Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 25, 1989 - June 9, 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: -U.S. Food and Drug Administration (1966). Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use.
Deviations:
not specified
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6-dimethylhept-5-enal
EC Number:
203-427-2
EC Name:
2,6-dimethylhept-5-enal
Cas Number:
106-72-9
Molecular formula:
C9H16O
IUPAC Name:
2,6-dimethylhept-5-enal
impurity 1
Chemical structure
Reference substance name:
6-methylhept-5-en-2-one
EC Number:
203-816-7
EC Name:
6-methylhept-5-en-2-one
Cas Number:
110-93-0
Molecular formula:
C8H14O
IUPAC Name:
6-methylhept-5-en-2-one
impurity 2
Chemical structure
Reference substance name:
(1R,2R,5R)-2-methyl-5-(prop-1-en-2-yl)cyclopentan-1-ol
Cas Number:
83059-39-6
Molecular formula:
C9H16O
IUPAC Name:
(1R,2R,5R)-2-methyl-5-(prop-1-en-2-yl)cyclopentan-1-ol
impurity 3
Chemical structure
Reference substance name:
(1R,2S,5R)-2-methyl-5-(prop-1-en-2-yl)cyclopentan-1-ol
Cas Number:
83026-65-7
Molecular formula:
C9H16O
IUPAC Name:
(1R,2S,5R)-2-methyl-5-(prop-1-en-2-yl)cyclopentan-1-ol
impurity 4
Reference substance name:
2-(3-methylcyclopent-2-en-1-yl)propan-2-ol
Molecular formula:
C9H16O
IUPAC Name:
2-(3-methylcyclopent-2-en-1-yl)propan-2-ol
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)BR
Sex:
female
Details on test animals or test system and environmental conditions:
---Test System ---
The Charles River Crl:CD®(SD)BR rat was selected for evaluation of B212 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.
On April 25, 1989, 130 female rats were received by the Test Facility from Charles River Breeding Laboratories, Inc., St. Constant, Canada. At receipt, the female rats were 72 days of age (birthdate of the female rats in the population used: February 12, 1989). Following arrival, these female rats weighed from 189 to 243 g. Forty (40) apparently healthy female rats were selected for use in this study. These female rats were acclimatized for approximately one week prior to the first day of intubation.
On April 11, 1989, 160 male rats of the same strain and source as the female rats were received by the Test Facility. At receipt, these male rats were 73 days of age (birthdate of the male rats in the population used: January 28, 1989). Following arrival, these male rats weighed from 238 to 336 g. Because the male rats were used only as breeders, and were not given the test article, they were not considered to be part of the test system. The test system consisted of the adult female rats and their litters.

---Experimental Design and Control of Bias---
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 130) that was used for identification during the acclimation period. Unique permanent numbers were assigned on the day the selected female rats were placed on study. Each female rat was individually identified with a Monel® self-piercing ear tag (Gey Band and Tag Co., Inc., No. MSPT 20101) inscribed with the rat's designated unique number. The male breeder rats were similarly randomized upon arrival and individually identified with a Monel® self-piercing ear tag (Gey Band and Tag Co., Inc., No. MSPT 20101) inscribed with the rat's designated unique number.
The 40 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 following dosage groups:
Group I : 0 mg/kg/d (vehicle) - 10 females - animal numbers 7166 to 7175
Group II : 300 mg/kg/d (vehicle) - 10 females - animal numbers 7176 to 7185
Group III : 1500 mg/kg/d (vehicle) - 10 females - animal numbers 7186 to 7195
Group IV : 3000 mg/kg/d (vehicle) - 10 females - animal numbers 7196 to 7205

Following seven days of test article administration, the female rats were assigned to cohabitation with untreated male rats. One male was paired with one female rat for a maximum of seven days. Female rats with spermatozoa observed in smears of vaginal contents and/or copulatory plugs observed in situ were considered to be at day 0 of presumed gestation and assigned to individual housing. All dams were permitted to deliver and nurse their litters during a four-day lactation period (day 1 of lactation/postparturition was defined as the day the first pup was delivered).
All dams and their pups were sacrificed with carbon dioxide on day 4 of lactation. All dams were examined for gross lesions of the thoracic and abdominal viscera. The same necropsy procedures were used for rats that were found dead or sacrificed moribund on the days these events occurred. (Dams that did not deliver a litter were sacrificed on day 25 of presumed gestation.) Pups that were stillborn or found dead were necropsied. Adult and pup tissues with gross lesions present were preserved in neutral buffered 10% formalin.

---Environmental Conditions---
The rats were housed individually in wire-bottomed stainless-steel cages suspended above absorbent paper liners, except during the postparturition period. 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, the 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. The study room was independently supplied with a minimum of ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters (Airo Clean® Room). Room temperature was recorded constantly throughout the study and was targeted to range between 70°F and 78°F. Humidity was recorded daily and was targeted to range between 35% and 65%. An automatically-controlled fluorescent light cycle was maintained at 12 hours light:12 hours dark, with the dark cycle beginning at 1900 hours EST (2000 EDT). No deviations occurred in the environmental conditions during the conduct of this study.
The rats were provided the meal form of Certified Rodent Chow® #5002 (Ralston Purina) ad libitum throughout the study. Routine analyses performed by the feed supplier did not reveal any contaminants in the. feed or deviations from expected nutritional requirements. Copies of the results of the feed analyses are available in the raw data.
Local water that had been passed through a reverse osmosis membrane was available to the rats ad libitum (individual sipper tubes) from an automatic watering system, when the rats were housed in stainless-steel hanging wire cages. The same water was supplied via individual bottles to rats housed in nesting boxes. Chlorine was added to the processed water as a bacteriostat. The water contained approximately 0.0 to 1.4 ppm of chlorine when analyzed and was determined to be suitable for consumption. The water is analyzed annually for possible chemical contamination (Lancaster Laboratories, Inc., Lancaster, Pennsylvania) and monthly for possible bacterial contamination (Purity- Standard Laboratories, Chalfont, Pennsylvania). Copies of the results of the water analyses are available in the raw data.
No agent present in either the feed or water was known to interfere with the results of this study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The oral route (gavage) was selected for use because it is one of the intended routes of human exposure of the test article, and the exact dosage can be administered. The Sponsor selected the dosages tested [0(Vehicle), 300, 1500 and 3000 mg/kg/day].
Details on mating procedure:
On April 11, 1989, 160 male rats of the same strain and source as the female rats were received by the Test Facility. At receipt, these male rats were 73 days of age (birthdate of the male rats in the population used: January 28, 1989). Following arrival, these male rats weighed from 238 to 336 g. Because the male rats were used only as breeders, and were not given the test article, they were not considered to be part of the test system. The test system consisted of the adult female rats and their litters.
Following seven days of test article administration, the female rats were assigned to cohabitation with untreated male rats. One male was paired with one female rat for a maximum of seven days. Female rats with spermatozoa observed in smears of vaginal contents and/or copulatory plugs observed in situ were considered to be at day 0 of presumed gestation and assigned to individual housing. All dams were permitted to deliver and nurse their litters during a four-day lactation period (day 1 of lactation/postparturition was defined as the day the first pup was delivered).
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
The female rats were given appropriate dosages of B212 for seven days prior to a seven-day cohabitation period. Daily administration of the test article to the female rats continued until scheduled sacrifice on: 1) day 25 of presumed gestation (rats that did not deliver a litter); or 2) day 4 of lactation (rats that delivered litters).
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/d (vehicle)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1500 mg/kg/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
3000 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
10 females
Control animals:
yes, concurrent vehicle

Examinations

Statistics:
Baseline maternal body weight data, body weight changes during the premating, gestation and lactation periods, feed consumption data, and litter averages for pup body weights and percentage of 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), 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.
Natural delivery parameters involving discrete data were evaluated using the Kruskal-Wallis Test procedures previously described.

Sokal, R.R. and Rohlf, F.J. (1969). Bartlett's test of homogeneity of variances. Biometry, W.H. Freeman and Co., San Francisco, pp. 370-371.
Snedecor, G.W. and Cochran, W.G. (1967). Analysis of Variance. Statistical Methods, 6th Edition, Iowa State University Press, Ames, Iowa, pp. 258-275.
Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Stat. Assoc. 50:1096-1129.
Sokal, R.R. and Rohlf, F.J. (1969). Kruskal-Wallis Test. Biometry, W.H. Freeman and Co., San Francisco, pp. 388-389.
Siege!, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York, pp. 96-104.
Dunn, O.J. (1964). Multiple comparisons using rank sums. Technometrics 6(3):241-252.

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 300 - < 1 500 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Significant decreases (P≤0.05) in pup body weights at birth and pup viability occurred for the middle dosage group, as compared with the control group values.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Results

The 3000 mg/kg/day dosage of B212 caused the moribund sacrifice or death of eight (P≤0.01) high dosage group rats on days 2 to 4 of the premating period. These events were preceded by clinical signs including decreased motor activity, excess salivation, oral exudate, urine-stained abdominal fur and chromodacryorrhea. Each (P≤0.01) of these rats had external observations at necropsy that were generally interrelated with antemortem clinical signs. Necropsy of one of these rats revealed liver changes.

 

Clinical observations attributed to the test article, in addition to the mortality previously described, occurred for each group given the test article. These signs included excess salivation (low, middle and high dosage groups), decreased motor activity, oral exudate, bradypnea, urine-stained abdominal fur (middle and high dosage groups), labored breathing, ataxia, impaired righting reflex and chromodacryorrhea (high dosage group). Decreased motor activity and excess salivation/oral exudate occurred for significant (P≤0.01) numbers of middle and high dosage group rats, and labored breathing for significant (P≤0.01) numbers of high dosage groups rats during the premating period. During gestation, excess salivation occurred for significant (P≤0.01) numbers of middle and high dosage group rats, and decreased motor activity and ataxia occurred for significant (P≤0.01) numbers of high dosage group rats.

 

Dosage-dependent inhibitory effects on body weight gains occurred during the premating period for each group given the test article, as compared with the control group values. As the result of these effects, significant (P≤0.05) decreases in average body weights occurred for the middle dosage group on days 6 and 7 of the study, and for the high dosage group on days 2 through 7 of the study. Body weight gains tended to be decreased during gestation for the middle and high dosage groups. Maternal body weight averages continued to be decreased for the middle and high dosage groups at the completion of the gestation period and on the first day of the lactation period, although the differences were not significant, as compared with the control group value. Average maternal body weight gains for the low and middle dosage groups were unaffected by the test article during lactation; death of the only high dosage group litter precluded these values from analyses at the high dose.

 

During the premating period, significant (P≤0.05 to ≤0.01) decreases in absolute and/or relative feed consumption values were produced by the low and middle dosages of B212; probably reflecting excessive spillage, feed consumption values were significantly increased (P≤0.01) for the high dosage group. Absolute and relative maternal feed consumption values for the middle and high dosage groups were generally increased during gestation, observations that were probably interrelated with excess salivation, an effect of these dosages of the test article [the relative feed consumption value was significantly increased (P≤0.01) for the middle dosage group on days 14 to 16 of gestation]. Decreased absolute and relative maternal feed consumption values occurred for the middle dosage group during the lactation period. The death of the only high dosage group litter precluded the values for this dam from analyses.

 

Mating and fertility incidences were similar for the control, low and middle dosage groups. One of the two high dosage group rats that survived the premating period mated and became pregnant. This high dosage group dam delivered a litter that died (P≤0.01) during the four-day lactation period.

Significant (P≤0.05 to P≤0.01) decreases in pup viability occurred for the middle and high dosage groups, as compared with the control group values. The middle dosage group litters weighed significantly less (P≤0.05) than the control group litters at birth; the only high dosage group litter weighed remarkably less than the control group value.

Dosages of B212 as high as 3000 mg/kg/day did not affect the averages for durations of cohabitation or gestation, implantation sites or pup sex ratios.

