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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 June to 05 August 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study conducted similarly to OECD test guideline No. 421. Read-across substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
No analytical data on preparations (homogeneity/stability); males not exposed to the test item; missing histopathological data and organ weight for reproductive organs; only one-week exposure of females before mating.
Qualifier:
equivalent or similar to
Guideline:
other: U.S. Food and Drug Administration (1966). Guidelines for reproduction studies for safety evaluation of drugs for human use.
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Justification for study design:
- Basis for dose level selection: The following dose levels i.e., 0 (vehicle), 250, 500 and 1000 mg/kg bw/day were selected based on toxicity studies conducted by the Sponsor.
- Route of administration: The oral route (gavage) was selected for use because it is one of the intended routes of administration of the test article to humans.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lorillard, Inc. / Lot. No. 1
- Physical state: Clear colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored protected from light under refrigeration

Test animals

Species:
rat
Strain:
other: Crl: CD® (SD) BR
Details on species / strain selection:
Crl: CD® (SD) BR rats were selected in this study because (1) this strain of rat has been demonstrated to be sensitive to reproductive and developmental toxins; (2) it has been widely used throughout industry for reproductive toxicity evaluations; and (3) historical data and experience exist.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan.
- Females nulliparous and non-pregnant: Yes
- Age at receipt: 71 days (females); 73 days (males)
- Weight at receipt: 187-232 g (females); 291-337 g (males)
- Housing: Rats were housed individually in wire-bottomed stainless steel cages suspended above absorbent paper liners, except during the acclimation, cohabitation and post-parturition periods. During cohabitation (a maximum of seven days), male and female rats were housed together, one male per female, in the same wire-bottomed cages. Beginning no later than day 20 of presumed gestation, female rats were individually housed in nesting boxes. Each dam and delivered litter were housed in a common nesting box during the four-day lactation period. Nesting boxes contained bed-o'cobs® bedding.
- Diet: Rats were provided with the meal form of Certified Rodent Chow 5002 (Ralston Purina), ad libitum
- Water: Local water that had been passed through a reverse osmosis membrane was available to the rats, ad libitum
- Acclimation period: ca. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 70-78 ° F
- Humidity: 35-65%:
- Air changes: Ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 28 June to 05 August 1988

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Mazola® Lots APR0789B, APR1489A and 80299
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test substance was dissolved in corn oil at concentrations of 0, 50, 100 and 200 mg/mL. The suspensions were prepared weekly at the Test Facility. All prepared suspensions were refrigerated and protected from light in amber glass bottles.

VEHICLE
- Concentration in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

DOSE VOLUME: 5 mL/kg bw/day
Details on mating procedure:
- M/F ratio per cage: 1:1
- Following seven days of test article administration, the female rats were assigned to cohabitation with breeder male rats.
- Length of cohabitation: One male was paired with one female rat for a maximum of seven days.
- Proof of pregnancy: Female rats with spermatozoa observed in smears of vaginal contents and/or plugs observed in situ were considered to be at day 0 of presumed gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (5 mL each) of each concentration were reserved on the first day of the dosage period and at the end of the dosage period and were shipped to the Sponsor for analysis.
Duration of treatment / exposure:
- Female rats were administered test substance for seven days prior to a seven-day cohabitation period. Daily dosage of the test substance to the female rats continued until scheduled sacrifice on: (a) day 25 of presumed gestation (rats assigned to natural delivery that did not deliver a litter); or (b) day 4 of lactation (rats that naturally delivered litters).
- Pups were possibly exposed to the test substance during maternal gestation or via maternal milk during the postparturition period. The offspring were not intentionally administered the test substance.
Note: Dams in the process of delivering pups were not given a daily dosage until later in the same day, when parturition had been completed.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 female rats/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The following dose levels i.e., 0 (vehicle), 250, 500 and 1000 mg/kg bw/day based on toxicity studies conducted by the Sponsor.
- Rationale for animal assignment: Upon arrival, the female rats were assigned two per cage on the basis of a computer-generated randomization procedure. Each female rat was assigned a temporary pretest number (1 through 240) that was used for identification during the acclimation period. Unique permanent numbers were assigned on the day the female rats were placed on study. The male breeder rats were similarly randomized upon arrival and assigned unique permanent numbers. Each rat was individually identified with a Monel® self-piercing ear tag inscribed with the rat's designated unique number.
The female rats were selected for study on the basis of physical appearance, and body weight. A second computer-generated (weight-ordered) randomization procedure was performed to assign these female rats to the dosage groups.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
F0 GENERATION FEMALE RATS

CLINICAL OBSERVATIONS: Yes
- Time schedule: All female rats were observed for viability at least twice each day of the study. These rats were also observed for clinical signs and general health one or two times during the acclimation period (recorded by exception) and for clinical signs, general health and/or pharmacotoxic effects daily during the dosage period (immediately prior to and then approximately 30 minutes after intubation).
Maternal behavior of the dams was evaluated daily when the pups were examined during the four-day lactation period. Specific maternal behavioural observations were recorded on days 1 and 4 post-parturition, when the pups were weighed. Variations from expected maternal behavior were recorded by exception during each day of the lactation period.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights for the female rats were recorded at least once weekly during the acclimation and pre-dosage periods and daily during the dosage period.

