Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, unpublished report available, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Kryocide (Cryolite); Sodium aluminofluoride
Supplier: Elf Atochem North America, Inc.
Batch number: 86.12
Purity: 96.0%

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Test animals: albino rat
Strain: CD (Sprague Dawldey derived)
Age at receipt: 30 days
Supplier: CharlesR iver Laboratories, Inc. Portage, Michigan 49081
Animals were acclimated for approximately two weeks.
Housing: Suspended, stainless steel, wire mesh bottom cages. Animals were housed individually throughout the study with the following exceptions: the first week of acclimation( two animals/sex/cage); mating (one male and one female/cage overnight); lactation( dam and litter/cage) and post-weaninogf the FI pups (one- two llttermates/sex/cage until formali nitiation of the pre-matingt reatmentp eriod which followed weaning of the last litters).
Feed and water: Ad libitum. Certified Rodent Diet No. 5002 (mash) was provided fresh at least weekly during the study. Analysis of each feed lot used during this study was performed by the supplier (Purina Mills, Inc. St. Lous, MO).

Environmental conditions: 12 hour light/dark cycle, actual temperature: 64-80°F (18-27°C), actual relative humidity: 24-79%.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Appropriate amounts of the test substance were mixed with the basal granulated diet to achieve the desired concentrations (prepared at a fixed concentration [ppm] in the diet and no correction for purity of the test substance was used). Fresh diets were prepared and presented to animals weekly during the study. Control animals received standard laboratory diet only. Unused portions of diet (control and treated) were stored at ambient temperature during intervals when not being presented to animals.

Prior to the initiation of the study, full-size (mock) batches of diets at the low- and high concentration levels( 200 and 1800 ppm, respectively) were evaluated to determine that diets prepared using the proposed mixing procedure were homogeneous. To determine homogeneity, three randomly drawn diet samples (approximately 100 grams each) were taken from each mix at each of three levels (top, middle, bottom - total of nine samples) in the mixer. If the data demonstrated that the mean of the values for the three levels were within ±15% of each other and ±20% of the nominal (desired) concentration, the batches were considered homogeneous.

In previous studies conducted in this Testing Facility with dietary administration of Kryocide, the stability of the Kryocide in the diet for 14 days post-preparation at ambient storage was established at dose levels that ranged from 50 to 5000 ppm. On the basis of these stability data, the only additional stability assessment performed for this study was a 21-day stability at ambient storage. These analyses were performed at the low- and high- concentrations (200 and 1800 ppm, respectively) for diets prepared to establish homogeneity. Following 21 days of storage at ambient temperature, two samples of approximately 100 grams each were collected at each concentration level and analyzed.
Details on mating procedure:
Initially, one male was co-housed with one female from the same treatment group nightly until evidence of mating was observed or for seven consecutive days. Each morning following cohabitation with the male, the female was evaluated for evidence of mating (microscopic observation of sperm in the vaginal smear and/or copulation plug in the vagina). The day on which evidence of mating was observed was defined as Day 0 of gestation. Once mated, females were removed from the mating unit and housed individually for the remainder of gestation. Females unmated after the initial seven-day mating interval were redistributed (randomly) to a different male within the same treatment group until evidence of mating was observed or for seven additional days. This same procedure was repeated for a third seven-day mating interval for unmated females. This same mating design was used for F1 generation. In the mating assignments of the F1 generation, care was taken in establishing the mating units to avoid brother-sister matings.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Two diet samples of approximately 100 grams each were collected for each test diet and control at the weekly preparation intervals through out the study. Analyses to confirm concentration levels of diets intended for use on study were performed for the first four mixes (Study Weeks 1-4) and subsequently for every fourth mix (Study Weeks 8, 12, 16, 20. 24. 28, 32, 36, 40, 44 and 48) for the remainder of the study. Samples collected from mixes not scheduled for analyses have been retained frozen at this testing facility until issuance of the final report.
Diet samples analyzed were evaluated in duplicate. The diets were considered acceptable for study if values for the duplicate assays were within ± 20% of each other and the mean of the two values were within ± 20% of the nominal (desired) concentration. All diet analyses (i.e., homogeneity, stability and confirmation of concentration levels) were performed by the Analytical Department of Pharmaco LSR, Toxicology Services North America.
Duration of treatment / exposure:
F0 generation animals received the appropriate treated diets for 14 weeks prior to initiation of mating (i.e., pre-mating period) and treatment continued until sacrifice. Following the formal initiation of the pre-mating period, F1 generation animals received the appropriate treated diets for 14 weeks prior to initiation of mating and treatment continued until sacrifice. F1 pups consumed diets at the treatment level of the dam late in lactation and selected F1 animals continued to consume diets at these concentration levels during the post-weaning period through to the formal initiation of the pre-mating period.
Frequency of treatment:
continuous
Details on study schedule:
Both parental generations received a 14-week pre-mating period and treatment continued during a 21-day mating period and post-mating interval (males and unmated females) until sacrificed. Mated females continued to be treated during the ensuing gestation, lactation and post-weaning periods until sacrifice.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200, 600 and 1800 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
14, 42, and 128 mg/kg bw/day for males and 16, 49, and 149 mg/kg bw/day for females, respectively, during premating
Basis:

No. of animals per sex per dose:
30
Control animals:
yes, plain diet
Details on study design:
Parental animals (F0, F1) were observed twice daily for mortality and unusual findings and each animal received a detailed physical examination weekly. Body weights and food consumption for the parental animals were recorded weekly during the pre-mating treatment periods and these parameters continued to be recorded weekly for males during the mating and post-mating period through to sacrifice. Body weights and food consumption were recorded for mated females at regular intervals during the gestation period and females with litters continued to be weighed at regular intervals during the lactation period. Each parental generation produced a single litter and pups were weaned on lactation day 21. On lactation day 4, litters with greater than eight pups were culled to that number so as to equalizes sex distribution (four/sex), when possible; litters with less than eight pups at Day 4 were not adjusted. During lactation, litter size, pup weights and pup sex distribution data were recorded. Randomly selected pups from the F1 litters (at least one pup/sex/litter) were chosen to become the F1 parental generation. At sacrifice, parental animals were given a gross postmortem examination and reproductive tissues, pituitary glands and gross lesions were taken and preserved in 10% neutral buffered formalin. During the sacrifice of the F1 parental males, testes and epididymal weights were also recorded. Reproductive tissues and pituitary glands were evaluated histomorphologically for control and high-dose animals from both parental generations and gross lesions with the exclusion of incisor observations, were evaluated for all animals.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (observations of mortality and gross clinical findings)
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes (Detailed physical examination for signs of local or systemic toxicity, pharmacological effects and palpation for tissue masses).
- Time schedule: weekly

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes. Compound intake was calculated using food consumption and the nominal concentration in the diet.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- Maximum of 8 pups/litter

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
-number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities


GROSS EXAMINATION OF DEAD PUPS:
-yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
At sacrifice, parental animals w ere given a gross postmortem examination and reproductive tissues, pituitary glands and gross lesions were taken and preserved in 10% neutral buffered formalin. During the sacrifice of the F1 parental males, testes and epididymal w eights w ere also recorded. Reproductive tissues and pituitary glands were evaluated histomorphologicallfy or control and high-dose animals from both parental generations and gross lesions w ith the exclusion of incisor observations, were evaluated for all animals.
Postmortem examinations (offspring):
The unselected F1 pups and all F2 pups were sacrificed at weaning or shortly thereafter, and evaluated for external and internal irregularities and only abnormal tissues were saved (10% neutral buffered formalin).
Statistics:
Several statistical methods were used, dependent on the parameter studied.
Reproductive indices:
Mating, pregnancy and fertility rates; mean number of days-to-mating
Offspring viability indices:
percentage of pups alive at day 4

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

200 and 600 ppm:
No adverse effects of treatment at dietary levels of 200 or 600 ppm were evident from parental or neonatal parameters.
During the detailed physical evaluations of the parental animals, white discoloration of the incisors (upper and/or lower) was seen in both generations at both concentration levels and at the 600 ppm level, additional observations involving the incisors i.e., presence of a beveled edge along the anterior margin and/or a mottled appearance, were seen with increased frequency. These effects which were considered indicative of a treatment-related response were almost identical to toxic effects noted with excessive fluoride exposure i.e., dental fluorosis. This was not entirely unexpected considering the chemistry of the test material. No changes in the critical reproductive indices were seen at the low- and mid-dose levels during either generation and the effects seen were clearly non-reproductive organ effects involving the teeth.

1800 ppm:
At the 1800 ppm dietary level, no adverse effects of treatment were evident in the parental generation animals (F0, F1). The only treatment-related responses seen in these parental-animals involved observations to the incisors similar to those discussed above. All of these types of observations were seen with increased frequency at this treatment level. Bevelled anterior edge of the lower incisor was observed in 67% of animals from both generations at 1800 ppm.
No adverse effect of treatment at the 1800 ppm dietary level was evident from the macroscopic evaluations of the parental animals. Likewise, microscopic evaluation of the reproductive tissues and pituitary glands of the parental animals (F0, F1) revealed no adverse effects. Additionally, microscopic evaluation of the incisors of several F1 males with findings at necropsy i.e., white discoloration, beveled edges, and other gross lesions for both parental generations revealed no adverse effect of treatment.

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
200 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: dental fluorosis
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
1 800 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: no effects on fertility were observed
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
600 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: decreased mean body weights during lactation for both the F1 and F2 litters and an increased incidence of F1 and F2 animals with pale/white livers and/or kidneys and enlarged hearts at weaning
Remarks on result:
other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
no effects observed

Details on results (F1)

At the 1800 ppm dietary level, a decrease in mean body weights during lactation for both the F1 and F2 litters (significantly decreased pup body weights during lactation days 7, 14, and 21 (82%-88% of control in F1 offspring) and days 4, 7, 14, and 21 (74%-89%) of control in F2 offspring) and an increased incidence of F1 and F2 animals with pale/white livers and/or kidneys and enlarged hearts at weaning.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion