Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
repeated dose toxicity: oral
Remarks:
other: One Generation Oral Dietary Reproduction Toxicity Study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Between 18 July 2001 and 17 April 2002.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, No.415 “One Generation Reproduction Toxicity Study” (adopted 26 May 1983).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description: clear amber coloured liquid
Date received: 6/7/2001, provided by Chevron Research and Technology Company
Expiry date: 05/01/2002

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: from Charles River Laboratories, Inc., Kingston, New York,
- Age and weight at study initiation: the males were approximately six weeks of age with body weights ranging from 134 to 166 grams, while the females were approximately seven weeks of age with body weights ranging from 163 to 204 grams.
- Fasting period before study: Not applicable
- Housing: Males were housed two or three per cage for three days to allow the animals to adapt to the automatic watering system. The males were housed individually for the remainder of acclimation and while on study (except during cohabitation) in suspended stainless steel cages. Females were housed individually during acclimation and while on study (except during cohabitation) in suspended stainless steel cages.
During the mating phase, animals were paired on a one male: one female basis within each dose group in polypropylene cages.
Following mating the males were re-housed in their original cages and the females were individually housed in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding for the gestation, birth and lactation phases of the study.
- Diet and water: PMI Certified Rodent Chow@ #5002 (Purina Mills, Inc.) and municipal tap water were provided to each animal ad libitum.
- Acclimation period: Males were acclimated to the laboratory conditions for a period of 16 days prior to in-life initiation. Females were acclimated to the laboratory conditions for a period of 22 days prior to in-life initiation.

ENVIRONMENTAL CONDITIONS
- Temperature: 65 to 79 °F (18 to 26 °C)
- Humidity: 30 to 70 %,
- Air changes (per hr): 10 to 15 air changes per hour.
- Photoperiod (hr dark / hrs light): 12 hours continuous light and 12 hours darkness

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Remarks:
Basal laboratory diet
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- For each test article dose group, a specified amount of HPV-1 (CAS #67124-09-8) was weighed into a pre-calibrated beaker. A sufficient quantity of corn oil was added to the beaker to achieve the desired volume and the mixture was stirred for 30 minutes. Each test article dosing mixture was prepared fresh weekly and stored at ambient conditions for a maximum of one week. During each preparation, corn oil was dispensed as received into daily aliquots for administration to control animals. The physical state of the control and each dosing solution was recorded during each preparation. The vehicle (group 1) was a clear yellow liquid, groups 2 and 3 were clear yellow solutions and group 4 was a clear dark yellow solution. The dosing solutions were stirred prior to dispensation and continuously until dosing was complete. The Study Director visually inspected the first preparation prior to initiation of dosing and found the mixtures acceptable for dosing.

DIET PREPARATION
- Not applicable

VEHICLE
- Concentration in vehicle: 50, 167 and 500 mg/kg/day groups
- Amount of vehicle (if gavage): 2.5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity, Stability and Concentration Analyses:
Prior to the first day of dosing, samples were collected for homogeneity and stability analyses from the three test article preparations. Three 5 mL samples each were collected from the top, middle and bottom of each concentration (for a total of 27 aliquots). Two of the three sets of samples were sent to Chevron Research and Technology Company, lntegrated Laboratory Technologies, under ambient conditions for analysis of homogeneity and stability over a seven-day period. The remaining set of samples was stored at SLI under ambient conditions. For each dosing preparation, including the vehicle control, two 5 mL samples were taken from each dose level (middle stratum) for concentration analysis. One sample from each dose level was sent to Chevron Research and Technology Company, lntegrated Laboratory Technologies, under ambient conditions for analysis of concentration. The remaining sample from each dose level was stored at SLI under ambient conditions.

