Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 1990 - June 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: EPA guideline with modifications as noted in the TSCA 4 test rule
Principles of method if other than guideline:
Persuant to the EPA Final Test Rule for MEKO under Section 4 of TSCA, this study was performed to evaluate the potential of MEKO administered by gavage to CD rats to produce alterations in parental fertility, maternal pregnancy and lactation, and growth and development of the offspring for two generations, one litter per generation for the F0 to F1 generation, and at least one litter per generation in two breedings for the F1 to F2 generation.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Butanone oxime
EC Number:
202-496-6
EC Name:
Butanone oxime
Cas Number:
96-29-7
Molecular formula:
C4H9NO
IUPAC Name:
(NE)-N-butan-2-ylidenehydroxylamine
Test material form:
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: (FO) 8 wks; (F1) 11 wks
- Weight at study initiation: (FO) Males: 263.2 - 264.7 g; Females: 198.1 - 200.5 g; (F1) Males: 169.5 - 194.6 g; Females: 136.6 - 162.8 g
- 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 (Ralston Purina Co., Richmond, IN); ad libitum
- Water (e.g. ad libitum): available ad libitum in water bottles
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23.9
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Amount of vehicle (if gavage): 2.0 mL/kg bw.
Details on mating procedure:
Male and female weanling rats (F0) were administered MEKO in deionised/distilled water by gavage at 0, 10, 100, 200 mg/kg bw/day at a dosing volume of 2.0 ml/kg bw/day, 30 animals/sex/dose, for 10 weeks. Animals were than randomly mated for a 3 week mating period to produce the F1 generation, with dosing continuing. Selected F1 weanlings, 30/sex/dose, were administered MEKO and mated as above.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the start of the study, a test formulation of the high and low dose levels of MEKO in vehicle were assayed for homogeneity, stability and dose level verification. To prepare dosing solutions, MEKO was dissolved in deionized/distilled water with the concentration determined by the following formula: Concentration (mg/ml) = Dose level (mg/kg)/Dose volume (2 mL/kg)
The concentration of MEKO in the dosing solutions varied with dose, and solutions for each concentration were formulated independently. The frequency of formulation depended on the stability data. For the first 4 formulations, aliquots o f all dosing solutions were analyzed for dose level verification prior to use. Dosing formulations with assayed value of 90-110% of the target concentration were considered to be suitable for use. If this criterion was not met, the solutions were reformulated prior to use. For subsequent formulations, aliquots for each dose level were retained and aliquots from all dose levels per formulation were analyzed (at least monthly).
Duration of treatment / exposure:
F0 generation: starting from 8 weeks of age during 10 week pre-mating, 3 weeks mating period (continued dosing).
F1 generation: starting from 11 weeks of age in the same regime.
Frequency of treatment:
5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
30 males/group and 30 females/group to yield at least 20 pregnant females/group at or near term.
Control animals:
yes, concurrent vehicle
Details on study design:
All animals assigned to each MEKO dosage level were dosed by gavage 5 days per week during the prebreed dosing periods and 7 days per week during the mating, gestational and lactational periods. F2 animals were not dosed with MEKO.
- Animals of the FO generation were approximately 8 weeks of age at the commencement of treatment. They were administered the dosing solutions by gavage at their respective formulations 5 days per week for at least 10 weeks prior to mating, i.e., until they were approximately 18 weeks of age.
- The animals were then mated on the basis of one male to one female selected randomly within each dose group for a period of 21 days. If mating had not occurred during the first week of cohabitation, the female was placed with a different male in the same dosage group (selected from non-successful males from the same dose group) for the remaining 14 days or until mating occurred. The observation of vaginal sperm was considered evidence of successful mating. Females were examined daily during the cohabitation period for the presence of sperm in the vaginal tract. The day vaginal sperm were observed was designated gestational day (gd) 0. Once vaginal sperm was observed, the male and female from that mating pair were housed. For any female which did not show evidence of successful mating after 21 days of cohabitation, the last scheduled mating day was considered gd 0 for that female and the animal was treated accordingly for subsequent events. Beginning on gd 20, each female was observed twice daily for evidence of littering. The dams were allowed to rear their young to day 21 postpartum. On day 21 postpartum, each litter was weaned. When each F1 litter reached day 21 postpartum, at least one male and one female pup per litter, if possible, were randomly selected to produce the F2 generation. These selected animals were then gavaged with the same concentration of MEKO as their parents. Each litter was represented at least once per sex, if possible, until a total of 30 per sex per treatment group were attained. Following this selection, ten weanlings/sex/dose were randomly selected for hematologic evaluation and necropsy. All pups not selected as parents were examined externally, euthanized (female mammary glands removed and fixed) and discarded.
- FO females continued to be dosed with MEKO for at least two (2) additional weeks following weaning of the F1 litters and vaginal smears were taken daily to determine presence or absence of estrous cycling and length of cycle.
- During the mating, gestational and lactational periods, the animals were gavaged daily, 7 days per week.
- Animals of the F1 generation were administered MEKO by gavage at their respective formulations 5 days per week for at least 11 weeks and were approximately 14 to 17 weeks of age at the initiation of the mating period. F1 females were evaluated for estrous cyclicity for the last 3 weeks prior to mating as described for the FO animals They were mated as described above for the FO animals, including, but not limited to, pairing one ma1e:one female, and cohabitation for 21 days or until vaginal sperm were observed, whichever came first (with change in pairing after 1 week if no evidence of successful mating was found). There were no brother-sister matings. F1 females continued to be dosed with MEKO for at least two (2) additional weeks following weaning of the F2 litters and vaginal smears were taken daily to determine presence or absence of estrous cycling and length of cycle. Because of the unexpectedly poor reproductive performance of the F1 animals to produce the initial F2 litters (designated F2a), unsuccessful F1 animals (females sperm-negative or sperm-positive with no resulting litters, and the males involved in the pairings) were re-paired, with a 2-week evaluation of estrous cyclicity prior to the remating, a 3-week mating period (with changes in pairing after each week if no evidence of successful mating was found and a 2-week post wean evaluation of vaginal cyclicity. For those interim periods, such as between matings, and from the end of mating to sacrifice for males, from the end of lactation (of F2a litters) until the rebreed, and from the end of the second lactation period (of F2b litters) until sacrifice for females, dosing continued and periodic body weights and daily clinical signs were recorded.

Examinations

Parental animals: Observations and examinations:
- Observations for mortality were made twice daily (a.m. and p.m.) and the general condition of all animals was checked daily.
- Clinical examinations were conducted and recorded daily throughout the course of the study. This record included the time of onset, degree and duration of symptoms. These cage-side observations included, but were not limited to, changes in: skin and fur, eyes, and mucous membranes, respiratory system, circulatory system, autonomic and central nervous system, somatomotor actiuity, and behavior pattern.
- The body weights of the male rats were determined and recorded initially and weekly through mating. The body weights of female rats were recorded in the same manner until confirmation of mating. During gestation, females were weighed on gestational days 0, 7, 14 and 20. Dams producing litters were weighed on lactational days (postnatal days) 0, 4, 7, 14 and 21. Body weight gains were computed.
- Feed consumption measurements were recorded weekly for all FO and F1 parental animals throughout the pre-breed treatment periods. During pregnancy of FO and F1 females, food consumption was recorded for gestational days (gd) 0-7, 7-14 and 14-20. During lactation of F1 and F2 litters, maternal food consumption was measured for lactational days 0-4, 4-7, 7-14 and 14-21, although maternal food consumption after lactational day 14 was confounded by the contribution from the pups since pups were self-feeding by this time. Feed consumption was not measured during the cohabitation period since two adult animals (breeding pair) were in the same cage.
- Hematologic evaluations, vaginal cytology evaluations for estrus cyclicity and histopathology of the liver, spleen, mammary glands, pituitary, ovaries, testes,epidiymides,seminal vesicles, prostate, vagina and uterus, were performed. Standard reproductive indices were determined such as mating index, fertility index, gestational length, implantation index, pups/liter, stillbirth and livebirth index and pup survival.
Oestrous cyclicity (parental animals):
Vaginal cytology evaluations for estrus cyclicity were performed.
Litter observations:
For F1 and F2 offspring. litter size, sex ratio, and body weights owere measured and recorded during lactation. On postnatal days 0, 1, 4, 7, 14 and 21, the number of pups/litter, % males/litter and body weights per litter were counted or measured and recorded.
Postmortem examinations (parental animals):
- At necropsy of parental males, body weights and organ weights (liver, spleen, testes and pituitary) were measured and recorded. At necropsy of parental females, body weights and organ weights (liver, spleen, ovaries and pituitary) were measured and recorded.
- For males and females the following hematology parameters were measured: red blood cells, number of nucleated red blood cells (per 100 white blood cells), reticulocytes (%), hemoglobin (g %), hematocrit (%), mean corpuscular volume, mean corpuscular hemoglobin, mean corpusular hemoglobin concentration (%), platelets, methemoglobin (%), and whtie blood cell count corrected for nucleated red blood cells.
-Histopathology of tissues from F0 and F1 males included epididymides, liver, pituitary, prostate, seminal vesicle, testes and spleen. Histopathology of tissues from F0 and F1 females included liver, mammary glands, ovaries, uterus, vagina and spleen.
Postmortem examinations (offspring):
Necropsy:
-All FO and F1 parental animals in all groups (both generations) were subjected to a complete gross necropsy. The gross necropsy included examination of the extemal surfaces; all orifices; cranial cavity; carcass; external and cut surfaces of the brain and spinal cord; the thoracic, abdominal, and pelvic cavities and their viscera; and cervical tissues and organs. All of the male and female adults from the control and high dose groups in both generations were subjected to a histopathologic examination. Sacrifice of the parental males occurred after the completion of the mating period. Sacrifice of the maternal animals occurred at least two weeks after the F2b litters had been weaned.
Histopathological Examination:
- Histopathologic evaluation was conducted on the following parental tissues from high dose and control groups: liver, spleen, pituitary, vagina, uterus, ovaries, mammary glands (also F1 and F2a and F2b weanling females), testes, epididymides, seminal vesicles and prostate.
- Additional histopathologic examinations as specified by the U.S. EPA Test Rule were as follows: Histologic examination of the testes was conducted on all FO and F1 adult males. Both testes per male were fixed in neutral buffered 10% formalin, embedded in plastic (glycol methacrylate; GMA) and one section per testis was cut through the center at 2-3p, and stained with hematoxylin/PAS (Periodic Acid Schiff reagent) and examined. Histological analyses included evaluations of the spermatogenic cycle, i.e., the presence and integrity of the 14 cell stages. Both ovaries of FO and F1 adult females in the high and control dose groups were serially sectioned at 5p, every tenth section mounted and stained, and ten (1 0) sections of each ovary examined to adequately detail oocyte and follicular morphology.
- Gross and histopathologic evaluations were conducted on the mammary glands in F1 and F2 (a and b) female pups sacrificed at weaning and in adult FO and F1 females. The entire ventral region of F1 adult females and F2 (a and b) weanling female pups were removed and fixed for all designated animals; representative mammary glands, the right and left inguinal mammary glands, of each female were histologically evaluated from high dose and control females. Male and female FO and F1 livers and spleens were evaluated microscopically for all dose groups since evaluation of high dose tissues indicated apparent possible treatment-related lesions in these tissues. A complete gross necropsy and histopathologic examination was conducted for any parental animals dying on test.
Statistics:
Both parametric and non parametric statistical procedures were applied to selected measures from the reproductive study (for details, refer to Tyl et al. Fund. Appl. Tox. 31, p. 51, 1996).
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, and livebirth index.
Offspring viability indices:
Survival indices: Day 4 (postnatal day 0-4; precull), Day 7 (postnatal day 4-7; precull), Day 14 (postnatal day 7-14; precull) , Day 21 (postnatal day 14-21; precull) , and lactation (postnatal day 4-21; precull).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Limited to the 100 and 200 mg/kg bw/day dose groups only, including rooting (in the bedding), salivation, slow respiration, mouth breathing, tremors, weaving, hypoactivity, stupor, lethargy, aggression, and three deaths (one male each on days 42, 49 and 53) at 200 mg/kg bw/day and rooting (in bedding), lethargy, and staggers walking at 100 mg/kg bw/day. The only apparent treatment-related signs observed at 10 mg/kg bw/day were rooting (in bedding) on day 12, rough coat on day 83 and aggression on days 79, 84 and 85. Rooting (in bedding) was observed in 1-26 males beginning on day 2 and observed almost every day thereafter at 200 mg/kg/day, in 1-19 males (beginning on day 9 and observed sporadically) at 100 mg/kg bw/day, and in two males on one day at 10 mg/kg bw/day.