No malformations or gross lesions identified in the pups were attributable to administration of B212 to the dams.

 

Conclusion

Based on the data in this study, the maternal no-observable-adverse-effect-level (NOAEL) for B212 was 300 mg/kg/day; minimal incidences of clinical signs, decreased body weights and significant (P≤0.01) decreases in absolute and relative feed consumption values occurred only during the premating period for the 300 mg/kg/day dosage group, these findings were not considered adverse. Significant (P≤0.01) numbers of 1500 and 3000 mg/kg/day dosage group rats had clinical signs and significant (P≤0.05 to P≤0.01) decreases in absolute and relative feed consumption values and body weight averages during the premating period. Eight (P≤0.01) of the high dosage group rats were moribund sacrificed or found dead on days 2, 3 and 4 of the premating period.

One (P≤0.01) of the two surviving high dosage group rats mated; this dam delivered a litter that died during the four-day lactation period. Mating and fertility of rats given B212 at dosages as high as 1500 mg/kg/day were similar to the control group values.

The NOAEL for B212 in the offspring was greater than 300 mg/kg/day and less than 1500 mg/kg/day. Significant decreases (P≤0.05) in pup body weights at birth and pup viability occurred for the middle dosage group, as compared with the control group values. These parameters were also affected for the only high dosage group litter during the period it lived.

 

Thus, B212 was not uniquely hazardous to reproductive performance of female rats or the growth and development of their offspring.

Applicant's summary and conclusion

Conclusions:
B212 was not uniquely hazardous to reproductive performance of female rats or the growth and development of their offspring.
Executive summary:

B212 was administered orally via gavage once daily to Crl:CD®(SD)BR virgin female rats (ten rats per group) at dosages of O(Vehicle, corn oil), 300, 1500 and 3000 mg/kg/day. All dosages were given at a dosage volume of 5 mL/kg/day and adjusted daily for body weight changes. Appropriate dosages of the test article were given to the female rats for seven days prior to and then through cohabitation (maximum of seven days), gestation, delivery and a four-day lactation/postparturition period (dams that delivered litters).

Mating/day 0 of presumed gestation was identified on the basis of spermatozoa present in a vaginal smear and/or a copulatory plug in situ. The rats were observed for clinical signs, death and/or delivery of a litter. Body weights were recorded daily during the dosage period; feed consumption observations were made weekly prior to mating, on days 0, 6, 14, 16, 21 and 25 of presumed gestation, and on days 1 and 4 of lactation (the day the first pup was born was defined as day 1 of lactation/postparturition). All dams were necropsied and examined for gross lesions. The 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 postparturition period. The pups were necropsied when found dead.

Based on the data in this study, the maternal no-observable-adverse-effect-level (NOAEL) for B212 was 300 mg/kg/day; minimal incidences of clinical signs, decreased body weights and significant (P≤0.01) decreases in absolute and relative feed consumption values occurred only during the premating period for the 300 mg/kg/day dosage group, these findings were not considered adverse. Significant (P≤0.01) numbers of 1500 and 3000 mg/kg/day dosage group rats had clinical signs and significant (P≤0.05 to P≤0.01) decreases in absolute and relative feed consumption values and body weight averages during the premating period. Eight (P≤0.01) of the high dosage group rats were moribund sacrificed or found dead on days 2, 3 and 4 of the premating period.

One (P≤0.01) of the two surviving high dosage group rats mated; this dam delivered a litter that died during the four-day lactation period. Mating and fertility of rats given B212 at dosages as high as 1500 mg/kg/day were similar to the control group values.

The NOAEL for B212 in the offspring was greater than 300 mg/kg/day and less than 1500 mg/kg/day. Significant decreases (P≤0.05) in pup body weights at birth and pup viability occurred for the middle dosage group, as compared with the control group values. These parameters were also affected for the only high dosage group litter during the period it lived.

 

Thus, B212 was not uniquely hazardous to reproductive performance of female rats or the growth and development of their offspring.