Results of homogeneity analyses indicate that the test article was homogeneous in the vehicle at concentrations of 20, 66.8 and 200 mg/mL and stable for more than one week when stored under ambient conditions. Results of concentration analyses verified the appropriate concentration of test article in the dosing mixtures.
Duration of treatment / exposure:
Oral administration of HPV-1 (CAS #67124-09-8) to F0 male rats for a minimum of 70 days prior to mating and until completion of parturition
F0 females were treated for a minimum of 14 days prior to mating through lactation day 20.
Frequency of treatment:
once daily oral gavage
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 167 and 500 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
28 animals per sex per group


Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected by the Sponsor Monitor in an attempt to produce graded responses to the test article. The high-dose level was expected to produce some toxic effects, but not excessive lethality. The mid-dose level was expected to produce none to minimal observable effects and the low-dose level was expected to produce no observable effects.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Random
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS:
- Yes
- Time schedule: Mortality/general health checks were performed twice daily, in the morning and afternoon. Detailed clinical observations were performed weekly and cage-side observations were performed daily approximately one-half hour to two hours following dosing. During gestation and lactation, detailed clinical observations were performed daily for FO females. Detailed clinical observations were also performed on the day of scheduled euthanasia.

NEUROBEHAVIOURAL EXAMINATION: Not applicable

BODY WEIGHT:
- Time schedule for examinations: Individual body weights were recorded weekly and on the day of scheduled euthanasia for the males. Individual body weights were recorded weekly for the females until evidence of mating was observed. When positive evidence of mating was detected, the females were weighed on gestation days 0, 7, 14 and 21. Following parturition, the females were weighed on lactation days 1, 4, 7, 14 and 21.

FOOD CONSUMPTION:
- lndividual food consumption was recorded on the same days as body weights for the males and females, except during cohabitation and lactation

OPHTHALMOSCOPIC EXAMINATION:
Not applicable

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes.
- Parameters: Erythrocyte count (RBC), Hematocrit (Hct), Hemoglobin concentration (Hgb), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Platelet count, Reticulocyte count (reticulocyte slides were prepared but were not examined since no test article changes were noted in the erythrocyte parameters), total and differential leukocyte counts
- Time schedule for collection of blood: Blood samples for hematology analysis were collected from ten males/group prior to scheduled euthanasia on study day 117/118 and from ten females/group prior to scheduled euthanasia on lactation day 21.
- Anaesthetic used for blood collection: Blood samples were obtained via the ocular orbital plexus while the animals were under light isoflurane anesthesia.
Sacrifice and pathology:
SACRIFICE
- Male animals: Surviving F0 males were euthanized by carbon dioxide inhalation followed by exsanguination and necropsied after completion of female parturition.
- Maternal animals: F0 females that delivered were euthanized by carbon dioxide inhalation followed by exsanguination and subjected to a complete gross necropsy examination on lactation day 21. Females that failed to deliver were euthanized 25 days after evidence of mating was first detected (post breeding day 25). Females with total litter loss were euthanized and necropsied on the day that no surviving pups remained.

GROSS NECROPSY / ORGAN WEIGHTS
- Scheduled Euthanasia and Necropsy
All necropsy examinations included examination of the external surfaces of the body, all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents. Uterine contents were examined and the number of implantation scars was recorded. Uteri with no macroscopic implantations were opened and placed in 10% aqueous ammonium sulfide solution for detection of early embryo lethality as described by Salewski et al [1964]. All abnormalities were recorded.
Fresh organ weights were obtained at scheduled euthanasia for the brain, liver, prostate and left cauda epididymis. Paired organ weights were obtained for the testes, epididymides, seminal vesicles and kidneys. The following organs were preserved for histopathological examination: Gross lesions, Liver, Kidneys, Brain, Ovaries, Pituitary, Prostate, Right epididymis (and corpus and caput of left epididymis), Testes (preserved in Bouin's fixative for 48 to 96 hours, rinsed and then retained in 10 % neutral buffered formalin), Seminal vesicles (with coagulating glands), Uterus (with oviducts and cervix), Vagina
- F0 Unscheduled Deaths and Necropsy
All animals found dead or euthanized for humane reasons were subjected to a complete gross necropsy examination at the time of death or euthanasia. When applicable, the animals were euthanized by carbon dioxide inhalation. The necropsy examination included evaluation of the external surfaces of the body, all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents. Uterine contents were examined and the number of implantation scars were recorded. All abnormalities were recorded and the following tissues were preserved in 10 % neutral buffered formalin for histopathological examination: Accessory genital organs (epididymides, seminal vesicles and prostate or uterus and vagina), Adrenals, all gross lesions Brain Cecum Colon Duodenum Esophagus Heart Ileum, Jejunum Kidneys Liver (three sections collected) Lungs (infused with formalin) with bronchi Mammary gland Pancreas Pituitary, spinal cord, spleen, stomach, testeslovaries, thymus, thyroidlparathyroid, trachea, urinary bladder