For F0 females, at 200 mg/kg bw/day, the signs included audible, irregular, raspy and labored respiration, tremors, rooting (in bedding), weaving, ataxia, gasping, nasal discharge, rust-colored fur, dehydration, dyspnea, convulsion, ataxia, stupor, excessive urination, bright yellow urine, and 11 deaths (one each on days 22, 41, 44, 48, 51, 55 and 57; two on day 58; and two animals sacrificed moribund, one each on days 45 and 56). Clinical observations at 100 mg/kg bw/day included weaving, tremors, rough coat, rooting (in bedding) and bright yellow urine. Rooting (in bedding) was observed in 2-27 females (beginning on day 2 and occuring almost daily) at 200 mg/kg bw/day, and in 1-1a females (beginning on day 1a and observed sporadically) at 100 mg/kg bw/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Four males died during the prebreed dosing period (including one from a dosing error) and one died during the breeding period for a total of four treatment-related deaths (out of 30, 13.3%) at 200 mg/kg bw/day. Eleven females died during the prebreed dosing period (out of 30,36.7%) at 200 mg/kg bw/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Beginning on the seventh week (day 49) and continuing through the end of the prebreed dosing period (day 70) and the three-week mating period, the mean body weights at 200 mg/kg bw/day were significantly lower at all these weekly time points. In addition, the mean value at 100 mg/kg bw/day on day 91 (week 13, end of the mating period) was significantly lower than the value at 0 mg/kg bw/day. F0 male weekly body weight changes exhibited the following statistically significant differences: reductions at 200 mg/kg bw/day for weeks 1. 6 and 7, at 100 mg/kg bw/day for weeks 7 and 10, and at 10 mg/kg bw/day for weeks 6 and 10.
For F0 females, the mean value at 200 mg/kg bw/day was significantly reduced only for week 8 (day 56). F0 female weekly body weight changes exhibited the following statistically significant differences: reductions at 200 mg/kg bw/day for weeks 1 and 8. In addition, a significant dose-related downward trend was observed for week 5 (days 28-35) with no significant pairwise comparison.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F0 male food consumption, expressed as g/day, was significantly lower at 200 mg/kg bw/day for all weekly timepoints except for week 2 during the 10-week prebreed dosing period; the value for the first week (days 0-7) was also significantly lower at 100 mg/kg bw/day.
F0 female food consumption during the 10-week prebreed dosing period, expressed as g/day, were significantly reduced at 200 mg/kg bw/day for weeks 1 (days 0-7), 8 (days 49-56) and 9 (days 56-63).
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):
Red blood cell (RBC) count was significantly reduced at all doses, 10, 100 and 200 mg/kg bw/day, in a dose-related manner. The number of nucleated RBCs was significantly increased at 200 mg/kg bw/day. Reticulocyte counts were significantly increased at 100 and 200 mg/kg bw/day, in a dose-related manner. Hemoglobin concentration was significantly decreased at all doses, 10, 100 and 200 mg/kg bw/day, in a dose-related manner. Hematocrit was significantly reduced at 200 mg/kg bw/day. Mean corpuscular volume (MCV; a measure of erythrocyte size and therefore maturity) and mean corpuscular hemoglobin (MCH) were significantly increased at 100 and 200 mg/kg bw/day. Methemoglobin concentration (as % of total hemoglobin) was significantly increased at 100 and 200 mg/kg bw/day. Corrected white blood cell count (corrected for the presence of nucleated erythrocytes) was significantly increased at 100 and 200 mg/kg bw/day. There were no significant differences among groups for the frequency of the following white blood cell types: segmented or band neutrophils, lymphocytes, monocytes, or eosinophils.
Hematologic evaluation of F0 females indicated significant decreases in red blood cell count, and significant increases in reticulocyte count, mean corpuscular volume and mean corpuscular hemoglobin at 100 and 200 mg/kg bw/day. In addition, hematocrit and hemoglobin concentrations were significantly reduced at 100 (but not 200) mg/kg bw/day. There was a significant dose-related upward trend for corrected WBC but no significant pairwise comparisons. There was no effect of treatment on WBC differential examination. There were no significant effects on nucleated RBCs, platelet counts, MCHC, or methemoglobin levels (although the methemoglobin level at 200 mg/kg bw/day was increased, but not statistically sjgnificantly).
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):
Histopathologic evaluation of selected tissues from F0 males indicated clear splenic effects at 10,100 and 200 mg/kg bw/day, including congestion, hematopoietic cell proliferation and pigment at all three doses and capsular fibrosis in two (2) males and necrosis in one (1) male at 200 mg/kg bw/day. In addition, livers exhibited a dose-related incidence of pigment deposition, and hematopoietic cell proliferation at 10,100 and 200 mg/kg bw/day. Testes exhibited a number of findings in one-five (1-5) males at 10,100 and 200 mg/kg bw/day with no dose-related incidence, including minimal to mild seminiferous tubule degeneration (uni- or bilateral) and atrophy (uni-or bilateral) and presence of giant cells in the seminiferous tubules. Epididymides, pituitary, prostate and seminal vesicles exhibited no treatment-related lesions.
Histopathologic evaluation of selected tissues from F0 females indicated splenic lesions at 10, 100 and 200 mg/kg bw/day, including congestion, hematopoietic cell proliferation and pigment deposition; livers at these dose levels also exhibited treatment- and dose-related incidences of hematopoietic cell proliferation and pigment deposition. F0 mammary glands, ovaries, pituitary, uterus and vagina exhibited no treatment-related lesions.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Vaginal cytology of F0 females for three weeks prior to mating and for two weeks subsequent to weaning of the F1 litters indicated no treatment-related effects on number of females cycling or cycle length.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
F0 female mating and fertility indices were equivalent across all groups; F0 male mating and fertility indices were also statistically equivalent across all groups; the mating index inhibited a significant dose-related downward trend in the absence of any significant pairwise comparisons. Gestational index, gestational length in days, number of implantation sites per dam, and number of total, live and dead pups per litter on postnatal day 0 were all equivalent across all groups.

