinical signs of toxicity, and anemia with concomitant extramedullary hematopoiesis and hemosiderosis in livers and spleens (and increased spleen weights). At 10 mg/kg/day, only adult liver and spleen histologic effects were present. There was no evidence of reproductive organ or mammary gland pathology or of reproductive or postnatal toxicity at any dose tested. Therefore, the NOAEL for reproductive and postnatal toxicity was at least 200 mg/kg/day for rats in this study.