HISTOPATHOLOGY
All tissues collected at necropsy from control and high-dose animals and from animals found dead or euthanized for humane reasons were processed for histopathological examination. The tissues were trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Histology was performed by HistoTechniques, Powell, Ohio, and the tissues were examined microscopically by Dr. William H. Baker, a board certified veterinary pathologist.
Other examinations:
Pregnancy and Parturition
The following was recorded for each female:
i) Date of pairing/mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

Litter Data
F1 Standardization of Litter Size
Observations and body weights on lactation day 4, each litter was randomly culled to a maximum of eight pups, four per sex per litter, when possible.
F1 Litter Data
Pup viability was determined daily throughout lactation. A detailed examination of each pup was petformed on lactation days 0, 4, 7, 14 and 21. The sex of each pup was determined on lactation day 0 and verified on lactation days 4, 7, 14 and 21. Individual pup weights were determined on lactation days 1, 4, 7, 14 and 21.


Sperm
Parameters examined in F0 male parental generations:
Sperm was collected from all males euthanized at scheduled necropsy for sperm count, concentration, motility and morphology assessment. All groups were analyzed. Sperm count, concentration and motility analyses were performed with a computer-assisted sperm analyzer (Hamilton-Thorne IVOS 10) and sperm morphology was assessed manually by trained personnel. The left cauda epididymis was thoroughly homogenized and a sample of the resulting homogenate was analyzed to determine cauda epididymal sperm number and concentration. The cauda sperm counts were expressed in terms of cauda weight, i.e., Millions (M)l(g) left cauda weight. Cauda sperm concentration was expressed as M/mL. Sperm motility (%) was assessed from sperm samples collected from the vas deferens. Samples for motility evaluation were recorded to an optical disk at the time of necropsy and subsequently analyzed. Sperm morphology was assessed by microscopic examination of a minimum of two hundred sperm per animal at 300-500x magnification. Morphological endpoints were based primarily on head and tail abnormalities, according to a modification of the classification systems described by Linder et al (1992) and Seed et al (1996) The mean percentage of normal sperm was calculated for each group.
Statistics:
Data, including body weights, body weight gain, food consumption, gestation length, mean live litter size and sperm parameters, were analyzed by ANOVA. If significance was observed with ANOVA, control to treatment group comparisons were performed using Dunnett's test. Count data were analyzed using R x C Chi-Square test followed by Fisher's Exact Test for copulation and fertility indices, pup sex ratios, the number of live and dead pups per group (on lactation day 0) and pup survival (after lactation day 0). Absolute and relative organ weights were analyzed for homogeneity of variance using Bartlett's test. If significance was detected with Bartlett's test (p < 0.01), multiple group comparisons proceeded using the Kruskal-Wallis non-parametric ANOVA, followed by Dunn's test, when p < 0.05. If significance was not detected with Bartlett's test, parametric procedures were used to analyze the data, i.e.,ANOVA followed by Dunnett's test when pc0.05. Hematology data were analyzed for homogeneity of variance using Levene's test (p < 0.01). If significance was detected with Levene's test (p<0.01), multiple group comparisons proceeded using the Kruskal-Wallis non-parametric ANOVA, followed by Dunn's test, when p < 0.05. If significance was not detected with Levene's test, parametric procedures were used to analyze the data, i.e., ANOVA followed by Tukey-Kramer test when p < 0.05. Statistical analyses regarding the hematology data were performed using the SLI Alpha GenTox computer system. All other statistical analyses were performed using the SLI Alpha ReproTox computer system. All analyses were two-tailed with a minimum significance level of 5 % (p < 0.05).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One F0 male (#5463) in the control group was euthanized for humane reasons due to swelling and impaired mobility of the left forelimb on study day 72 and one F0 male (#5413) in the 167 mg/kg/day group was euthanized for humane reasons due to severe malalignment on study day 65. All other F0 males in the control, 50, 167 and 500 mg/kg/day groups survived to scheduled euthanasia at study termination.
One F0 female (#1347) in the 50 mg/kg/day group was found dead on gestation day 22. Two F0 females (#I420 and #1423) in the 50 mg/kg/day group had total litter loss and were euthanized on lactation day 0. Total litter loss has been observed in control animals at SLI and was not considered treatment related since it did not occur in the 167 and 500 mg/kg/day groups. In addition, three F0 females (#1379, #I404 and #1421) in the control group, one F0 female (#1400) in the 50 mg/kg/day group, one F0 female (#1434) in the 167 mg/kg/day group and two F0 females (#I353 and #1439) in the 500 mg/kg/day group had evidence of mating but failed to deliver and were euthanized 25 days after evidence of mating was detected. All other F0 females in the control, 50, 167 and 500 mg/kg/day groups survived to scheduled euthanasia.
Prior to death on gestation day 22, clinical signs for F0 female #I347 in the 50 mg/kg/day group included ocular discharge, dark material around the eyes and nose, reddish colored urine stain, fecal stain and piloerection. The death of this animal was not considered to be test article related since no mortality occurred for F0 females in the 167 and 500 mg/kg/day groups. However, a definitive cause of death could not be determined.
The most remarkable clinical signs observed for surviving F0 males included a low incidence of salivation prior to dosing and a dose-related increase in post-dose salivation in the 167 and 500 mg/kg/day groups. An increased incidence of hairloss was also observed for F0 males in the 50, 167 and 500 mg/kg/day groups compared to controls; however, the finding did not follow a dose response. A low incidence of clinical signs including scab(s), dark material around the eyes, and dark material around the nose was observed for F0 males in each study group, including controls.
Clinical signs were observed for surviving F0 females in the 50, 167 and 500 mg/kg/day groups. The clinical signs included a low incidence of reddish vaginal discharge, swelling (mammary glands), dark material around the eyes and dark material around the nose in the 50, 167 and 500 mg/kg/day groups; a low incidence of salivation prior to dosing, an increased incidence of urine stain and a dose-related increase in post-dose salivation in the 167 and 500 mg/kg/day groups; and a low incidence of ocular discharge in the 500 mg/kg/day group. A low incidence of clinical signs including soft stools and scab(s) was also observed for F0 females in each study group, including controls.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights of F0 males in the 500 mg/kg/day group were approximately 5-7 % lower than controls beginning on study day 42 and continuing throughout the study (day 119); the differences were statistically significant on study days 42-1 12. In addition, mean body weight change of F0 males in the 500 mg/kg/day group was statistically lower than controls (approximately 17-60 %) on study days 14-21, 28-42, 91-98 and 112-1 19. In contrast to the above changes, mean body weight change of F0 males in the 500 mg/kg/day group was statistically higher than controls during study days 70-77.
There were no toxicologically meaningful differences in mean body weights or body weight change for F0 males in the 50 and 167 mg/kg/day groups. Mean body weights of F0 males in the 50 and 167 mg/kg/day groups were comparable to controls throughout the study. In addition, with the exception of statistically lower mean body weight change for F0 males in the 50 mg/kg/day group during days 63-70 and statistically higher mean body weight change for F0 males in the 50 and
167 mg/kg/day groups during days 70-77, mean body weight change of F0 males in the 50 and 167 mg/kg/day groups was comparable to controls throughout the study. A graph of mean body weights for F0 males is included below:
There were no toxicologically meaningful differences in mean body weights or body weight change for F0 females in the 50, 167 and 500 mg/kg/day groups prior to mating or during gestation or lactation. With the exception of statistically higher mean body weight change for F0 females in the 500 mg/kg/day group during study days 0-7, mean body weights and body weight change of F0 females in the 50, 167 and 500 mg/kg/day groups were comparable to controls throughout the study.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute and relative (to final body weight) kidney and liver weights of F0 males in the 167 and 500 mg/kg/day groups were statistically higher than controls at study termination. These differences were not considered to be toxicologically meaningful since they did not correlate with any toxicologically significant test article-related microscopic changes in the kidney or liver. However, the higher kidney weights may be associated with nephropathies occurring in the rat that were not considered toxicologically meaningful.
Absolute and relative (to final body weight) liver weights of F0 females in the 500 mg/kg/day group were statistically higher than controls at study termination. These differences were not considered to be toxicologically meaningful since they did not correlate with any toxicologically significant test article-related microscopic changes in the liver.
No statistically significant or toxicologically meaningful differences in absolute or relative weights were noted in the F0 male or female reproductive or accessory organs.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Gross necropsy findings for F0 male #5463 in the control group euthanized for humane reasons due to impaired mobility of the left forelimb included swollen left forelimb with subcutaneous edema (no fracture) and whitish-yellow mucoid material in the jejunum. Gross necropsy findings for F0 male #5413 in the 167 mg/kg/day group euthanized for humane reasons due to severe malalignment included fractured rostrum, including nasal bones and hard palate, dark brown material mixed with normal ingesta in the stomach and dark red thymus.
Gross necropsy findings for F0 female #I 347 in the 50 mg/kg/day group found dead on gestation day 22 included mottled lungs (dark red and red), white foam the entire length of the trachea, multiple black foci on the glandular stomach mucosa and yellow mucoid material in the small intestine and colon. A definitive cause of death could not be determined.
Gross necropsy findings for F0 female #I353 in the 500 mg/kg/day group that failed to deliver and was euthanized 25 days after evidence of mating was detected included dark brownish-red fluid in the uterus and vagina and one small placenta and two nonviable pups in the vagina. No remarkable findings were observed at necropsy for the other F0 females that failed to deliver and were euthanized post breeding day 25. Gross necropsy findings for two F0 females in the 50 mg/kg/day group that were euthanized due to total litter loss (#I420 and #1423) included fetal tissue mixed with ingesta in the stomach, red mucoid material in the vagina and two retained pups each (one in each horn of each female).
No remarkable gross necropsy findings were noted for F0 males or females in the control, 50, 167 and 500 mg/kg/day groups that survived to scheduled euthanasia at study termination.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No toxicologically meaningful microscopic lesions were observed in any of the tissueslorgans examined from this study.
Microscopic evaluation revealed minimal to mild hyaline droplet accumulation in 27 group 4 males. Hyaline droplets can be induced by a variety of chemical compounds in certain strains of male rats and are frequently associated with the accumulation of a*,-globulin within the hyaline droplets. A progressive nephropathy accompanies hyaline droplet accumulation, highlighted early on by tubular epithelial degenerationlregeneration and tubular dilatation with granular casts. Hyaline droplet accumulation and the associated nephropathy in male rats are not considered toxicologically significant for humans. Renal tubular degenerationlregeneration, tubular dilatation and inflammation were observed in control animals and group 4 females. These findings are consistent with early signs of chronic progressive nephropathy, a spontaneous, progressive, age-related entity.

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
167 mg/kg bw (total dose)
Based on:
test mat.
Remarks:
systemic toxicity
Sex:
male/female
Basis for effect level:
other: based on decreased body weights in the 500 mg/kg bw dose group.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Histopathologic Findings

No toxicologically significant test article-related lesions were noted in the tissues examined.

Although kidney observations are listed separately under left and right kidneys, findings are presented as combined data for both kidneys. Minimal to mild hyaline droplet accumulation was present in 27 Group 4 males (96%). Other prominent renal changes including tubular epithelial degeneration/regeneration, tubular dilatation and inflammation were noted in males and females from Groups 1and 4. The incidences of these lesions varied by sex and dose group.

A nephroblastoma was noted in the left kidney of a Group 4 female (1353). The mass was approximately 5 x 8 mm, was well-delineated and occupied the cortical and medullary space at one pole of the kidney, well within the limits of the normal structure.

A dermal mass observed grossly in a Group 4 female (1403) was diagnosed as a mammary carcinoma.

SUMMARY

The renal lesions noted in the control and treated animals represent changes associated with hyaline droplet nephropathy (HDN) and/or chronic progressive nephropathy (CPN).

Hyaline droplets, as noted in the Group 4 males, can be induced by a variety of chemical compounds in certain strains of male rats and is frequently associated with the accumulation of a2m-globulin within the hyaline droplets. A progressive nephropathy accompanies hyaline droplet accumulation, highlighted early on by tubular epithelial degeneration/regeneration and tubular dilatation with granular casts. Although histological findings in this study are consistent with a2m -globulin, specific identification of a,,-globulin has not been done. Definitive diagnosis of a2m - globulin is academic in that its presence and the associated nephropathy (HDN) In male rats is not considered toxicologically significant for humans according to Environmental Protection Agency Risk Assessment Forum (Baetck K.P, et al 1991).

The renal tubular degeneration/regeneration, tubular dilatation and inflammation, as observed in control animals and Group 4 females, are consistent with the constellation of lesions representing early signs of Chronic Progressive Nephropathy (CPN), a spontaneous, progressive, age-related entity. CPN is most common and prominent in male rats, as noted in this study.

The increased incidence of tubular changes and inflammation in Group 4 males is interpreted and the dual expression of both HDN and CPN.

Nephroblastomas occur rarely in young adult rats, with an incidence of 0 -0.4% (Mesfin, G.M. 1999). This was interpreted as an incidental finding and not considered treatment related.

The most common spontaneous neoplasm in aged Sprague-Dawley females are of mammary gland origin. Incidence of mammary carcinomas is 8.8% according to Chandra, et. al.(Chandra M, 1992). This lesion was interpreted as spontaneous and was not considered test article related.

Other changes noted were considered to be normal findings in rats of this age and strain.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, a dosage level of 167 mg/kg/day was considered to be the NOAEL for parental (F0) toxicity.
Executive summary:

Introduction

This study was conducted to evaluate the potential effects of oral administration of HPV-1 (CAS #67124-09-8) on the integrity and performance of the reproductive system in male and female rats, including gonadal function, mating behavior, conception, gestation, parturition, lactation and weaning. This study was also conducted to provide preliminary information concerning the effects of the test substance on neonatal moribundity, mortality and postnatal developmental toxicity. This standardized procedure was conducted according to OECD guidance 415.

Methods

The study consisted of a vehicle control and three treatment groups, with 28 males and 28 females in each group. HPV-1 (CAS #67124-09-8) was dissolved in corn oil and administered at dosage levels of 50, 167 and 500 mg/kg/day, by once daily oral gavage, to F0 parental animals. All doses were given at a constant volume of 2.5 mL/kg. Control animals were administered corn oil under the same experimental conditions at an equivalent dose volume.F0 males were treated for a minimum of 70 days prior to mating and until completion of parturition. F0 females were treated for a minimum of 14 days prior to mating through lactation day 20.

Both F0 parental animals and F1 offspring were closely examined for indications of toxicity. Experimental endpoints for F0 animals included clinical observations, body weights, food consumption, mating, parturition, lactation, hematology determinations and offspring growth and viability. All F0 and F1 animals were subjected to a gross necropsy examination at the time of death or terminal euthanasia.

Results

Oral administration of the test substance to F0 male rats for a minimum of 70 days prior to mating and until completion of parturition produced clinical signs including salivation prior to dosing and a dose-related increase in post-dose salivation in the 167 and 500 mg/kg/day groups and lower mean body weights (reduced approximately 5-7 % compared to controls) in the 500 mg/kg/day group.

Mean food consumption of F0 males in the 50, 167 and 500 mg/kg/day groups was comparable to controls throughout the study. There were no toxicologically meaningful differences between the control, 50, 167 and 500 mg/kg/day groups with respect to the F0 male mating and fertility indices. The F0 male hematology parameters, F0 male gross necropsy observations, F0 male absolute or relative organ weights or the F0 male sperm parameters were evaluated and no statistically significant differences were observed. In addition, no toxicologically meaningful microscopic lesions were observed in any of the F0 male tissues/organs examined from this study.

Oral administration of the test substance to F0 female rats for a minimum of 14 days prior to mating and throughout lactation produced clinical signs for F0 females in the 50, 167 and 500 mg/kg/day groups. The clinical signs included a low incidence of reddish vaginal discharge, swelling (mammary glands), dark material around the eyes and dark material around the nose in the 50, 167 and 500 mg/kg/day groups; a low incidence of salivation prior to dosing, an increased incidence of urine stain and a dose-related increase in post-dose salivation in the 167 and 500 mg/kg/day groups; and a low incidence of ocular discharge in the 500 mg/kg/day group.

There were no toxicologically meaningful differences between the control, 50, 167 and 500 mg/kg/day groups with respect to F0 female mean body weights, body weight change, food consumption, mating and fertility indices, precoital intervals, gestation lengths or the hematology parameters evaluated. Gross necropsy findings for one F0 female in the 500 mg/kg/day group euthanized on post breeding day 25 included dark brownish-red fluid in the uterus and vagina and one small placenta and two nonviable pups in the vagina. No other remarkable findings were noted in the F0 females at necropsy and no toxicologically meaningful microscopic lesions were observed in any of the F0 female tissues/organs examined from this study.

No toxicologically meaningful differences were noted in F1 pup viability during lactation. Mean F1 pup weights were statistically lower than controls in the 167 mg/kg/day group on lactation day 14 and in the 500 mg/kg/day group on lactation days 14 and 21; however, mean F1 pup weights in these groups remained within the range of the test laboratory historical control data. With the possible exception of a slight increase in the incidence of pups cool to the touch, no remarkable F1 pup observations were noted during lactation. No remarkable findings were noted at necropsy for F1 pups stillborn, found dead, culled on day 4 or euthanized on lactation day 21.

Summary of all observed toxicological effects were summarized in the following table.

 

F0 – Systemic Effects

F0 - Reproduction

F1 - Development

Control

-

-

-

50 mg/kg/day

Nothing remarkable

Nothing remarkable

Live born pups is lower

167 mg/kg/day

Post dosing salivation, urine stain

Nothing remarkable

Mean pup weights were lower (P < 0.05), but remained within the range of the test facilities historical control data.

500 mg/kg/day

Post dosing salivation, lower body weight, oculars discharge

Nothing remarkable

Mean pup weights were lower (P < 0.05), but remained within the range of the test facilities historical control data

Conclusions

According to the study director, a dosage level of 50 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for parental (FO) toxicity. There were no indications of impaired fertility or other reproductive effects in the F0 males or females at dosage levels up to 500 mg/kg/day. A dosage level of 50 mg/kg/ay was considered the no-observed-adverse-effect level (NOAEL) for developmental effects, as a result of decreased pup weights noted for the 167 and 500 mg/kg/day group pups during the latter half of lactation.

The study results were re-examined and new NOAELs for for parental and developmental toxicity were determined. Comparing to 50 mg/kg/day, the additional clinical observations included salivation and urine stain in females treated with 167 mg/kg/day test substance. Because 1) salivation was considered to be a reflex to administration of the test substance, 2) urine staining did not correlate with urination pattern or kidney gross necropsy, and 3) the severity of the effects is limited.

Based on the decreased body weight gains in F0 animals treated with 500 mg/kg/day, a dosage level of 167 mg/kg/day was considered to be the NOAEL for parental (F0) toxicity.

A dosage level of 167 mg/kg/day was considered the NOAEL for developmental effects, since the decreased pup weights noted for the 167 and 500 mg/kg/day group pups during the latter half of lactation were within the range of historical control data.