FOOD CONSUMPTION:
- Food consumption was recorded weekly during the premating/pre-administration period; on days 0, 6, 10, 14, 16, 20 and 25 during the presumed gestation period; and on days 1 and 4 of the lactation period.

OTHER:
- Mating performance was evaluated daily during the cohabitation period and/or confirmed by observation of pregnancy (implantation sites present in utero on day 25 of presumed gestation, or natural delivery of a litter).
- The female rats were evaluated for duration of gestation (day 0 of presumed gestation to the day the first pup was delivered), litter size (live and dead pups and live pups only) and pup viability.
Litter observations:
LITTERS
- Each litter was evaluated for viability a minimum of twice each day during the four-day lactation period.
- The pups present in each litter were counted once each day.
- Physical signs (including nursing behavior and gross external physical anomalies) were recorded for the pups once each day during the four-day lactation period.
- Pup body weights were recorded on days 1(birth) and 4 post-parturition.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Pups that either appeared stillborn or that died before initial examination of the litter for viability were examined for vital status at birth. Each litter was subsequently examined daily for pup viability.

GROSS EXAMINATION OF DEAD PUPS:
- Dead pups were necropsied and examined for the cause of death. Tissues with observed gross lesions were preserved in neutral buffered 10% formalin for possible future evaluation.
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: All surviving animals; Dams that did not deliver litters were sacrificed with carbon dioxide on day 25 of presumed gestation and examined for gross lesions and implantation sites. On day 4 or 5 of lactation, as described previously, all dams that delivered litters were sacrificed with carbon dioxide.

GROSS NECROPSY
- Dams were examined for gross lesions and the number and placement of implantation sites. Ovaries from all dams and any observed gross lesions were preserved in neutral buffered 10% formalin for possible future evaluation.
Postmortem examinations (offspring):
When not precluded by autolysis and/or cannibalization by the dam, dead pups were necropsied and examined for the cause of death . Tissues with observed gross lesions were preserved in neutral buffered 10% formalin for possible future evaluation .
Statistics:
Maternal and pup incidence data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.
Baseline maternal body weight data, body weight changes during gestation and lactation, feed consumption data, duration of cohabitation, litter averages for percent mortality, pup body weight and percent male pups were analyzed using Bartlett's Test of Homogeneity of Variances and the Analysis of Variance, when appropriate (i.e., Bartlett's Test was not significant, P>0.05). If the Analysis of Variance was significant (P≤0.05), then Dunnett's Test was used to identify the statistical significance of individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett's Test was significant (P≤0 .05)], the Kruskal-Wallis Test was used when 75% or fewer ties were present; when more than 75% ties were present, the Fisher's Exact Test was used. In cases where the Kruskal-Wallis Test was statistically significant (P≤0.05), Dunn's Method of Multiple Comparisons was used to identify statistical significance of individual groups.
The Analysis of Covariance was used to evaluate average body weight changes from day 1 of dosage, day 0 of presumed gestation and day 1 of lactation (F0 Generation female rats).
Natural delivery parameters involving discrete data were evaluated using the Kruskal-Wallis Test procedures previously described.

See the statistical analysis in "Attached background material" section

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations that occurred at dosage-dependent incidences and were considered to be effects of the test substance included excess salivation (250, 500 and 1000 mg/kg bw/day dosage groups), urine-stained abdominal fur, ataxia, and decreased motor activity (1000 mg/kg bw/day dosage group). Excess salivation occurred for the low and middle dosage group rats during the premating and gestation periods. High dosage group rats had excess salivation occur during the premating, gestation and lactation periods, as well as additional clinical observations occur during the premating and gestation periods. The number of rats with excess salivation was significantly increased (P≤0.05 to P≤0.01) in the middle and high dosage groups, as compared with the control group number. At 1000 mg/kg bw/day dosage group, the number of rats that also had urine stained abdominal fur, in addition to excess salivation, was significantly increased (P≤0.01), in comparison with the control group number during the premating period.
All other clinical observations were considered unrelated to the test substance. Red vaginal exudate, noted for one high dosage group rat on day 0 of gestation, was considered to be the result of trauma incurred during mating. A mass in the left inguinal area, that was probably a galactocele, was evident for one middle dosage group rat from day 0 of gestation through necropsy on day 4 post-parturition. Limited areas of alopecia occurred at biologically comparable incidences in the rats (one rat in each of the low, middle and high dosage groups).
Mortality:
no mortality observed
Description (incidence):
No deaths occurred during the conduct of this study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Premating:
Administration of the 1000 mg/kg bw/day dosage of test substance to the rats inhibited body weight gain prior to cohabitation. Significant (P≤0.01) weight loss occurred for the 1000 mg/kg bw/day dosage group after the first dosage was given. Between days 1 and 2 of study, rats in the control, low, middle and high dosage groups had average body weight changes of +2.8, +3.8, +2.7 and -6.0++ g, respectively. In these same respective groups, average body weight gains between days 1 and 7 of study were +17.2, +18.7, +19 .1 and +13 .1 g. Body weights on day 7 of study averaged 246.8, 247.1, 246.0 and 244.4 g in the four respective groups.