Details on results (P0)

F0 Gestation:
Maternal gestational body weights were equivalent across all groups for all timepoints evaluated; gestational weight change (gd 0-20) was also equivalent across all groups. Matemal gestational food consumption, expressed as g/day or g/kg/day, was also equivalent across all groups; a significant downward dose-related trend was observed for the mean value for gd 0-7, when expressed as g/day, with no significant pairwise comparisons. Clinical observations of F0 dams during gestation included ataxia in one dam at 200 mg/kg bw/day on gd 2 and in one dam at 100 mg/kg bw/day on gd 14, rooting (in bedding) in all groups except 10 mg/kg bw/day including the control group, observed in one dam per group on occasional days, audible breathing in one dam at 200 mg/kg bw/day on gd 1, and weaving and labored breathing in one dam at 100 mg/kg bw/day on gd 14.

F0 Lactation and F1 Pups:
Matemal lactational body weights were equivalent across all groups for all timepoints evaluated; there was a significant dose-related downward trend for body weight on postnatal day 7 but no significant pairwise comparisons. Lactational weight changes (postnatal days 0-21) were also statistically equivalent across all groups. Maternal lactational food consumption, expressed as g/day or g/kg bw/day, was equivalent across all groups for all timepoints evaluated.
Clinical observations of F0 dams during lactation indicated black feces. audible breathing, rooting (in bedding), and lethargy observed in 1-2 dams per day on occasional days at 200 mg/kg bw/day; rooting (in bedding) was also observed in one dam each on occasional days at 100 mg/kg bw/day. Clinical observations during the two-week postwean vaginal cytology evaluation included the death of one (1) dam at 10 mg/kg bw/day on day 2, and rooting (in bedding) at 100 and 200 mg/kg bw/day each with an incidence of one or a few females per designated group(s) on occasional days.



Adult toxicity was observed in both generations and in both sexes at all doses of MEKO with clear dose-related incidences and severity of findings. Consistent evidence of treatment related anemia was observed at 200 mg/kg bw/d with concomitant histologic evidence of extramedullary hematopoiesis and hemosiderosis in adult livers and spleens; spleen weights were increased at this dose level as well. At 100 mg/kg bw/day, these effects were also seen. At 10 mg/kg bw/day only histologic evidence of extramedullary hematopoiesis and hemosiderosis was observed in spleens and livers of F0 and F1 males and females. Therefore, the NOAEL for adult toxicity could not be established. There was no evidence of reproductive organ or mammary gland histopathology at any dose tested. There was no evidence of reproductive or postnatal toxicity at any dose tested. There was no "no observable adverse effect level" (NOAEL) detected for adult toxicity in this study due to histologic evidence of hepatic and splenic involvement at the low dose. A low incidence of minimal to mild testicular degeneration/atrophy occurred at all dose levels in the parental and F1 generation males. These effects also occurred in the F1 generation control males. These findings were unaccompanied by any changes in testes weight. The histological findings did not occur in a dose related manor and are commonly found in CD rats of this age. The testicular findings were not considered to be related to the treatment with MEKO. There were also no treatment related findings in the prostate and seminal vesicles. No compound related lesions were found in the ovaries, uterus, vagina or mammary glands in either the parental or filial generation females. There were no significant effects of treatment on the estrus cycle with regard to the number of parental or filial females cycling or the length of the cycle. The NOAEL for this reproductive and postnatal toxicity was at least 200 mg/kg bw/day under the conditions of this study.

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
> 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P0)

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical observations of P1 males during the prebreed and mating periods were limited to piloerection, rough coat and bloody urine at all doses, 1-2 males per group on occasional days, audible breathing (one male, day 72) and pica (one male, day 49) at 100 mg/kg bw/day, 100 mg/kg bw/day (1-4 males), and 200 mg/kg bw/day (1-8 males) observed sporadically, and tremors (one male on day 14) and deaths (one male each on days 0, 17, and 78) at 200 mg/kg bw/day. (One male at 10 mg/kg bw/day was euthanized due to a mass on the left shoulder.)
Clinical observations of P1 females during the prebreed dosing period included rooting (in bedding) observed only at 100 mg/kg bw/day (one female sporadically) and 200 mg/kg bw/day (1-4 females regularly), rough coat in a few animals at all dose levels, occasional lethargy in all groups (Including the control group in one or a few animals), and piloerection, labored, raspy breathing, and gasping in one animal on a given day only at 200 mg/kg bw/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Fifteen (of 30, 50.0%) males died at 200 mg/kg bw/day, over half of these during the prebreed dosing period. A total of eight (8) females died in the 200 mg/kg bw/day group (out of 30, 26.7%), half of those during the prebreed dosing period and apparently half during the postwean period. None of the deaths were due to dosing errors.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
P1 male weekly body weights were significantly reduced at 200 mg/kg bw/day at weeks 13 and 14 (days 91 and 98). Weekly P1 male body weight changes, for week 7 (days 42-49), were significantly increased at 10 and 100 mg/kg bw/day but not at 200 mg/kg bw/day. For weeks 11 and 13, the mean weight change at 200 mg/kg bw/day was statistically significantly reduced. For week 14, there was a significant dose related downward trend but no significant pairwise comparisons.
P1 female body weights during the 11-week prebreed dosing period were consistently and significantly reduced at 100 and 200 mg/kg bw/day for the first six weeks (days 0 through 42); for weeks 7 (day 49),8 (day 56), and 9 (day 63), there were statistically significant dose-related downward trends but no significant pairwise comparisons. For weeks 10 (day 70) and 11 (day 77), the mean body weight values at 200 mg/kg bw/day were significantly reduced. Body weights during the mating period (for females not yet found sperm-positive) were significantly reduced at 200 mg/kg/day for both weekly weights (weeks 12 and 13, days 84 and 91) and at 100 mgfkg/day for week 13 (day 91) with decreasing numbers of females included (as more females became sperm-positive and were weighed and recorded based on gestational days).
P1 female weekly body weight changes was statistically increased with no significant trend for week 8 at 100 mg/kg bw/day. For week 10, weight change was significantly reduced at 200 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
For P1 males, food consumption was significantly reduced at 100 and 200 mg/kg bw/day for week 3 (days 14-21); for week 6 (days 35-42), food consumption was significantly reduced at 200 mg/kg bw/day. For week 8 (days 49-56), food consumption was significantly increased at 10 (but not 100) and 200 mg/kg bw/day. For week 11 (days 70-77), food consumption was significantly increased at 10 mg/kg/day and significantly reduced at 200 mg/kg bw/day.
For P1 females, food consumption was significantly reduced for week 3 (days 14-21) at 100 and 200 mg/kg bw/day; for week 10 (days 63-70), food consumption at 200 mg/kg bw/day was significantly reduced; and for week 11 (days 70-77), food consumption at 100 mg/kg bw/day was significantly reduced.
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):
For P1 males, hematologic findings included significantly reduced red blood cell count, hemoglobin concentration and hematocrit at 100 and 200 mg/kg bw/day, significantly increased reticulocytes, mean corpuscular volume, mean corpuscular hemoglobin and corrected white blood cell count at 100 and 200 mg/kg bw/day, a significant reduction in mean corpuscular hemoglobin concentration at 200 mg/kg bw/day and significant increases in nucleated red blood cells and methemoglobin concentration at 200 mg/kg bw/day. The white blood cell differential examination indicated significant increases in segmented neutrophils and significant decreases in lymphocytes at 100 and 200 mg/kg bw/day. There were significant trends associated with the percentages of band neutrophils (increasing) and monocytes (decreasing), but no statistically significant pairwise comparisons. There were no effects on platelets.
Hematology on the P1 females indicated significant decreases in red blood cell counts, hemoglobin concentration, and hematocrit at 100 and 200 mg/kg bw/day. Nucleated red blood cell count, corrected white blood cell count (WBC), and mean corpuscular hemoglobin concentrations (MCHC) were significantly increased at 200 mg/kg bw/day. Reticulocyte counts, mean corpuscular volume and mean corpuscular hemoglobin were significantly increased at 100 and 200 mg/kg bw/day. Platelet count was unchanged across groups. Methemoglobin concentration was statistically equivalent across all groups. Differential WBC examination indicated a significant upward trend for segmented neutrophils but no significant pairwise comparisons; all other WBC types exhibited equivalent distributions across all treatment groups.
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):
At necropsy at the end of the second mating period (to produce F2b litters), P1 male body weights were significantly reduced at 200 mg/kg bw/day. Absolute spleen weights were significantly increased and absolute pituitary weights were Significantly reduced at 100 and 200 mg/kg bw/day. Relative liver weight was significantly increased at 200 mg/kg bw/day, and relative spleen weights were significantly increased at 100 and 200 mg/kg bw/day. Relative testes weights exhibited no significant pairwise comparisons but there was a significant dose-related upward trend. Relative pituitary weight was significantly reduced at 10 and 100 mg/kg bw/day but not at 200 mg/kg bw/day.
At necropsy, matemal P1 body weights were significantly reduced at 100 and 200 mg/kg bw/day. Absolute liver weight was significantly reduced at 100 (but not 200) mg/kg bw/day. Absolute spleen weights were significantly increased at 100 and 200 mg/kg bw/day. Absolute ovarian and pituitary weights were equivalent across all groups. Relative liver weight was significantly increased at 200 mg/kg bw/day. Relative spleen weights were significantly increased at 100 and 200 mg bw/kg/day. Relative ovaries and pituitary weights were equivalent across all groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
For P1 males, the only treatment- and dose-related findings concernd the spleen: slightly enlarged and dark spleen was observed in two males at 10 mg/kg bw/day, slightly enlarged spleen was observed in one male at 10 and one male at 100 mg/kg/day, an enlarged and pale spleen was observed in two males at 100 mg/kg/day and an enlarged and dark spleen was observed in 24 males at 100 and 14 males (out of 15) evaluated at scheduled necropsy at 200 mg/kg bw/day.
Gross necropsy observations for P1 females indicated that only findings relating to spleen size exhibited a treatment- and dose-related incidence and severity. One (1) F1 female at 10 mg/kg bw/day exhibited a slightly enlarged spleen. At 100 mg/kg bw/day, 13 females exhibited slightly enlarged spleens and 12 exhibited enlarged spleens. At 200 mg/kg bw/day, 18 females exhibited enlarged spleens and four (4) exhibited slightly enlarged spleens.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic evaluation of selected tissues from P1 males indicated treatment-related and dose-related lesions in the spleen and liver, including hematopoietic cell proliferation and pigment deposition in both organs and congestion in the spleen at 10, 100 and 200 mg/kg bw/day. The epididymides, pituitary, prostate, seminal vesicles and testes exhibited no treatment-related lesions.
There was an increase in the incidence of immature testes at 200 mg/kg bw/day (four at 200 mg/kg bw/day versus none at 0 or 100 mg/kg bw/day, and one at 10 mg/kg bw/day) due to the unscheduled deaths of P1 males at 200 mg/kg bw/day early in the prebreed dosing period; i.e., they were young animals with appropriately immature testes.
Histopathologic evaluation of selected tissues from P1 females indicated only splenic and hepatic findings as treatment related. In the liver, at 10 mg/kg bw/day, two females exhibited pigment deposition and 15 exhibited hematopoietic cell proliferation. At 100 mg/kg bw/day, 17 females exhibited pigment deposition, 26 exhibited hematopoietic cell proliferation, two (2) exhibited clear cell foci, and one (1) each exhibited basophilic cell foci and bile duct hyperplasia (the latter two findings were not observed at any other dose). At 200 mg/kg bw/day, 17 females exhibited pigment deposition, 18 exhibited hematopoietic cell proliferation, one (1) exhibited a centilobular fatty change and one (1) exhibited a clear cell focus. In the spleen, almost all to all of the females at 10, 100 and 200 mg/kg bw/day exhibited hematopoietic cell proliferation, congestion and pigment deposition.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Vaginal cytology of P1 females for the last three weeks of the prebreed dosing period and two weeks post-wean indicated no differences among groups for any parameters, including number (%) cycling, cycle length in days, number not cycling or with abnormal cycles. There was a significant trend across groups during the prebreed evaluation period for longer cycle length, but no significant pairwise comparisons and no evidence at all of possible lengthened cycle in the postwean period of evaluation. Females not cycling or with abnormal cycles, in general, produced live F2a or F2b litters.
Vaginal cytology for P1 females prior to the breed and subsequent to the wean of F2b litters exhibited no significant changes among groups for any parameter, including number (%) cycling, cycle length, number not cycling or with abnormal cycles. Of the 22 females which exhibited no or abnormal cycles, 20 did not produce litters and two (one each at 100 and 200 mg/kglday) produced F2b (but not F2a) litters.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproductive and lactational indices for P1 parents and F2a litters exhibited no treatmentrelated changes, including no changes in mating index, fertility index (either sex), gestational index, gestational length in days, number of implantation sites per litter, number of total, live or dead pups on postnatal day 0, prenatal mortality index of F2a offspring, stillbirth index, live birth index, 4, 7, 14 and 21 survival indices or in lactational index.
Reproductive and lactational indices of F2b litters exhibited no treatment- or dose-related changes across groups; if anything, indices were better at 200 mg/kg bw/day than at lower doses or at 0 mg/kg bw/day. No effects of treatment were observed on mating index, fertility index (males or females), gestational length, number of implantation sites, total, live or dead pups per litter, gestational index, prenatal mortality index, stillbirth index, live birth index, day 4, 7, 14, and 21 survival indices and lactational index.

Details on results (P1)

Gestation - P1 females for F2a litters:
P1 matemal gestational body weights (during the first mating) were equivalent across all groups at all timepoints evaluated; matemal gestational weight change (gd 0-20) was similarly equivalent across groups. Maternal food consumption during gestation, expressed as g/day, exhibited a significant reduction at 100 (but not 200) mg/kg bw/day for gd 0-7, no significant differences for gd 7-14 or 14-20 and significant reductions at 100 and 200 mg/kg bw/day for the gestation period, gd 0-20.
P1 clinical observations during gestation (of F2a litters) which were treatment- or dose-related were limited to rooting (in bedding) in 1-6 dams at 100 and 200 mg/kg bw/day almost daily and chromodacryorrhea in one female each at 100 and 200 mg/kg bw/day.

Lactation - P1 females for F2a litters:
Maternal P1 body weights during lactation of their F2a litters exhibited significant reductions at 100 and 200 mg/kg bw/day on postnatal day 0 and a significant reduction at 200 mg/kg bw/day on postnatal day 7, with no significant differences among groups on postnatal days 4, 14 and 21. Maternal lactational weight change exhibited a significant upward trend across groups but no significant pairwise comparisons. Maternal P1 lactational food consumption, expressed as g/day,
was equivalent across all groups for all intervals evaluated throughout the lactational period (postnatal days 0-21). Maternal F1 lactational food consumption, expressed as g/kg bw/day, was significantly increased at 200 mg/kg bw/day for postnatal days 0-4 and equivalent across all groups for all other intervals; consumption for the lactational period (postnatal days 0-21) exhibited a significant upward trend across groups but no significant pairwise comparisons.
Maternal P1 clinical observations during the lactation period and vaginal cytology evaluation period (of F2a litters) indicated only rooting (in bedding) at 100 (1-2 animals occasionally) and 200 mg/kg bw/day (1-5 animals almost daily), and diarrhea/pica, audible, labored respiration and gasping (one or a few animals on occasional days) and two deaths (one female each on day 13 and 14 during vaginal cytology evaluation period) at 200 mg/kg/day as treatment-related.

Gestation - P1 females for F2b litters:
P1 females who did not produce F2a litters were rebred to P1 males who were not successful sires to produce F2b litters. A total of 16, 12, 11 and six (6) F1 females were rebred at 0, 10, 100 and 200 mg/kg bw/day, respectively. A total of two (2), four (4), three (3) and three (3) litters at 0, 10, 100 and 200 mg/kg bw/day, respectively, resulted from this rebreed.
P1 maternal gestational body weights, gestational weight gain (gd 0-20) and maternal gestational food consumption. expressed a glday or glkg/day were all statistically equivalent across all groups for all timepoints or intervals evaluated.
Clinical observations of P1 females during the rebreed indicated only rooting (in bedding); post dosing (one female occasionally) as treatment related, observed only at 200 mg/kg bw/day.

Lactation - P1 females for F2b litters:
P1 matemal lactational body weights, lactational weight change, and food consumption (g/day and glkg/day) were all statistically equivalent across all groups for all timepoints or intervals evaluated. P1 maternal treatment-related clinical signs during lactation were limited to dams at 200 mg/kg/day, including audible, labored respiration, gasping, pale and demise of one female (sacrificed in extremis) on lactation day 3.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
> 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (P1)

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations of F1 pups during the lactation period indicated one (1) cyanotic male pup on postnatal day 0 at 200 mg/kglday, five (5) females and three (3) males on postnatal day 14 with thin fur at 100 mg/kg bw/day, and one (1) male with a missing tail (most probably traumatic) on day 1 at 10 mg/kg bw/day
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects 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:
effects observed, treatment-related
Description (incidence and severity):
Hematologic evaluation of F1 weanlings, 10/sex/dose, incieated, for F1 males, a significant reduction in platelet count, and a significant increase in mean corpuscular volume at 200 mg/kg bw/day. In addition, an apparent increase in methemoglobin concentration was observed at 100 (but not 200) mg/kg bw/day; hemoglobin concentration was significantly reduced at 100 (but not 200) mg/kg bw/day, and mean corpuscular volume was significantly increased at 10 (but not 100) mg/kg bw/day. There were no effects on any other hematologic parameters, including WBC differential. For F1 females, there were no significant treatment-related findings; corrected white blood cell count was significantly increased at 10 (but not 100 or 200) mg/kg/day.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross necropsy findings of F1 pups which died during the lactation period indicated no apparent treatment-related findings; most deaths occurred during the first four days of life,
predominantly with primary atelectasis (defective expansion of the pulmonary alveoli at birth), patent ductus arteriosus and no milk in stomach.
Histopathological findings:
no effects observed
Description (incidence and severity):
Histopathologic examination of inguinal mammary glands from unselected F1 female weanlings indicated no lesions observed.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

The number of litters are clearly reduced at 200 mg/kg bw/day due to the demise of 11 F0 females and six F0 males prior to the mating period. Prenatal mortality index and stillbirth index were statistically equivalent across all groups; the values for these parameters were apparently increasad at 200 mg/kg bw/day and possibly at 100 mg/kg bw/day for prenatal mortality. Live birth index and day 4 survival index were statistically equivalent across all groups with an apparent decrease at 200 mg/kg bw/day.
Day 7, 14, and 21 survival indices and lactation index were equivalent across all groups. Litter size on postnatal days 0, 1, 4, 7, 14 and 21 and sex ratio (% males) per litter and pup body weights per litter for all pups and males and females separately on postnatal days 1, 4, 7, 14. and 21 were all statistically equivalent across all groups. Pup body weights per litter on days 14 and 21 (all pups and males and females .separately) exhibited significant downward trends with no significant pairwise comparisons.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
reproductive and postanatal toxicity
Generation:
F1
Effect level:
> 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects at highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical observations of F2a pups during lactation indicated no treatment-related signs.
Clinical observations for F2b pups during lactation indicated no treatment- or dose-related findings.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
For F2b pups, at 100 mg/kg bw/day, five pups died during lactation and two pups died at 200 mg/kg bw/day.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
F2a pup body weights per litter (all pups, or males and females separately) were all equivalent across groups for all timepoints evaluated, on postnatal days 0,1,4, 7, 14 and 21.
F2b pup body weights per litter (total or separately by sex) were all equivalent across all groups for F2b litters at all timepoints evaluated during the three week lactation period.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Hematology data from F2a weanlings, 10/sexldose, exhibited no significant treatment-related changes in any parameter in either sex; there was a significant dose-related downward trend for platelet count in both males and females and a significant upward trend for mean corpuscular volume in females but no significant pairwise comparisons for either parameter. There was also an apparent increase in methemoglobin concentration in F2a female weanlings at 200 mg/kg bw/day (and possibly at 100 mg/kg bw/day), which were not statistically significant.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross necropsy observations of F2a pups which died during lactation also indicated no treatment-related findings.
Gross necropsy observations of F2b pups were limited to pups which died during lactation at 100 (five pups) and 200 (two pups) mg/kg/day. These pups exhibited no dose related, unique or unusual findings.
Histopathological findings:
no effects observed
Description (incidence and severity):
Histopathology of inguinal mammary glands from F2a and F2b female weanlings indicated no lesions present in any evaluated animal.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

F2a litter sizes and sex ratio (% male pups per litter) were all equivalent across groups for all timepoints evaluated, on postnatal days 0,1,4, 7, 14 and 21.
F2b litter sizes and sex ratio (% male pups per litter) were all equivalent across all groups for F2b litters at all timepoints evaluated during the three week lactation period.

Effect levels (F2)

Dose descriptor:
NOAEL
Remarks:
reproductive and postnatal toxicity
Generation:
F2
Effect level:
> 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects at highest dose tested

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the presence of overt parental toxicity at 100 and 200 mg/kg bw/day and of minimal parental toxicity at 10 mg/kg bw/day, there were no treatment-related effects on rat parental reproductiive activity or reproductive organ histology or on any offspring parameters assessing pre- and postnatal survival and growth for two generations. In this study, a NOAEL for adult rat toxicity was not established. However, the rat NOAEL for reproductive and postnatal toxicity was at least 200 mg/kg bw/day.