Gestation
Although the effect was not clearly dosage-dependent, possibly reflecting differences in litter sizes, biologically remarkable increases in average body weight gains occurred for pregnant rats in each group given test substance, as compared with the control group value. These increases in average maternal body weight gains were statistically significant (P≤0 .05) for the low and high dosage group rats between days 6 and 14 of gestation and resulted in significantly (P≤0.05 to P≤0.01) increased average maternal body weights on days 14 and 16 of gestation.
In the control, low, middle and high dosage groups, respectively, maternal body weights averaged 251.5, 255.3, 252.4 and 259.5 g on day 0 of gestation, 326.7, 347.3**, 339.6 and 351.3** g on day 14 of gestation, and 347.8, 364.8*, 353.7 and 368.4* g on day 16 of gestation. On day 21 of gestation, maternal body weights averaged 419.1, 450.8, 434.6 and 442.8 g in these same respective groups. Between days 6 and 14 of gestation, maternal body changes averaged +44.3, +55.0*, +47.3 and +53.1* g in the control, low, middle and high dosage groups, respectively. Maternal body weight changes between days 0 and 21 of gestation averaged +167.9, +195.6, +182.1 and +183.3 g in these same respective groups.

Lactation
At the beginning of the lactation period, each group given test substance weighed more than the control group. During the four-day post-parturition period, the effect of the test substance that caused weight gain and increased feed consumption during gestation decreased in severity, although it remained present. Maternal body weights averaged 305.2, 328.2, 330.6 and 333.1 g, in the four respective groups on day 1 of lactation, and 310.5, 332.0, 326 .9 and 326.3 g on day 4 of lactation. Between days 1 and 4 post-parturition, dams in the control, low, middle and high dosage groups, respectively, gained +5.3, +3.8, -3.7 and -4.6 g.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Premating:
Feed consumption values were unaffected by administration of test substance to the rats during the premating period. The differences that occurred were neither biologically important nor statistically significant among the four groups (P>0.05). In the four respective groups, feed consumption averaged 16.1, 16.5, 15.6 and 15.6 g/day. Feed consumption relative to body weight values averaged 67.4, 69.1, 66.0 and 65.6 g/kg/day for the rats in these same respective dosage groups.

Gestation:
Feed consumption values (g/day and g/kg/day) were significantly (P≤0.05 to P≤0.01) increased for the pregnant rats in each group given the test substance, as compared with the control group values. These increases in feed consumption during gestation were the possible cause of the increased weight gain during gestation that occurred for the rats given the test substance, as compared with the control group rats. It is noteworthy that not only were absolute feed consumption values (g/day) increased, but that the feed consumption relative to body weight values (g/kg/day) were also increased, indicating that the rats consumed even more feed than was necessary to maintain their body weights.
In the control, low, middle and high dosage groups, respectively, dams consumed 20.5, 23.1**, 22.6* and 23 .5** g/day between days 0 and 20 of gestation. In these same respective groups, feed consumption relative to weight values averaged 65.5, 70.7*, 70.6* and 71.4** g/kg/day between days 0 and 20 of gestation.

Lactation:
Feed consumption was increased for the middle and high dosage groups during the four-day lactation period, as compared with the control group values. Between days 1 and 4 post-parturition, absolute maternal feed consumption averaged 25.0, 25.0, 29.3 and 27.8 g/day in the four dosage groups, respectively. Maternal feed consumption relative to body weight values averaged 81.6, 75.3, 88.5 and 84.2 g/kg/day during the four-day post-parturition period for these same respective groups.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Dosages of test substance as high as 1000 mg/kg bw/day did not adversely affect the reproductive performance of the female rats. In the 0(vehicle), 250, 500 and 1000 mg/kg bw/day dosage groups, respectively, 10, 8, 9 and 10 rats mated and were pregnant. These incidences of mating and pregnancy are within the range observed historically.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Administration of the 1000 mg/kg bw/day dosage of test substance to the dams increased in utero and postnatal deaths of the offspring, as compared with the control group values.
All pregnant dams delivered one or more live pups. Implantation averages were similar for all groups (there were averages of 16.9, 17.0, 16.3 and 16.4 implantations per pregnant dam in the control, low, middle and high dosage groups, respectively). Delivered live litter sizes averaged 15.3, 16.0, 15.0 and 12.8 pups in the four respective groups, indicating that in utero deaths (resorptions) were increased for the high dosage group.
Pup mortality was significantly (P≤0.01) increased for litters of dams given the 1000 mg/kg bw/day dosage of test substance, as compared with the control group value. One dam had each of its five live born pups die before weighing on day 1 of lactation (the dam had a total of 16 implantation sites in utero). In the control, low, middle and high dosage groups, respectively, 97.4, 98.4, 97.8 and 87.5%** of the liveborn pups survived four days. By day 4 post-parturition, there were averages of 14.9, 15.8, 14.7 and 11 .2 surviving pups per litter. The increased incidences of clinical observations for the high dosage group pups were related to morbidity.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Dosages of test substance as high as 1000 mg/kg bw/day did not adversely affect pup body weights.
Sexual maturation:
no effects observed
Description (incidence and severity):
Dosages of test substance as high as 1000 mg/kg bw/day did not adversely affect the durations of gestation and pup sex ratios.
Other effects:
no effects observed
Description (incidence and severity):
No anatomical malformations or variations were revealed by external examination or necropsy of the pups in this study.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
viability

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the NOAEL for maternal toxicity was 500 mg/kg bw/day based on altered body weight and food consumption at 1000 mg/kg bw/day. Increases in bodyweight and food consumption, not considered to represent a relevant adverse toxicological effect of the treatment, were observed at 250 mg/kg bw/day. The NOEL for developmental toxicity was 500 mg/kg bw/day based on the smaller live litter sizes (in utero deaths) at 1000 mg/kg bw/day.
Executive summary:

In a Reproduction / Developmental Toxicity Screening Test conducted similarly to OECD Guideline 421 and in compliance with GLP, test substance was administered to groups of Crl: CD® (SD) BR rats (10 females/dose) at 0, 250, 500 and 1000 mg/kg bw/day by oral (gavage) for seven days prior to and then through cohabitation (maximum of seven days), gestation, delivery and a four-day lactation/post-parturition period. Clinical signs, body weight change, food consumption were monitored during the study. All animals were subjected to a gross necropsy examination. All litters were examined for number, viability, body weight, sex ratio and external morphology of the pups. Delivered pups were additionally examined for viability, clinical observations and body weight during a four-day post-parturition period.

No female rats died during the conduct of this study. All treated group rats showed excessive salivation; this observation was statistically significant at 500 and 1000 mg/kg bw/day when compared with the control group. At 1000 mg/kg bw/day, urine-stained abdominal fur occurred during the premating period, and one or two rats in this dosage group had ataxia and/or decreased motor activity occur infrequently during the premating and/or gestation periods. No other clinical or necropsy observations for the rats in this study were considered effects of the test substance.

 

Biologically remarkable decreases in body weight gain and food consumption occurred at 1000 mg/kg bw/day during the premating period, with significant weight loss evident for this group after the first dosage was given. During gestation, biologically remarkable increases in weight gain and food consumption occurred in all treated groups when compared with the control group. Statistically significant increases in body weight gain at 250 and 1000 mg/kg bw/day, and statistically significant increases in absolute (g/day) and relative (g/kg/day) food consumption in all treated groups when compared with the control group. These effects remained present but decreased in severity during the lactation period.

 

No adverse effect on the reproductive performance was observed at up to 1000 mg/kg bw/day. There were no dosage-dependent or statistically significant differences in duration of cohabitation, pregnancy incidences or implantation averages among the four groups. All pregnant dams delivered one or more live pups. At 1000 mg/kg bw/day, in utero deaths, evident as a biologically remarkable decrease in delivered live litter size, and a statistically significant increase in pup mortality, with associated observations of pup morbidity were observed.

 

At 1000 mg/kg bw/day, no adverse effect on the duration of gestation, pup sex ratios, pup body weights or the gross morphology of the pups were observed.

 

Under the test conditions, the NOAEL for maternal toxicity was 500 mg/kg bw/day based on altered body weight and food consumption at 1000 mg/kg bw/day. Increases in bodyweight and food consumption, not considered to represent a relevant adverse toxicological effect of the treatment, were observed at 250 mg/kg bw/day. The NOEL for developmental toxicity was 500 mg/kg bw/day based on the smaller live litter sizes (in utero deaths) at 1000 mg/kg bw/day.